A Novel Motif Essential for SNARE Interaction with the K+
Channel KC1 and Channel Gating in Arabidopsis W
Christopher Grefen, Zhonghua Chen, Annegret Honsbein, Naomi Donald, Adrian Hills, and Michael R. Blatt1
Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cellular and Systems Biology-Plant Sciences, University
of Glasgow, Glasgow G12 8QQ, United Kingdom
The SNARE (for soluble N-ethylmaleimide–sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/
PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane,
and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K+
channel subunit KC1 and forms a tripartite complex with a second K+ channel subunit, AKT1, to control channel gating and
K+ transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We
made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein–
protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with
KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this
motif is the minimal requirement for SNARE-dependent alterations in K+ channel gating when heterologously expressed,
and that rescue of KC1-associated K+ current of the root epidermis in syp121 mutant Arabidopsis plants depends on
expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously
unidentified motif required for SNARE–ion channel interactions and lead us to suggest a mechanistic framework for
understanding the coordination of vesicle traffic with transmembrane ion transport.
INTRODUCTION
The superfamily of SNARE (for soluble N-ethylmaleimide–sensi-
tive factor protein attachment protein receptor) proteins, found in
all eukaryotes, is essential in later stages of vesicle targeting and
fusion. They help match vesicles with their target membranes for
delivery of specific membrane proteins and cargo, and they
overcome the dehydration forces associated with lipid bilayer
fusion in an aqueous environment. These processes sustain
cellular homeostasis in yeast (Ungar and Hughson, 2003), they
are essential for synaptic transmission between nerves (Jahn
et al., 2003), and they underpin growth and development in
plants (Campanoni and Blatt, 2007; Grefen and Blatt, 2008).
Complementary SNAREs, located at the vesicle and target
membranes, interact to draw the membrane surfaces together
for fusion. SNAREs comprise theminimal set of proteins required
to accelerate fusion in reconstituted vesicle preparations (Weber
et al., 1998; Parlati et al., 1999; Hu et al., 2003), although other
components, including the N-ethylmaleimide–sensitive factor,
Sec1, Munc18, and their homologs (Burgoyne and Morgan,
2007; Sudhof and Rothman, 2009), affect SNARE conforma-
tions and their interactions (Brunger, 2005; Lipka et al., 2007;
Bassham and Blatt, 2008).
In plants, SNAREs play important roles in vesicle traffic asso-
ciated with defense against fungal pathogens (Collins et al.,
2003; Pajonk et al., 2008) and the response to bacterial elicitors
(Robatzek et al., 2006). In addition, they contribute to events
beyond their canonical roles in membrane targeting and vesicle
fusion (Grefen and Blatt, 2008). The vacuolar SNAREs SYP22
and VTI11, for example, have surfaced as components essential
for gravitopism (Kato et al., 2002; Surpin et al., 2003; Yano et al.,
2003), plausibly linking sensory processing to vacuolar mem-
brane structure or composition (Saito et al., 2005). A fewSNAREs
are known also to interact with ion channels and affect their
regulation. In neuromuscular and neuroendocrine tissues, bind-
ing of the SNARESyntaxin 1A to K+ and Ca2+ channels is thought
to facilitate neurotransmission and hormone secretion (Leung
et al., 2007). In the model plants tobacco (Nicotiana tabacum)
and Arabidopsis thaliana, the SNARE SYP121 (=SYR1/PEN1)
has been implicated in hormonal regulation of Ca2+, Cl2, and K+
channels in guard cells (Leyman et al., 1999; Sokolovski et al.,
2008), in the latter case independent of its role in delivery and
recycling of the ion channel proteins (Sutter et al., 2006, 2007).
Recently, we reported that SYP121 of Arabidopsis, originally
identified with its tobacco homolog in a screen for signaling
elements associated with abscisic acid and drought (Leyman
et al., 1999; Geelen et al., 2002), interacts directly with the
regulatory K+ channel subunit KC1 and forms a tripartite complex
with a second K+ channel subunit, AKT1 (Honsbein et al., 2009).
KC1 interaction proved highly selective for SYP121. We found
the SYP121-KC1-AKT1 complex to be required for the activity
of inward-rectifying K+ currents at the plasma membrane of
root epidermal cells and for K+ nutrient acquisition and growth
when channel-mediated K+ uptake was limiting. These results
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Michael R. Blatt ([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.110.077768
The Plant Cell, Vol. 22: 3076–3092, September 2010, www.plantcell.org ã 2010 American Society of Plant Biologists
demonstrated an unexpected role for the SNARE analogous to
SNARE-ion channel complexes of mammals but unrelated to
signaling or its coupling to vesicle traffic. Thus, they raised
fundamental questions about the mechanics of interaction,
notably about the relationship of K+ channel binding to the
domains required for SNARE core complex assembly that drive
vesicle fusion. We have since identified the minimal sequence
motif harbored by SYP121 and prerequisite for its interaction
with KC1.We report here that themotif is unique to SYP121 and
localized to a region of canonical SNARE structure not previ-
ously associated with ion channel interactions. The results
show that the motif is essential for the interaction of SYP121
with KC1, and it is necessary for the SNARE to facilitate gating
in the K+ channel when expressed heterologously and in the
plant. Significantly, the identity and position of the motif within
the primary SNARE sequence points to a novel mechanism for
mutual control of SYP121-dependent membrane vesicle traffic
and of the K+ channels. Thus, the findings lead us to suggest a
novel framework for understanding the coordination of vesicle
traffic with transmembrane ion transport.
RESULTS
SYP121/SYP122 Chimeras Identify the N Terminus of
SYP121 as Essential for K+ Channel Interaction
We made use of a mating-based split-ubiquitin assay for
interacting proteins (Grefen et al., 2007, 2009) employed previ-
ously to identify SYP121 interaction with KC1 and their formation
of a tripartite complex with AKT1 (Honsbein et al., 2009). The
mating-based assay gives Met-sensitive rescue of yeast growth
on minimal medium. Because the assay relies on reassembly of
the N- and C-terminal halves (Nub and Cub) of the ubiquitin
moiety and cleavage of a LexA-VP16 transactivator, which then
migrates to the nucleus, it permits work with full-length, integral
membrane proteins. Our previous studies showed that KC1
bound selectively with SYP121 and not with SYP122, the closest
homolog to SYP121, with 64% amino acid sequence identity
with which it shares partial functional redundancy in vivo (Assaad
et al., 2004; Zhang et al., 2007; Bassham and Blatt, 2008). To
identify the binding domain responsible for SYP121 interaction,
we therefore made initial use of complementary sets of con-
structs to generate chimeric proteins in which segments of the
chimeric SNAREs corresponded to different combinations of the
SYP121 or SYP122 residue sequences.
Figure 1 shows the yeast mating-based split-ubiquitin assay
and supporting immunoblot data from one of three independent
experiments, each of which yielded equivalent results. Like other
Qa-SNAREs (Lipka et al., 2007; Grefen and Blatt, 2008; Sudhof
and Rothman, 2009), both SYP121 and SYP122 are type-II
integral membrane proteins comprising a C-terminal membrane
anchor, an H3 coiled-coil domain that binds with its cognate
SNARE partners to drive vesicle fusion, and an N-terminal set of
coiled-coil domains (Ha, Hb, and Hc) that folds back on the H3
domain and regulates its accessibility for binding. For purposes
of preliminary analysis, we divided the SNAREs between four
regions: N corresponding to the N-terminal 39–amino acid
Figure 1. SNARE Interaction with the KC1 K+ Channel Depends on the
Presence of the N Terminus of SYP121.
Yeast mating-based split-ubiquitin assay for interaction of the SNARE
chimeras with KC1-Cub as the bait. SNARE chimeras of SYP121 and
SYP122 were constructed by exchange of four domains designated N, H,
Q, and C, indicated above, corresponding to the N-terminal 39–amino acid
residues, the Ha, Hb, and Hc a-helices, the H3 or Qa a-helix, and the
transmembrane a-helix and C-terminal extension, respectively. Segment
alignments (above) for the two SNAREs at the junction points are shown
with arrows indicating the domain breaks. Yeast diploids created with
NubG fusion constructs of each chimera (left) and with SYP121 (in gray
font) and SYP122 (in black font) for reference together with controls
(negative, NubG; positive, wild-type Nub) spotted (left to right) on CSM
medium without Trp, Leu, and uracil (CSMwlu) to verify mating, CSM
without Trp, Leu, uracil, adenine, His, and Met (CSMwluahm) to verify
adenine- and His-independent growth (second panel), and on CSMwluahm
with the addition of 0.2 mM Met to verify interaction at lower KC1-Cub
expression levels (Obrdlik et al., 2004; Grefen et al., 2009). Diploid yeast
was dropped at 1.0 and 0.1 OD600 in each case. Immunoblot analysis
(5 mg total protein/lane) of the haploid yeast used for mating is included
(right) using commercial VP16 antibody for KC1 and both SYP121 and
SYP122 polyclonal antibodies for the SNARE chimeras, the latter showing
association of the principle epitopes with the Ha-Hb-Hc domains of the
SNAREs.
SNARE-K+ Channel Interaction Motif 3077
residues preceeding the Ha a-helix; H corresponding to the Ha,
Hb, andHca-helices (residues 40 to 192); Q (residues 193 to 283)
corresponding to the H3 a-helix terminating with the conserved
Thr residue at position 283; and C corresponding to the remaining
C-terminal sequence (residues 284 to 346) that incorporated the
transmembrane a-helix and an extended stretch of residues that
are predicted to reside outside the cell (Blatt et al., 1999; Leyman
et al., 1999). As before (Honsbein et al., 2009), a readout of
interactionwas evidenced by growth of diploid yeast on selective
media when they expressed both the KC1-Cub and Nub-
SYP121 fusion proteins, and growth was retained in the pres-
ence of 0.2 mMMet to repress transcription of the KC1-Cub bait
construct. Little or no growth was recovered on selective media
and in the presence of Met when yeast carrying KC1-Cub and
Nub-SYP122 were mated. Among the SYP121-SYP122 chi-
meras, growth was recovered in all matings incorporating fusion
constructs with the N domain of SYP121, even when the H, Q,
and C domains were derived from SYP122, and substitution with
the SYP122 N domain virtually eliminated growth when mated
with yeast carrying the KC1-Cub bait. Immunoblot analysis in
every case showed expression of the chimeric SNAREs, al-
though, as expected, the efficacy of the polyclonal antibodies
depended on the relative distribution of epitopes between the
two SNARE sequences in the chimeras. Expression of KC1 was
verified by rescue of diploid yeast growth with the wild-type Nub
and by immunoblots for the VP16 epitope of the fusion protein in
the THY.AP4 yeast prior to mating. Thus, we concluded that the
N-terminal 39 amino acids harbor a motif that is both sufficient
and necessary for SNARE interaction with the KC1 K+ channel.
Alignment of SYP121 and SYP122 shows that the N-terminal
amino acid sequences of the SNAREs diverge principally in three
short segments of 4 to 10 residues each, denoted in the lower-
case suffixes n1, n2, and n3 (Figure 2). We used these segments
as the basis for constructing a second round of chimeras. In the
first instance, we substituted the n1, n2, and n3 segments from
SYP122 into the corresponding positions in the backbone of the
Nub-SYP121 fusion construct. In a second set of experiments,
we used as a backbone the fusion construct comprising the N
domain of SYP121 and the H, Q, and C domains of SYP122 that
retained the capacity to rescue yeast growth in conjunction with
KC1-Cub (Figure 1). Figure 2 shows the readout from experi-
ments using each of these backbones along with supporting
immunoblot analyses. In both cases, an interactionwith KC1was
indicated for constructs that incorporated the n1 segment of
SYP121 by the rescue of yeast growth on selective media and in
the presence of Met, whereas substitutions with the n1 segment
of SYP122 showed a loss of yeast growth. Substitutions with
either the n2 or n3 segment from SYP122 had no appreciable
effect on the rescue of yeast growth. In short, these results
indicated that residues within the N-terminal 20 amino acids
were essential for SYP121 interaction with KC1.
The SYP121 N Terminus Is Essential for K+ Channel Gating
When Heterologously Expressed
KC1 is a so-called silent K+ channel subunit; expressed on its
own, it does not yield measurable K+ currents, but it interacts
with different inward-rectifying K+ channel subunits including
AKT1 (Obrdlik et al., 2004; Duby et al., 2008) to affect the voltage
dependence of channel gating (Geiger et al., 2009). AKT1 and
KC1 preferentially assemble in heteromers (Duby et al., 2008),
and in vivo these assemblies coalesce with SYP121 to yield
functional K+ currents (Honsbein et al., 2009). When expressed
Figure 2. SNARE Interaction with the KC1 K+ Channel Depends on the
Presence of the n1 Segment of the SYP121 N-Terminal Domain,
Corresponding to Amino Acid Residues Ser10-Pro17.
Yeast mating-based split-ubiquitin assay for interaction of the N-terminal
domain chimeras with KC1-Cub as the bait. N-terminal domain chimeras
were constructed by exchange of three segments designated n1, n2, and
n3, each comprising a sequence of 4 to 10 residues of SYP121 and
SYP122. Chimeras were constructed both in SYP121 and in the N1HQC2
domain chimera of SYP121 and SYP122 (see Figure 1). Alignment of the
segments for the two SNAREs within the N-terminal domain is shown
(above) with the segments as indicated. Yeast diploids created with
NubG-fusion constructs of each chimera and with controls (negative,
NubG; positive, wild-type Nub [NubWt]) spotted (left to right) on CSM
medium without Trp, Leu, and uracil (CSMwlu) to verify mating, CSM
without Trp, Leu, uracil, adenine, His, and Met (CSMwluahm) to verify
adenine- and His-independent growth (second panel), and with the
addition of 0.2 mMMet to verify interaction at lower KC1-Cub expression
levels (Obrdlik et al., 2004; Grefen et al., 2009). Diploid yeast was
dropped at 1.0 and 0.1 OD600 in each case. Immunoblot analysis (5 mg
total protein/lane) of the haploid yeast used for mating are included on
the right in each case using commercial VP16 antibody for KC1 (below)
and both SYP121 and SYP122 polyclonal antibodies for the SNARE
chimeras as in Figure 1.
3078 The Plant Cell
heterologously on its own or with KC1, AKT1 does yield an
inward-rectifying K+ current (Gaymard et al., 1996; Duby et al.,
2008). However, the gating of channels assembled as heteromers
of AKT1 and KC1, like that of the homomeric AKT1 channels,
differs fundamentally from that of the K+ channels in vivo, a
difference evident in the midpoint for channel activation (V1/2) and
sensitivity to a change in voltage (the gating charge, d) (Dreyer and
Blatt, 2009), unless coexpressed with SYP121 (Honsbein et al.,
2009).
We used these characteristics to explore the functional con-
sequences of selected SYP121 chimeras identified by the split-
ubiquitin experiments. Electrophysiological recordings were
performed under voltage clamp after coexpressing KC1 and
AKT1 together with the SYP121 mutants in Xenopus laevis
oocytes and verifying expression of the SNAREs. To ensure
activation of AKT1 in the oocytes, all combinations of channel
subunits and SNAREs were coexpressed with the protein kinase
CIPK23 and calcineurin-like activator CBL1 (Li and Luan,
2006; Xu et al., 2006). We extracted the gating charac-
teristics V1/2 and d in each case by jointly fitting the steady state
K+ currents to a Boltzmann function of the form
IK ¼ gmax
�V2EK
�=�1þ edðV2V1=2Þ=RT� ð1Þ
where gmax is the conductance maximum, V is the membrane
voltage, EK is the equilibrium voltage for K+, and R and T have
their usual meanings.
As before (Honsbein et al., 2009), we found that expressed
alone, AKT1 yielded an anomalous K+ current measurable at
voltagesnegative of250mV; expressingAKT1 togetherwithKC1,
with or without (data not shown) the noninteracting SNARE
SYP122, gave a K+ current measurable only at voltages negative
of 2140 mV (Figure 3). To avoid substantial indetermination in
fitted parameters obtained from these data and from subsequent
analyses (see also Figures 11 to 14),we applied standardmethods
of joint fittings with key parameters held in common between data
sets (curves) (Press et al., 1992) and introduced the minimal
assumption of parameters for known (control) data sets consistent
with previous analyses and observations that KC1 coexpression
does not affect significantly the saturation current or gating charge
of the K+ channels, only the value for V1/2 (Duby et al., 2008;
Honsbein et al., 2009). On analysis, these currents werewell-fitted
jointly to the Boltzmann function in every case with a gating
charge, d, near a value of unity andwith onlyV1/2 differing between
the two circumstances (Figures 3 and 4). Fittings of currents
Figure 3. Coexpression with SYP121 Selectively Rescues AKT1-KC1 K+
Current in Xenopus Oocytes.
(A) Current traces and steady state current–voltage curves recorded
under voltage clamp in 96 mM K+ from oocytes expressing KC1 alone
(squares), AKT1 alone (diamonds), AKT1 with KC1 (molar ratio 1:1) alone
(downward-facing open arrowheads), and with the SNAREs and SNARE
chimeras (molar ratio to KC1, 2:1; see Honsbein et al., 2009) SYP121
(upward-facing open arrowheads), N1HQC2 (upward-facing closed ar-
rowheads), N2HQC1 (downward-facing closed arrowheads), and
SYP121n1 incorporating the n1 sequence from SYP122 (circles). Data
for SYP122 omitted for clarity (see Figure 4). Insets: Corresponding
whole-cell currents cross-referenced by symbol. Clamp cycles: holding
voltage, �20 mV; voltage steps, 0 to �190 mV. Scale: 4 mA and 3 s.
Currents from oocytes injected with water and with SYP121 cRNA only
gave results similar to those for KC1 alone. All measurements were
performed in coexpression with CBL1 and CIPK23, which are essential
for AKT1 function in oocytes, using cRNA injections in 1:1:1 molar ratios
with AKT1 (Xu et al.,.2006). Solid curves are the results of joint, nonlinear
least squares fitting of the K+ currents (IK) to the Boltzmann function
(Equation 1) and are summarized in Figure 4. The characteristic voltage
dependence (V1/2) indicates the midpoint of the voltage range for gating,
and the apparent gating charge (d) is a unique property of the gate, its
sensitivity to voltage changes and the associated conformations. Best
fittings were obtained with gmax held in common (gmax for the data
shown, 48 6 9 nS) and with separate, joint values for d between SNARE
chimeras that showed interaction with KC1 and those that did not (see
Figures 1 and 2). Similar results were obtained in each of four indepen-
dent experiments (>24 oocytes for each set of constructs). Both analysis
and visual inspection showed an increase in d and shift in V1/2 on
inclusion of SYP121 and the interacting chimeras with AKT1 and KC1.
(B) Verification of SNARE protein expression. Immunoblot analysis of
total membrane protein (10 mg/lane) extracted from oocytes collected
after electrical recordings and detected with aSYP121 and aSYP122
antibodies (Tyrrell et al., 2007; Honsbein et al., 2009).
SNARE-K+ Channel Interaction Motif 3079
recorded on coexpression of KC1with AKT1 generally yieldedV1/2
values near the limit of clamp voltages achievable in oocytes.
CoexpressingAKT1andKC1withwild-typeSYP121 that interacts
with KC1 (Figure 1; see also Honsbein et al., 2009), by contrast,
yielded a K+ current at voltages negative of 2100 mV that was
well-fitted to the same Boltzmann function, but with values for d
near 2 and V1/2 close to 2150 mV (Figures 3 and 4). These
characteristics were similar to those reported previously on het-
erologous expression in oocytes andSf9 insect cells and compare
favorably with the characteristics of K+ currents obtained in vivo
(Gassmann and Schroeder, 1994; White and Lemtirichlieh, 1995;
Buschmann et al., 2000; Honsbein et al., 2009).
We found a similar and strong divergence of gating character-
istics that paralleled the SNARE–KC1 interactions when compar-
ing the gating parameters of the currents recorded with the
SNARE chimeras (Figures 3 and 4). Coexpressing AKT1 and KC1
with the SYP121-SYP122 chimera N1HQC2 yielded K+ currents
with V1/2 and d values similar to those associated with the wild-
type SYP121, whereas coexpressing AKT1 and KC1 with the
N2HQC1chimera gavecurrent characteristics that aligned closely
with those obtained on coexpressing AKT1 and KC1 alone. Much
the same separation of gating characteristics was observed on
expressing the SYP121 incorporating the n1 segment substitution
fromSYP122. In this case, values for V1/2 and d aligned with those
derived from currents on expressing AKT1 with KC1 alone or with
SYP122. Thus, analysis of channel gating yielded unequivocal
evidence of the functional requirement for the SYP121 N terminus
to affect gating of the K+ channels and paralleled the results of the
yeast mating-based split-ubiquitin screen.
The SYP121 N Terminus Determines SYP121–KC1
Interaction in Vivo
Interactions on heterologous expression in yeast and in Xenopus
oocytes do not rule out the possibility that additional compo-
nents unique to the plant might be important in stabilizing or
directing other domain interactions between SYP121 and KC1.
We therefore made use of a bimolecular fluorescence comple-
mentation (BiFC) assay (Walter et al., 2004) to test these inter-
actions in vivo. Constructs incorporating the open reading
frames for KC1, SYP121, SYP122, and selected SYP121-
SYP122 chimeras fused to the N- and C-terminal halves of
yellow fluorescent protein (YFP) were used to transform Agro-
bacterium tumefaciens and were transiently expressed in Arabi-
dopsis root epidermis by cocultivation (Grefen et al., 2010). We
transformed both the wild type and the syp121 (=pen1-1/
syp121-1) mutant Arabidopsis as a check against possible
interference by higher levels of expression of the native SNARE.
The syp121 mutation introduces a premature stop codon within
the coding sequence of the gene that results in a loss of SNARE
protein expression (Zhang et al., 2007; Pajonk et al., 2008).
Because plant ion channels generally express at levels too low
for detection by fluorescence microscopy, expression was
driven by the constitutive Arabidopsis Ubiquitin-10 gene pro-
moter (Grefen et al., 2010). There is a potential for mistargeting
when a protein is overexpressed. Nonetheless, ion channel dis-
tributions, and that ofmost SNAREs, generally alignwith the native
Figure 4. Coexpression with SYP121-SYP122 Chimeras That Interact
with KC1 Selectively Rescue the Gating Parameters Associated with
SYP121 and the AKT1-KC1 K+ Current in Xenopus Oocytes.
Summary of K+ channel gating parameters of gating charge (A) and V1/2 (B)
recorded from oocytes expressing AKT1 with KC1 in combinations with
SYP121, SYP122, and their chimeras. Data are means6 SE obtained from
joint fittings to Equation 1 of four or more independent data sets in each
case, including the data of Figure 3. Fittings performed by nonlinear least
squares minimization (Marquardt, 1963) with the conductance maximum
gmax held in common within each data set and the apparent gating charge
(d) allowed to vary between SNAREs and chimeras that interact with KC1
and those that do not. Data for SYP121 and the chimera N1HQC2 are
statistically different from the other data sets with P < 0.01.
3080 The Plant Cell
protein in vivo when driven by the constitutive promoter (Uemura
et al., 2004; Sutter et al., 2006, 2007; Grefen et al., 2010).
We used confocal laser scanning microscopy to quantify and
compare fluorescence signals and their distributions obtained
on expressing the KC1-cYFP fusion with different combina-
tions of SNARE fusions. Confocal stacks were used to derive
three-dimensional image projections, and these images were
then analyzed for YFP fluorescence intensity after background
subtraction. As before (Honsbein et al., 2009), we found pro-
nounced YFP fluorescence over background in Arabidopsis
epidermal cells, both in wild-type and syp121 plants, when
transformed with complementary BiFC constructs fused to KC1
and to thewild-type SYP121, but not to SYP122 (Figures 5 and 6;
see Supplemental Movies 1 to 3 online). In each of three,
independent experiments, cotransformations of KC1-nYFP
with the SYP121-SYP122 chimera N1HQC2 fused to cYFP
yielded a fluorescence signal comparable with that of the wild-
type SYP121-cYFP fusion; cotransformations of KC1-nYFP with
cYFP fusions of the N2HQC1 chimera resulted in fluorescence
signals close to background, although expression of the fusion
constructs in these instances was confirmed by immunoblot
analysis (see Figure 10). In principle, BiFC could yield false-
positive interactions as a consequence of overexpression. How-
ever, moderate expression driven by the Ubiquitin-10 promoter
(Grefen et al., 2010) and the absence of an interaction, among
others with the close homolog of SYP121 and the noninteracting
SYP121-SYP122 chimeras, militates against this idea. Addition-
ally, we found the YFP fluorescence was restricted to the cell
periphery and failed to recover after local photobleaching (Figure
7; see Supplemental Movie 4 online), indicating that, like the KC1
complex with wild-type SYP121, interacting assemblies with the
SYP121 mutants were not mobile within the cytosol or within a
circulating endomembrane compartment. Thus, we conclude
that the N terminus of SYP121 is a primary determinant of the
physical interaction between KC1 and SYP121 in vivo.
Figure 6. Analysis of the Interaction of KC1 with SYP121 N Terminus.
Mean fluorescence intensity (arbitrary units) 6 SE for BiFC of KC1 with
SYP121, SYP122, and SYP121-SYP122 chimeras (Figures 1 to 4) ex-
pressed as fusion constructs with N- and C-terminal halves of YFP after
correction for background of (nontransformed) control measurements.
Data from six independent experiments (n > 18 seedlings for each set of
constructs) derived from three-dimensional reconstructions of fluores-
cence image stacks, including those of Figure 5. No appreciable differ-
ence was observed in data from wild-type and syp121 mutant
Arabidopsis, and the results have been pooled. Expressing KC1-nYFP
on its own yielded a fluorescence signal statistically equivalent to the
background and signals from KC1-nYFP coexpressed with SYP122-
cYFP and N2HQC1-cYFP were only marginally higher. Coexpression
with N1HQC2-cYFP gave fluorescence indistinguishable from that
obtained on coexpression with SYP121-cYFP. See Figure 10 for immu-
noblot analysis of cYFP fusion constructs. Data for SYP121 and the
chimera N1HQC2 are statistically different from the other data sets with
P < 0.01.
Figure 5. Interaction of KC1 with SYP121 in Vivo Depends on the N
Terminus of the SNARE.
BiFC of KC1 with SYP121 and the SYP121-SYP122 chimeras N1HQC2
and N2HQC1 expressed in the root epidermis of syp121 mutant
Arabidopsis plants as fusion constructs with the N- and C-terminal
halves of YFP (nYFP and cYFP), respectively. Similar results were
obtained in each of three independent experiments in both mutant and
wild-type Arabidopsis (pooled data summarized in Figure 6). Images for
each combination of constructs are single-plane bright-field ([A], [E], and
[I]), bright-field plus YFP fluorescence ([B], [F], and [J]) and YFP
fluorescence only ([C], [G], and [K]), and three-dimensional projections
from fluorescence image stacks ([D], [H], and [L]). Bar = 50 mm. See also
Supplemental Movies 1 to 3 online.
SNARE-K+ Channel Interaction Motif 3081
The FxRF Residues Define a Minimal Motif for
SYP121–KC1 Interaction
To identify the residues critical for KC1 interaction, we sequen-
tially mutated each of the eight amino acids between Ser-10 and
Pro-17, corresponding to the n1 domain, of the wild-type Nub-
SYP121 fusion to Ala before retransforming yeast with the
site-mutated construct for mating and interaction analysis with
KC1-Cub. Immediately preceding the N terminus of the n1
segment, the Phe at position 9 is highly conserved among Qa-
SNAREs, not only those of Arabidopsis and other plants but also
of yeast and mammals (see Figure 15 and Discussion). There-
fore, we extended the analysis to include the Phe residue at
position 9. Figure 8 summarizes the readouts for KC1–Cub
interaction with these Nub-SYP121 single-site mutants and the
corresponding immunoblot analysis verifying expression of the
SNARE and KC1 fusion proteins. Of the nine SNARE mutants,
yeast growthwas lost following site substitutionswith Ala at Phe-
9, Arg-11, and Phe-12, indicating that substitutions at each of
these residues interfered with the interaction between the
SNARE and KC1. Significantly, an equivalent substitution of
Arg-13 had no effect on yeast growth, nor was growth sup-
pressed following replacements affecting the peptide backbone
angle and negative charge with P17A and E16A, respectively.
To assess the requirement for the FxRFmotif in vivo, we made
use of BiFC as before by transient transformation with A.
tumefaciens. Again, no differences in BiFC signal were observed
in syp121 mutant Arabidopsis compared with the wild type, and
these data were therefore pooled. Fluorescence signals were
quantified by confocal laser scanning microscopy and their
distributions obtained on expressing the KC1-nYFP fusion with
different combinations of SNARE fusions was compared. The
results (Figures 9 and 10) showed a pronounced YFP fluores-
cence over background in Arabidopsis epidermal cells when
transformed with complementary BiFC constructs fused to KC1
and to the wild-type SYP121, but not to SYP122. In each of three
independent experiments, cotransformations of KC1-nYFP with
the SYP121-S10A mutant fused to cYFP yielded a fluorescence
signal comparable with that of the wild-type SYP121-cYFP
fusion, but cotransformations with cYFP fusions of the other
SYP121 site mutants resulted in fluorescence signals that were
statistically indistinguishable from background. Expression of
the fusion constructs in each casewas confirmed by immunoblot
analysis. Thus, we conclude that the FxRF motif of SYP121 is a
primary determinant of the physical interaction betweenKC1 and
SYP121 in vivo.
The FxRF Motif Determines SNARE Control of Channel
Gating and K+ Current Rescue in the Plant
We used the Ala substitutionmutants in each of the four residues
from F9 to F12 of SYP121 to assess their functional impact on K+
channel gating, quantified using the parameters of V1/2 and the
gating charge d. As before, electrophysiological recordings were
performed under voltage clamp after coexpressing KC1 and
AKT1 together with the SYP121 mutants in Xenopus oocytes,
including the protein kinase CIPK23 and calcineurin-like activa-
tor CBL1 (Li and Luan, 2006; Xu et al.,2006), and gating
Figure 7. The Interacting Complex of SYP121-SYP122 Chimera N1HQC2
with the K+Channel KC1 Is Localized to theCell Periphery and IsNonmobile.
Fluorescence recovery after photobleaching in Arabidopsis syp121 mu-
tant root epidermis expressing N1HQC2-cYFP and KC1-nYFP to yield a
BiFC signal.
(A) Bright-field (left column), composite (middle column), and YFP
fluorescence (right column) images taken at time points before and after
local photobleaching (area indicated by dotted rectangle, center row,
left) of the fluorophore. Images collected at times indicated in each frame
relative to the time of photobleach. Bar = 10 mm. See Supplemental
Movie 4 online for the complete time series.
(B) Fluorescence recovery after photobleaching analysis of the fluores-
cence signal taken from the regions indicated (inset) after normalization
and correction for fluorescence decay. Gray bar indicates the period of
photobleaching. Solid curves are the results of nonlinear least squares
fitting of the postbleach fluorescence signals to single exponential
functions yielding an immobile fraction of 0.94. Similar results were
obtained in each of four separate experiments and with BiFC fluores-
cence signals obtained with SYP121n2-cYFP and wild-type SYP121-
cYFP paired with KC1-nYFP (data not shown; see Honsbein et al., 2009)
yielding a mean immobile fraction of 0.92 6 0.04.
3082 The Plant Cell
characteristics were extracted from steady state K+ current–
voltage curves by jointly fitting to a Boltzmann function (Equation
1). As with the SYP121-SYP122 chimeras, we found a strong
divergence in gating characteristics of the SYP121 site mutants
that paralleled their ability to interact with KC1. When expressed
together with AKT1 and KC1, the SYP121-S10Amutant yielded a
K+ current similar to that of the wild-type SNARE, showing an
appreciable steady state current amplitude at voltages negative
of 2100 mV. Fitted to the Boltzmann function, these data gave
values for d near 2 andV1/2 close to2150mV (Figures 11 and 12),
statistically equivalent to those for the wild-type SNARE. By
contrast, coexpressionwith the SYP121 sitemutants F9A, R11A,
and F12A each yielded K+ currents similar to those recorded on
expressing AKT1 and KC1 alone and gave corresponding values
for V1/2 negative of –200 mV and d values close to unity. Analysis
Figure 8. SYP121 Interaction with the KC1 K+ Channel Depends on
Residues Phe-9, Arg-11, and Phe-12.
Yeast mating-based split-ubiquitin assay for interaction between the
KC1-Cub bait and Nub-SYP121 prey carrying substitutions with Ala at
the sites indicated (left) and with controls (negative, NubG; positive,
wild-type Nub [NubWt]) and with wild-type SYP121 and SYP122 for
reference. The N-terminal amino acid sequences of SYP121 and
SYP122 included with the n1 segment indicated (above) for reference.
Diploid yeast spotted (left to right) on CSM medium without Trp, Leu,
and uracil (CSMwlu) to verify crossing, CSM without Trp, Leu, uracil,
adenine, His, and Met (CSMwluahm) to verify adenine- and His-inde-
pendent growth, and with the addition of 0.2 mM Met to verify
interaction on suppressing KC1-Cub expression levels (Obrdlik et al.,
2004; Grefen et al., 2009). Diploid yeast was dropped at 1.0 and 0.1
OD600 in each case. Immunoblot analysis (5 mg total protein/lane) of the
haploid yeast used for mating is included on the right in each case
using commercial VP16 antibody for KC1 and SYP121 polyclonal
antibodies for the SNARE mutants. Note the presence of growth under
Met suppression for the SYP121 site mutants S10A, R13A, S14A,
G15A, E16A, and P17A.
Figure 9. Interaction of KC1 with SYP121 in Vivo Depends on Residues
of the FxRF Motif of SNARE.
BiFC analysis of KC1 association with SYP121 site mutants expressed
in Arabidopsis root epidermis as fusion constructs with the N- and
C-terminal halves of YFP (nYFP and cYFP), respectively. Similar results
obtained in each of four independent experiments (see Figure 10).
Images shown for combinations of KC1-nYFP with the SYP121 site
mutants F9A, S10A, and R11A are single-plane bright-field ([A], [E], and
[I]), bright-field plus YFP fluorescence ([B], [F], and [J]) and YFP
fluorescence only ([C], [G], and [K]), and three-dimensional reconstruc-
tions from fluorescence image stacks ([D], [H], and [L]). Bar = 50 mm.
See also Supplemental Movies 5 to 7 online.
SNARE-K+ Channel Interaction Motif 3083
of channel gating thus showed the functional requirement for the
FxRF motif of SYP121 to alter the gating of the K+ channels.
To validate the impact of the SYP121 FxRF motif on the K+
currents and channel gating in the plant, we made use of voltage
clamp recordings from syp121 mutant Arabidopsis plants. Our
previous studies (Honsbein et al., 2009) showed that the SNARE
mutant, like the null mutants kc1-2 and akt1-1, resulted in a loss
of inward-rectifying K+ current in the root epidermis; the K+
current was recovered in the syp121 mutant background when
complemented with the wild-type transgene under control either
of its own or a constitutive promoter. Therefore, we reasoned that
the K+ current should similarly recover in the syp121 background
when complemented with the SYP121 single-site mutant S10A,
which retained the ability to interact with KC1, but not with the
F9A, R11A, and F12A mutants that failed to interact with KC1.
Measurements were performed using two-electrode voltage
clamp methods on root epidermis of Arabidopsis. We compared
K+ currents from wild-type and syp121 mutant plants, and
syp121 mutant seedlings transformed with the four SYP121
single-site mutants under control of a constitutive promoter. We
also used syp121 mutant seedlings transformed with wild-type
SYP121 and SYP122 as positive and negative controls, respec-
tively. To confirm transformations on a cell-by-cell basis, each of
the SNARE constructs was cloned into a bicistronic binary vector
with two, independent expression cassettes, both localized on
the same T-DNA and each under the control of the Arabidopsis
Ubiquitin-10 gene promoter, one of which was used to drive the
expression of a cytosolic green fluorescent protein (GFP)marker.
Epidermal cells expressing the GFP marker were identified
visually under epifluorescence illumination and were impaled
using multibarrelled microelectrodes for standard two-electrode
voltage clamp recordings (Blatt, 1987; Blatt and Gradmann,
1997). Mature epidermal cells of Arabidopsis roots are not
coupled through plasmodesmata (Duckett et al., 1994), thus
enabling in situ recordings from these single cells. Nonetheless,
root hairs can introduce a substantial and local current sink in
electrical recordings (Meharg et al., 1994). Therefore, to avoid
problems of voltage control associated with nonhomogeneous
current spread, we made use of epidermal cells in files lacking
root hairs. Finally, to check against mistargeting of the mutant
SNAREs, we also performed parallel transformations using
fluorescently tagged constructs.
Figure 13 shows representative measurements fromwild-type
and syp121 mutant plants and from the various complementa-
tions of the syp121mutant line. Similar results were obtained in at
least six independent experiments in every case and are sum-
marized in Figure 14. Wild-type plants showed currents under
voltage clamp typical of the inward-rectifying K+ channels in the
root epidermis (Honsbein et al., 2009). This current, relaxed with
half-times of 300 to 400 ms, was evident principally at voltages
near and negative of 2120 mV and, on analysis, yielded the
characteristic gating parameters for d near 2 and V1/2 close to
2160 mV (Figure 14). The inward-rectifying K+ current was not
evident in recordings from the syp121 mutant nor in syp121
mutant plants transformed with constructs encoding SYP122
(data not shown; seeHonsbein et al., 2009) or the SYP121 single-
site mutations F9A and F12A, except at voltages negative of
2200 mV; a small but persistent current was recorded from
syp121 mutant plants transformed with the R11A mutant of
SYP121 but, again, only when the clamp range was extended to
voltages negative of2200 mV. By contrast, an inward-rectifying
K+ current similar to that of the wild-type plants was observed in
every case in recordings from syp121 mutant plants when
Figure 10. Interaction of KC1 with SYP121 in Vivo Depends on Residues
of the FxRF Motif of SNARE.
(A) Mean fluorescence intensity (arbitrary units)6 SE for BiFC of KC1 with
SYP121, and SYP121 single-site mutations (Figures 8 and 9) expressed as
fusion constructs with N- and C-terminal halves of YFP after correction for
background of (nontransformed) control measurements. Data from three
independent experiments (n > 16 seedlings for each set of constructs)
derived from three-dimensional projections of fluorescence image stacks,
including those of Figure 9. Expressing KC1-nYFP on its own yielded a
fluorescence signal statistically equivalent to the background, and signals
from KC1-nYFP coexpressed with the SYP121 site mutants apart from
S10A-cYFP were similar. Coexpression with S10A-cYFP gave fluores-
cence indistinguishable from that obtained on coexpression with SYP121-
cYFP in the same experiments. Data for SYP121 and the S10A mutant are
statistically different from the other data sets with P < 0.01.
(B) Verification of SNARE protein expression. Immunoblot analysis of
total membrane protein (10 mg/lane) extracted from syp121 mutant
Arabidopsis seedlings expressing the various cYFP fusion constructs
and detected with aSYP121 and aSYP122 antibodies (Tyrrell et al., 2007;
Honsbein et al., 2009). Membrane protein extracted from untransformed
wild-type Arabidopsis seedlings included as a control. The wild-type,
chimera, and single-site mutant fusion proteins run close to the pre-
dicted 47-kD molecular mass. The native SYP121 protein yields a band
consistent with its 38-kD molecular mass.
3084 The Plant Cell
complemented with the construct encoding the S10A mutant of
SYP121. Furthermore, analysis of currents from plants trans-
formed with the S10A mutant showed gating parameters that
aligned closely with those derived from the wild-type plants and
from syp121 mutant plants transformed with the wild-type
SYP121 gene. These recordings also yielded outward-rectifying
K+ currents (data not shown), much as previously described in
mesophyll and guard cells (Very and Sentenac, 2003; Dreyer and
Blatt, 2009) and in Arabidopsis root epidermal cells (Honsbein
et al., 2009), indicating that effects of the experimental manip-
ulations were restricted to the inward-rectifying current. Finally,
parallel transformations using the fluorescently tagged SNARE
mutants (see Supplemental Figure 1 online) showed their local-
ization to the cell periphery, not the cytosol or tonoplast, with an
accumulation around the tip of the root hair much as has been
reported for the wild-type SYP121 (Enami et al., 2009; Grefen
et al., 2010). Thus, we conclude that the FxRF motif is a primary
determinant not only of the physical interaction between KC1
and SYP121 but of the functional expression of K+ current
determined by channel assembly with SYP121 in vivo.
DISCUSSION
Membrane vesicle traffic and the SNARE proteins that drive it are
increasingly recognized as important players in plant cell devel-
opment and growth, as well as signaling and defense. Vesicle
traffic affects the steady state complement of membrane pro-
teins and their tissue distributions during development and at the
cellular level it drives the turnover of ion channels, transporters,
and receptor proteins, contributing to their modulation by hor-
mones and environmental factors (Bassham and Blatt, 2008;
Grefen and Blatt, 2008). These processes play a part in coordi-
nating the ensemble of transport activities at the plasma mem-
brane, although many details are only beginning to come to light.
Additionally, there is now unequivocal evidence of other roles for
SNAREs in the control of ion channels in plants. Our recent
demonstration of a direct and selective interaction of the Arabi-
dopsis SNARE SYP121 with the KC1 K+ channel established a
role for the SNARE in mineral nutrition (Honsbein et al., 2009).
This discovery and its seeming independence from signaling and
vesicle traffic led us to propose its physiological function as a
molecular governor coupling K+ transport with cell surface area
in volume control (Grefen and Blatt, 2008). Thus, the discovery
raised a fundamental question about the identity of the
interacting domain on the SNARE and its relationship to the H3
coiled-coil that assembles in the SNARE core complex and to the
mechanics of vesicle fusion. We have now isolated the KC1
binding site of SYP121, taking advantage of chimeras with the
closely related SNARE SYP122 that does not interact with KC1.
We report here (1) that interaction of SYP121 with KC1, when
expressed in yeast and in situ in the plant, is associated with a
Figure 11. Coexpression with SYP121 and Interacting SYP121 Single-
Site Mutants Selectively Rescues AKT1-KC1 K+ Current in Xenopus
Oocytes.
(A) Current traces and steady state current–voltage curves recorded
under voltage clamp in 96 mM K+ from oocytes expressing KC1 alone
(diamonds), AKT1 alone (circles), AKT1 with KC1 (molar ratio 1:1) alone
(closed squares) and with the SNARE, and SNARE mutants (molar
ratio to KC1, 2:1; see Honsbein et al., 2009) SYP121 (upward-facing
closed arrowheads), SYP121-F9A (downward-facing open arrowheads),
SYP121-S10A (downward-facing closed arrowheads), SYP121-R11A
(open squares), and SYP121-F12A (upward-facing open arrowheads).
Clamp cycles: holding voltage, �20 mV; voltage steps, 0 to �180 mV.
Scale = 3 mA and 4 s. Insets: Corresponding whole-cell currents cross-
referenced by symbol. Currents from oocytes injected with water and
with SYP121 cRNA only gave results similar to those for KC1 alone. All
measurements performed in coexpression with CBL1 and CIPK23,
which are essential for AKT1 function in oocytes, in 1:1:1 molar ratios
with AKT1 (Xu et al., 2006). Solid curves are the results of joint, nonlinear
least squares fitting of the K+ currents (IK) to the Boltzmann function
(Equation 1) and are summarized in Figure 13. Best fittings were obtained
with gmax held in common (gmax for the data shown, 49 6 3 nS) and with
separate, joint values for d between SYP121 and SNARE mutants that
showed interaction with KC1 and those that did not (see also Figures 3
and 4). Similar results were obtained in each of three separate exper-
iments (>12 oocytes for each set of constructs). Both analysis and visual
inspection showed an increase in d and shift in V1/2 on inclusion of
SYP121 and the interacting mutants with AKT1 and KC1.
(B) Verification of SNARE protein expression. Immunoblot analysis of
total membrane protein (10 mg/lane) extracted from oocytes collected
after electrical recordings and detected with aSYP121 antibody (Tyrrell
et al., 2007; Honsbein et al., 2009).
SNARE-K+ Channel Interaction Motif 3085
previously unidentified motif situated within the first 12 residues
of the SNARE sequence, (2) that this same motif is the minimal
requirement for SNARE-dependent alterations in the gating
characteristics of K+ channels assembled with KC1, and (3)
that rescue of the K+ current in syp121 mutant Arabidopsis
depends on complementation with SYP121 that retains this
motif. The KC1 binding site of SYP121 is situated immediately
adjacent Ser residues thought to be phosphorylated in response
to pathogen attack (Pajonk et al., 2008), and, as we note below, it
overlaps with a consensus domain recognized to be important in
regulating vesicle traffic. Thus, the site of KC1 interaction marks
Figure 12. Coexpressionwith SYP121 and Interacting SYP121Single-Site
Mutants Selectively Rescues AKT1-KC1 K+ Current in Xenopus Oocytes.
Summary of K+ channel parameters of gating charge (A) and V1/2 (B)
recorded from oocytes expressing AKT1 with KC1 in combinations with
SYP121, SYP122, and the SYP121 site mutants F9A, S10A, R11A, and
F12A. Data are means 6 SE obtained from joint fittings to Equation 1 of
four or more independent data sets in each case, including the data of
Figure 3. Fittings were performed by nonlinear least squaresminimization
(Marquardt, 1963) with the conductance maximum gmax held in common
within each data set and the apparent gating charge (d) allowed to vary
between SNAREs and chimeras that interact with KC1 and those that do
not. Data for SYP121 and the S10A mutant are statistically different from
the other data sets with P < 0.01.
Figure 13. Coexpression with Interacting SYP121 Single-Site Mutants
Selectively Rescues AKT1-KC1 K+ Current in Vivo.
Current traces and steady state current-voltage curves recorded under
voltage clamp from root epidermal cells of Arabidopsis wild-type plants
(closed circles), syp121 mutant plants (open circles), and syp121 mu-
tant plants expressing the transgenes for SYP121-F9A (upward-facing
open arrowheads), SYP121-S10A (upward-facing closed arrowheads),
SYP121-R11A (downward-facing open arrowheads), and SYP121-F12A
(squares). Data for syp121 plants expressing SYP121 and SYP122 were
omitted for clarity. Clamp cycles: holding voltage,�20mV; voltage steps,
0 to �220 mV. Scale = 2 mA and 1 s. Insets: Corresponding whole-cell
currents cross-referenced by symbol. Solid curves are the results of joint,
nonlinear least squares fitting of the K+ currents (IK) to the Boltzmann
function (Equation 1) and are included in Figure 14. Similar results were
obtained in each of five separate experiments (>12 seedlings for each
construct). Both analysis and visual inspection showed a rescue of the
K+ current with SYP121 and the SYP121-S10A mutant that retains
SNARE-KC1 interaction and functionality in yeast and oocytes.
3086 The Plant Cell
the N terminus of SYP121 as being of special importance: it sug-
gests a close functional overlap between SYP121-dependent
membrane vesicle traffic and control of the K+ channels, and it
discounts a direct competition between SNARE core complex
formation and channel binding. Furthermore, it offers a mecha-
nistic framework from which to explore the coordinate control of
vesicle traffic and transmembrane ion transport.
AMinimal Motif for K+ Channel Interaction and Control
Three observations point to the FxRF motif as an essential and
unique determinant of KC1 binding with SYP121 and to its
functional impact. The first and most important of these derives
fromchimeric substitutionswith domains from the closely related
SNARE SYP122. We found that replacing the N-terminal 39
amino acids of SYP121 with the corresponding sequence from
SYP122 eliminated KC1 interaction in yeast and SNARE action
on K+ channel gating in oocytes; conversely, SNARE–KC1 inter-
action and its impact on channel gating were recovered so long
as this same sequence from SYP121 replaced the N-terminal
domain of SYP122 (Figures 1, 3, and 4 to 6). Second, within this
N-terminal domain a similar pattern of results was obtained on
exchanging eight-residue segments between the two SNAREs
(Figures 2 to 4 and 6), and Ala-scanning mutagenesis within and
adjacent the critical segment identified the core FxRF sequence
determining both KC1 binding and the action of SYP121 on
gating of the K+ channel (Figures 8 to 12). Finally, the same
N-terminal sequence proved essential for functional interaction
of the SNARE with KC1 when expressed in vivo (Figures 13 and
14). These results do not rule out additional sites of interaction
with the K+ channel subunit, but they demonstrate that the
N-terminal FxRF motif is necessary for the physical and func-
tional interaction of SYP121 with KC1.
A key to interpreting the impact of the SYP121 motif can be
drawn from the electrophysiological analyses of SNARE expres-
sion in the syp121 mutant Arabidopsis background that blocks
the inward K+ channel current. We showed previously that
SYP121, through its association with KC1, assembles with a
third protein, the AKT1 K+ channel subunit, and that all three
proteins are essential for functional expression of the inward K+
current in vivo (Honsbein et al., 2009): eliminating SYP121
expression, as in the syp121 mutant (Collins et al., 2003; Zhang
et al., 2007), effectively suppressed the inward K+ current, and
complementing the syp121 mutant with the full-length protein
restored the current, both with SYP121 under control of its own
promoter andwhen constitutively expressed. These effects were
selective for the inward-rectifying K+ current and could be shown
to arise from direct interaction with these channels rather than an
effect mediated through channel protein traffic in vivo (Honsbein
et al., 2009). It follows that SYP121-SYP122 chimeras and
mutant SYP121 constructs that complement the syp121 Arabi-
dopsis plants should retain a capacity to interact functionally with
KC1 in order to affect channel gating. Thus, the parallel between
the readout for interaction based on yeast growth (Figures 1, 2,
and 8) and K+ current rescue (Figures 13 and 14), as well as the
absence of any impact on the outward K+ currents in vivo, argues
strongly that KC1 binding associated with the SYP121 FxRF
motif is a prerequisite for its functional impact on gating.
Figure 14. Coexpression with Interacting SYP121 Single-Site Mutants
Selectively Rescues AKT1-KC1 K+ Current in Vivo.
Summary of K+ current parameters of gating charge (A) and V1/2 (B)
recorded from Arabidopsis expressing AKT1 with KC1 in combinations
with SYP121. Data are means 6 SE obtained from joint fittings of at least
six independent data sets for each construct, in each case with the K+
equilibrium voltage set to �30 mV and the conductance maximum gmax
held in common (Equation 1). Parameter values for noninteracting
constructs were determined assuming a gmax in common with the
mean wild-type K+ current and 0.8 # d # 1.2, consistent with results
from heterologous expression studies (see above and Honsbein et al.,
2009) and should therefore be viewed as estimates only. Data for wild-
type Arabidopsis and for syp121 mutant plants complemented with the
SYP121-S10A mutant are statistically different from the other data sets
(for d, P < 0.01; for V1/2, P < 0.02).
SNARE-K+ Channel Interaction Motif 3087
The same conclusion can be drawn from the heterologous
expression studies in Xenopus oocytes. Furthermore, analysis of
channel gating in this case provides additional detail to the
molecular consequences of the SYP121-KC1 association. As
before (Honsbein et al., 2009), we found coexpression of AKT1
with KC1 only to give a small current near the negative voltage
extreme, whereas coexpression of the channel subunits together
with SYP121 yielded substantial K+ current near and negative of
2100 mVwith values for V1/2 close to2150 mV (Figure 4). Unlike
the situation in the plant, expression of AKT1 alone also yields an
inward K+ current in oocytes, but with anomalous gating char-
acteristics (Duby et al., 2008; Geiger et al., 2009; Honsbein et al.,
2009). Currents obtained with AKT1 alone and with KC1 were
well-fitted to Boltzmann functions with d near unity. By contrast,
coexpression of AKT1 andKC1with SYP121 gave currentswith d
near 2, similar to values returned from analysis of the inward K+
current in the plant (see Figures 3, 4, and 11 to 13; Honsbein
et al., 2009). The K+ currents obtained with the SNARE chimeras
and SYP121 mutants followed this same dichotomous distribu-
tion, the pattern corresponding directly with SNARE–KC1 inter-
action in yeast: chimeras and mutants that failed to rescue yeast
growth also showed currents similar to those recorded from
oocytes expressing AKT1 and KC1 alone or with the noninter-
acting SNARE SYP122; SNARE mutants and chimeras that
rescued yeast growth also gave K+ currents with gating param-
eters that matched closely those on coexpression of AKT1 and
KC1 with the wild-type SYP121. Because the gating parameters
V1/2 and d together encapsulate the intrinsic voltage range and
sensitivity for gating—in short, the capacity for membrane volt-
age to affect channel protein conformations (Dreyer and Blatt,
2009)—this dichotomy in gating characteristics underscores the
functional importance of the FxRF motif in the conformational
changes effected by the SNARE on the K+ channel.
A Unique N-Terminal Domain with Overlapping Functions?
A few examples of SNARE–ion channel interactions are known
and have been associated with other cellular functions. Notably,
themammalianQa-SNARESyntaxin 1Ahasbeen reported tobind
a numberof K+ andCa2+ channels and to subtly affect their gating.
Interactions of Syntaxin 1A with the SUR1-KATP complex of ATP-
sensitive K+ channels and the voltage-gatedKv2.1 K+ channel are
thought to help regulate insulin secretion (Michaelevski et al.,
2003; Cui et al., 2004), and its associations with L- and N-type
Ca2+ channels (Wiser et al., 1996;Bezprozvanny et al., 2000; Arien
et al., 2003) have been suggested similarly to coordinate neuro-
transmission and neuroendocrine secretion (Leunget al., 2007). In
all of these examples, the interacting domains of SNARE appear
to be situated within the C-terminal a-helix that anchors the
protein in the plasma membrane and the adjacent H3 a-helix that
normally assembles as part of the SNARE core complex during
vesicle fusion. The H3 a-helix also binds the CFTR Cl2 channel
(Naren et al., 1997; Ganeshan et al., 2003), although the associ-
ation with vesicle fusion and possible roles for this interaction are
less obvious. One difficulty in each of these examples rests with
the amphipathic properties of the Qa-SNARE H3 a-helix: its
seemingly promiscuouscapacity for protein interaction has raised
concerns about interpreting the physiological significance of
SNARE binding with the channel proteins (Fletcher et al., 2003).
It remains an unprecedented feature of SYP121, therefore, that its
binding with KC1 depends not on the H3 or membrane-spanning
a-helices, but on a previously unidentified motif isolated at the
cytosolic N terminus of the SNARE and unique to SYP121.
Indeed, the FxRF motif appears unique to plants and, among
Arabidopsis Qa-SNAREs, to SYP121 (Figure 15).
KC1 interaction with the SYP121 N terminus now offers a
structural framework to explore its regulation and association
with other physiological phenomena in vivo. There is good
evidence that Ser residues near the N terminus of SYP121 and
its close homolog SYP122 are phosphorylated in response to
pathogen challenge (Nuhse et al., 2003; Heese et al., 2005),
although their significance for any underpinning mechanisms
remains unknown. In SYP121, phosphorylation is thought to
occur at the Ser-7 position, while in SYP122, the adjacent Ser-6
and Ser-8 residues appear the major targets for kinase action
(Benschop et al., 2007). Furthermore, we note that residues
within and adjacent to the FxRF motif of SYP121 are likely to be
important in regulating SNARE core complex formation and
vesicle traffic. The N termini of several Qa-SNAREs, including
mammalian Syntaxin 1A and Syntaxin 5, and the yeast SNAREs
Sed5p, Tlg1p, and Tlg2p, are now recognized to form binding
sites for so-called Sec1/Munc18 (SM) proteins that facilitate
assembly of the SNARE core complex in yeast and mammalian
tissues (Burgoyne and Morgan, 2007; Sudhof and Rothman,
2009). Many details of SM protein binding are still unresolved at
this time. Nonetheless, it is clear that their interaction with the
N-terminal domains of the cognate SNAREs greatly accelerates
fusion, probably by stabilizing protein conformations and vesi-
cle positioning during core complex formation (Sudhof and
Rothman, 2009). At the heart of the binding site on the SNAREs
is a motif comprising highly conserved Asp and Phe residues
separated by four to five amino acids, roughly one a-helical turn,
Figure 15. Conserved Phe Residue Associated with Sec1/Munc18
Protein Binding Overlaps the FxRF Motif of SYP121.
Alignment of representative Qa-SNAREs fromDrosophila,Homo sapiens,
Caenorhabditis elegans, Saccharomyces cereviseae, and Arabidopsis
showing the highly conserved Asp and Phe residues associated with
Sec1/Munc18 protein binding (dark gray), adjacent and largely con-
served residues (light gray), and the overlap with the KC1 interaction
motif of SYP121 (boxed).
3088 The Plant Cell
close to the N terminus of the protein. These residues fit within a
minor groove on the surface of the SM proteins (Bracher and
Weissenhorn, 2002) when the SNARE is in one or more open
(active) conformations, and binding of the SNARE at this site on
the SM proteins is thought to facilitate SM binding with the core
complex (Carpp et al., 2006; Sudhof and Rothman, 2009).
Aligning the corresponding Arabidopsis Qa-SNAREs (Figure 15)
shows that the conserved Asp and Phe residues critical for SM
binding are found in SYP121 and, furthermore, that the Phe
residue is incorporated within the FxRF motif of SYP121, imply-
ing an overlap and, plausibly, competition for binding between
KC1 and one or more Arabidopsis SM proteins. The observation
thus raises entirely new questions that will bear future explora-
tion, notably whether KC1 competes for SYP121 binding with
Arabidopsis SM proteins or KC1 interaction with SYP121 might
substitute in the role of an SM partner and whether KC1 binding
affects SYP121-mediated vesicle traffic to the plasma mem-
brane. Addressing these questions will require knowledge of the
SNARE binding domain of KC1. Regardless of the answers, the
identity of the SYP121 FxRF motif defines a unique site for K+
channel interaction and gating control, and it presents important
and novel perspectives on the concept of the SNARE-K+ channel
association as a molecular governor (Grefen and Blatt, 2008;
Honsbein et al., 2009) to coordinate membrane traffic with
osmotically active solute transport in the plant.
METHODS
Molecular Biology
Open reading frames forAKT1,KC1,SYP121, andSYP122were amplified
with gene-specific primers including Gateway attachment sites (attB1/
attB2). A subsequent BP reaction in pDONR207 (Invitrogen) yielded Entry
clones that were verified via sequencing. The AKT1 and KC1 sequences
were obtained without stop codon to allow C-terminal fusions, whereas
SYP121 and SYP122 were amplified to include their native stop codons
and facilitate correct localization as type-II membrane proteins.
Chimeric cloneswere constructedusing restriction endonucleases, site-
directed mutagenesis, PCR amplification, and DNA ligation. For the
N2HQC1 chimera, SYP121-pDONR207 was cut by sequential digestion
with ApaI and AgeI. The 227-bp fragment incorporating the attL1 site and
the first 39 codons of SYP121 was discarded. A PCR product was
amplified using SYP122-pDONR207 as template to contain the attL1-site
and the first 38 codons of SYP122 with ApaI and AgeI restriction sites and
was inserted at the ApaI and AgeI restriction site. For the NH2QC1
chimera, codon 190 of SYP122 in pDONR207 was mutated to create an
AgeI site through the translationally silent point substitution of ACA with
ACT. Sequences corresponding to theH3 and transmembrane domains of
SYP122 were excised using AgeI and PvuII and replaced with the
corresponding PCR fragment of SYP121 including the same restriction
sites. The NHQ2C1 chimera was constructed by transcriptionally silent,
site-directed mutagenesis at codons 282 and 283 of SYP122 in
pDONR207 to generate a MluI site (mutation of ACACGG to ACGCGT).
Sequences coding for the transmembrane domain of SYP122 were then
excised by digestions with MluI and PvuII, and the corresponding PCR
fragment of SYP121 was inserted. The N1HQC2 chimera was generated
byAgeI andPvuII digestion ofSYP121-pDONR207, and the excised 1035-
bp fragment was discarded and replaced with a corresponding PCR
fragment of SYP122. The NH1QC2 chimera was constructed by mutation
of SYP121-pDONR207 to eliminate an AgeI site at codons 39 to 40.
Thereafter, sequencescorresponding to theN-terminal andHabcdomains
of SYP121 were PCR amplified with primers to add 59 ApaI and 39 AgeI
sites. The PCRproduct was used to replace the corresponding domains in
SYP122-pDONR207. TheNHQ1C2 chimerawas created byApaI andMluI
digestion to excise a fragment containing the attL1 site, N, Habc, and H3
domains of SYP122 from theMluI mutagenized SYP122-pDONR207 (see
above). This site was then replacedby ligationwith the corresponding PCR
product from SYP121. Point mutants were generated by site-directed
mutagenesis as described by Qi and Scholthof (2008) using primer
sequences listed in Supplemental Table 1 online. Finally, Gateway Des-
tination clones were generated using LR Clonase II (Invitrogen) by LR
reaction according to the manufacturer’s instructions. For BiFC and split-
ubiquitin assays, coding sequences for AKT1 and KC1 were cloned in
pMetYC-Dest and pUBC-nYFP, and coding sequences for the SNAREs
SYP121 and SYP122, the chimeras, and point mutants were cloned in
pNX32-Dest and pUBN-cYFP (Grefen et al., 2009, 2010). For electrophys-
iological analysis in oocytes, these constructs were subcloned into pGT-
Dest (see below), and for subcellular localization analysis, single residue
mutants of SYP121 were cloned in pUBN-EOS (Grefen et al., 2010).
The bicistronic vector was prepared by digesting pUB-Dest (Grefen
et al., 2010) using the unique SbfI restriction site, which is situated
between the 35S terminator and the Basta resistance gene. A PCR
reaction was performed with pUBN-GFP-Dest as template to create a
PUBQ10:GFP fragment flanked by SbfI sites using the primers TTCCT-
GCAGGTACCCGACGAGTCAGT (59 to 39) and TTCCTGCAGGTTACTTG-
TACAGCTCGTCCATGC (39 to 59). This product was introduced in the
linearized vector to create the bicistronic vector pUB-Bic-Dest. pUB-
Bic-Dest was maintained and amplified using ccdB survival cells (Invi-
trogen) grown in the presence of the selection markers chloramphenicol
(30 mg/L) and spectinomycin (100 mg/L), and the Gateway Entry and
Destination clones were amplified using Top10 cells (Invitrogen) and the
appropriate antibiotic (gentamycin [20 mg/L] for Entry clones and spec-
tinomycin [100 mg/L] for Destination clones).
Split-Ubiquitin Assays
For yeast mating-based split-ubiquitin assays, the haploid yeast strains
THY.AP4 and THY.AP5 (Obrdlik et al., 2004; Grefen et al., 2007) were
transformed as described previously (Grefen et al., 2009). Pools of 10 to
15 single colonies were selected after 3 d and were inoculated in
selectivemedia for overnight growth. The liquid cultureswere harvested
at OD600 2 to 3 and were resuspended in YPD. Matings were performed
by mixing equal aliquots of cultures containing KC1-Cub in THY.AP4
with the appropriate NubG-SNARE in THY.AP5. Aliquots of 4 mL from
each mixture were dropped on YPD plates and incubated at 308C
overnight. Diploid colonies were selected and inoculated in vector-
selective media lacking Leu, Trp, and uracil (CSMwlu) and were grown to
OD600 of 2 to 3. Thereafter, the yeast was harvested and resuspended in
sterile water. Serial dilutions at OD600 1.0 and 0.1 in water were
dropped, 7 mL per spot, onto plates without and with additions of 200
mM Met on interaction-selective media additionally lacking His, Met,
and adenine (CSMwluham). Growth was monitored at 2, 3, and 4 d, and
images used were taken on the final day. Yeast was also dropped on
CSMwlu media as a control for mating and cell density, and images were
taken after 24 h. To verify expression, yeast was harvested in aliquots
equal to those used for the dilution series and was extracted for protein
gel blot analysis using polyclonal antibodies against SYP121 and
SYP122 as before (Grefen et al., 2009; Honsbein et al., 2009). KC1-
Cub expression was verified in THY.AP4 yeast prior to mating using the
VP16 antibody (Abcam).
Electrophysiology
For electrical recordings using Xenopus laevis oocytes, constructs
with AKT1, KC1, CIPK23, CBL1, SYP121, and SYP122 were used as
SNARE-K+ Channel Interaction Motif 3089
described previously (Honsbein et al., 2009). The SNARE chimeras and
point mutants were cloned in pGT-Dest, which we created using the
oocyte expression vector pBSXG1 (Groves and Tanner, 1992) and
introducing a Gateway cassette with the Gateway conversion kit (Invi-
trogen). Plasmids were linearized and capped cRNA was synthesized in
vitro using T7 mMessage mMachine (Ambion). cRNA quality as a single
band was confirmed by denaturing gel electrophoresis. cRNA was mixed
to ensure equimolar ratios unless otherwise noted. To ensure uniform
injections of AKT1 transcript, mixtures were made up to a standard
volume as necessary with RNase-free water.
Stage V and VI oocytes were isolated from mature Xenopus, and the
follicular cell layer was digested with 2 mg/m collagenase (type 1A;
Sigma-Aldrich) for 1 h. Injected oocytes were incubated in ND96 (96 mM
NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES-NaOH,
pH 7.4) supplemented with gentamycin (5 mg/mL) at 188C for 3 d before
electrophysiological recordings. Whole-cell currents were recorded un-
der voltage clamp using an Axoclamp 2B two-electrode clamp circuit
(Axon Instruments) as described previously (Leyman et al., 1999; Sutter
et al., 2006). Measurements were performed under continuous perfusion
either with 30 mM KCl and 66 mM NaCl or with 96 mM KCl, in each case
with the addition of 1.8 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES-
NaOH, pH 7.2. Oocytes yielding currents were collected and total
membrane protein isolated according to Sottocornola et al. (2006) using
20 mL of extraction buffer per oocyte. Polyclonal antibodies against
SYP121 and SYP122were used at dilutions of 1:5000 in combination with
the ECL Advance Detection Kit (GE Healthcare). Immunoblots were
quantified and normalized to the Ponceau S stain using ImageJ (http://
rsbweb.nih.gov/ij/).
Recordings from Arabidopsis thaliana root epidermal cells were carried
out on wild-type and syp121 mutant seedlings 6 to 8 d postgermination
following transformation by Agrobacterium tumefaciens cocultivation
(Grefen et al., 2010) with the SNAREs and SNARE mutant constructs in
pUB-Bic-Dest. Seedlings were bathed in solutions of 10 and 30 mM KCl
and 5 mM Ca2+-MES, pH 6.1 (adjusted with CaOH, free [Ca2+] = 1 mM)
and voltage clamp records performed using standard, two-electrode
methods (Meharg et al., 1994; Honsbein et al., 2009; Chen et al., 2010).
Measurements were performed on mature epidermal cells in non-root-
hair cell files to avoid electrical coupling and clamp–current dissipation by
root hairs and between cells (Duckett et al., 1994; Meharg et al., 1994). All
recordings were analyzed and leak currents subtracted using standard
methods (Leyman et al., 1999; Sutter et al., 2006) with Henry III software
(Y-Science, University of Glasgow).
Confocal Microscopy
Arabidopsis seedlings were grown in 0.53Murashige and Skoog, pH 7.2,
for 3 to 5 d and transformed by cocultivation with A. tumefaciensGV3101
(Grefen et al., 2010), methods developed from those previously described
for Agrobacterium rhizogenes (Campanoni et al., 2007) and including
0.003% Sylwet (Duchefa) in the cocultivation medium to aid transforma-
tion (Li et al., 2009). Confocal images were obtained as before (Sutter
et al., 2006) on a Zeiss LSM510-UV microscope. GFP fluorescence was
excited with the 458- or 488-nm argon laser lines; YFP fluorescence was
excited with the 514-nm laser line. Emitted light was collected through a
NFT515 dichroic and 505- to 530-nm (GFP) and 535- to 590-nm (YFP)
band-pass filters. Pinholes were set to 1 airy unit. Bright-field images
were collected with a transmitted light detector. Laser intensity, photo-
multiplier gain, and offset were standardized.
Statistics
Statistical analysis of independent experiments is reported as means 6
SE as appropriate with significance determined by Student’s t test or
analysis of variance. Joint nonlinear least squares fittings were performed
using a Marquardt-Levenberg algorithm (Marquardt, 1963) implemented
in SigmaPlot v.11 (SPSS).
Sequences, Vectors, and Maps
Sequences and maps for all vectors described above are available at
www.psrg.org.uk, and vectors are available on request.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: AKT1 (At2g26650), KC1 (At4g32650), SYP121 (At3g11820),
and SYP122 (At3g52400).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The Cellular Distribution in Vivo of SYP121 Is
Unaffected by the SYP121-R11A Mutation.
Supplemental Table 1. Oligonucleotides That Were Designed to
Construct the Clones Used in This Study.
Supplemental Movie 1. 3D Projection of Transient Expression in
Arabidopsis Root Epidermis of the BiFC Pair cYFP-SYP121 and KC1-
nYFP.
Supplemental Movie 2. 3D Projection of Transient Expression in
Arabidopsis Root Epidermis of the BiFC Pair cYFP-N1HQC2 and
KC1-nYFP.
Supplemental Movie 3. 3D Projection of Transient Expression in
Arabidopsis Root Epidermis of the BiFC Pair cYFP-N2HQC1 and
KC1-nYFP.
Supplemental Movie 4. FRAP Time Series of Transient Expression in
Arabidopsis Root Epidermis of the BiFC Pair cYFP-N1HQC2 and
KC1-nYFP.
Supplemental Movie 5. 3D Projection of Transient Expression in
syp121 Mutant Arabidopsis Root Epidermis of the BiFC Pair cYFP-
SYP121-F9A and KC1-nYFP.
Supplemental Movie 6. 3D Projection of Transient Expression in
syp121 Mutant Arabidopsis Root Epidermis of the BiFC Pair cYFP-
SYP121-S10A and KC1-nYFP.
Supplemental Movie 7. 3D Projection of Transient Expression in
syp121 Mutant Arabidopsis Root Epidermis of the BiFC Pair cYFP-
SYP121-R11A and KC1-nYFP.
Supplemental Movie Legends.
ACKNOWLEDGMENTS
We thank W.-H. Wu (Beijing) and Hans Thordahl for constructs and
Arabidopsis lines. George Boswell and Amparo Ruiz-Pardo helped with
Xenopus and plant maintenance. This work was supported by grants BB/
H001630/1 and BB/H001673/1 from the UK Biotechnology and Biological
Sciences Research Council and by a Wellcome VIP award to M.R.B.
Received July 3, 2010; revised September 2, 2010; accepted September
13, 2010; published September 30, 2010.
3090 The Plant Cell
REFERENCES
Arien, H., Wiser, O., Arkin, I.T., Leonov, H., and Atlas, D. (2003).
Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca
(v)1.2) in a cooperative manner. J. Biol. Chem. 278: 29231–29239.
Assaad, F.F., Qiu, J.L., Youngs, H., Ehrhardt, D., Zimmerli, L., Kalde,
M., Wanner, G., Peck, S.C., Edwards, H., Ramonell, K., Somerville,
C.R., and Thordal-Christensen, H. (2004). The PEN1 syntaxin de-
fines a novel cellular compartment upon fungal attack and is required
for the timely assembly of papillae. Mol. Biol. Cell 15: 5118–5129.
Bassham, D.C., and Blatt, M.R. (2008). SNAREs: Cogs and coordina-
tors in signaling and development. Plant Physiol. 147: 1504–1515.
Benschop, J.J., Mohammed, S., O’Flaherty, M., Heck, A.J.R., Slijper,
M., and Menke, F.L.H. (2007). Quantitative phosphoproteomics of
early elicitor signaling in Arabidopsis. Mol. Cell. Proteomics 6: 1198–
1214.
Bezprozvanny, I., Zhong, P.Y., Scheller, R.H., and Tsien, R.W. (2000).
Molecular determinants of the functional interaction between syntaxin
and N-type Ca2+ channel gating. Proc. Natl. Acad. Sci. USA 97:
13943–13948.
Blatt, M.R. (1987). Electrical characteristics of stomatal guard cells: The
contribution of ATP-dependent, “electrogenic” transport revealed by
current-voltage and difference-current-voltage analysis. J. Membr.
Biol. 98: 257–274.
Blatt, M.R., and Gradmann, D. (1997). K(+)-sensitive gating of the K+
outward rectifier in Vicia guard cells. J. Membr. Biol. 158: 241–256.
Blatt, M.R., Leyman, B., and Geelen, D. (1999). Molecular events of
vesicle trafficking and control by SNARE proteins in plants. New
Phytol. 144: 389–418.
Bracher, A., and Weissenhorn, W. (2002). Structural basis for the Golgi
membrane recruitment of Sly1p by Sed5p. EMBO J. 21: 6114–6124.
Brunger, A.T. (2005). Structure and function of SNARE and SNARE-
interacting proteins. Q. Rev. Biophys. 38: 1–47.
Burgoyne, R.D., and Morgan, A. (2007). Membrane trafficking: Three
steps to fusion. Curr. Biol. 17: R255–R258.
Buschmann, P.H., Vaidyanathan, R., Gassmann, W., and Schroeder,
J.I. (2000). Enhancement of Na(+) uptake currents, time-dependent
inward-rectifying K(+) channel currents, and K(+) channel transcripts
by K(+) starvation in wheat root cells. Plant Physiol. 122: 1387–1397.
Campanoni, P., and Blatt, M.R. (2007). Membrane trafficking and polar
growth in root hairs and pollen tubes. J. Exp. Bot. 58: 65–74.
Campanoni, P., Sutter, J.-U., Davis, C.S., Littlejohn, G.R., and Blatt,
M.R. (2007). A generalized method for transfecting root epidermis
uncovers endosomal dynamics in Arabidopsis root hairs. Plant J. 51:
322–330.
Carpp, L.N., Ciufo, L.F., Shanks, S.G., Boyd, A., and Bryant, N.J.
(2006). The Sec1p/Munc18 protein Vps45p binds its cognate SNARE
proteins via two distinct modes. J. Cell Biol. 173: 927–936.
Chen, Z.H., Hills, A., Lim, C.K., and Blatt, M.R. (2010). Dynamic
regulation of guard cell anion channels by cytosolic free Ca2+ con-
centration and protein phosphorylation. Plant J. 61: 816–825.
Collins, N.C., Thordal-Christensen, H., Lipka, V., Bau, S., Kombrink,
E., Qiu, J.L., Huckelhoven, R., Stein, M., Freialdenhoven, A.,
Somerville, S.C., and Schulze-Lefert, P. (2003). SNARE-protein-
mediated disease resistance at the plant cell wall. Nature 425:
973–977.
Cui, N.G., Kang, Y.H., He, Y., Leung, Y.M., Xie, H.L., Pasyk, E.A.,
Gao, X.D., Sheu, L., Hansen, J.B., Wahl, P., Tsushima, R.G., and
Gaisano, H.Y. (2004). H3 domain of syntaxin 1A inhibits KATP
channels by its actions on the sulfonylurea receptor 1 nucleotide-
binding folds-1 and -2. J. Biol. Chem. 279: 53259–53265.
Dreyer, I., and Blatt, M.R. (2009). What makes a gate? The ins and outs
of Kv-like K+ channels in plants. Trends Plant Sci. 14: 383–390.
Duby, G., Hosy, E., Fizames, C., Alcon, C., Costa, A., Sentenac, H.,
and Thibaud, J.B. (2008). AtKC1, a conditionally targeted Shaker-
type subunit, regulates the activity of plant K+ channels. Plant J. 53:
115–123.
Duckett, C.M., Oparka, K.J., Prior, D.A.M., Dolan, L., and Roberts, K.
(1994). Dye coupling in the root epidermis of arabidopsis is progres-
sively reduced during development. Development 120: 3247–3255.
Enami, K., Ichikawa, M., Uemura, T., Kutsuna, N., Hasezawa, S.,
Nakagawa, T., Nakano, A., and Sato, M.H. (2009). Differential
expression control and polarized distribution of plasma membrane-
resident SYP1 SNAREs in Arabidopsis thaliana. Plant Cell Physiol. 50:
280–289.
Fletcher, S., Bowden, S.E.H., and Marrion, N.V. (2003). False inter-
action of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed
by co-immunoprecipitation and pull-down assays: Implications for
identification of protein-protein interactions. Neuropharmacology 44:
817–827.
Ganeshan, R., Di, A., Nelson, D.J., Quick, M.W., and Kirk, K.L. (2003).
The interaction between syntaxin 1A and cystic fibrosis transmem-
brane conductance regulator Cl- channels is mechanistically distinct
from syntaxin 1A-SNARE interactions. J. Biol. Chem. 278: 2876–2885.
Gassmann, W., and Schroeder, J.I. (1994). Inward-rectifying K+ chan-
nels in root hairs of wheat (a mechanism for aluminum-sensitive low-
affinity K+ uptake and membrane potential control). Plant Physiol. 105:
1399–1408.
Gaymard, F., Cerutti, M., Horeau, C., Lemaillet, G., Urbach, S.,
Ravallec, M., Devauchelle, G., Sentenac, H., and Thibaud, J.B.
(1996). The baculovirus/insect cell system as an alternative to Xen-
opus oocytes. First characterization of the AKT1 K+ channel from
Arabidopsis thaliana. J. Biol. Chem. 271: 22863–22870.
Geelen, D., Leyman, B., Batoko, H., Di Sansebastiano, G.P., Moore,
I., Blatt, M.R., and Di Sansabastiano, G.P. (2002). The abscisic acid-
related SNARE homolog NtSyr1 contributes to secretion and growth:
evidence from competition with its cytosolic domain. Plant Cell 14:
387–406 Erratum Plant Cell 14: 963.
Geiger, D., Becker, D., Vosloh, D., Gambale, F., Palme, K., Rehers,
M., Anschuetz, U., Dreyer, I., Kudla, J., and Hedrich, R. (2009).
Heteromeric AtKC1{middle dot}AKT1 channels in Arabidopsis roots
facilitate growth under K+-limiting conditions. J. Biol. Chem. 284:
21288–21295.
Grefen, C., and Blatt, M.R. (2008). SNAREs—Molecular governors in
signalling and development. Curr. Opin. Plant Biol. 11: 600–609.
Grefen, C., Donald, N., Schumacher, K., and Blatt, M.R. (2010). A
Ubiquitin-10 promoter-based vector set for fluorescent protein tag-
ging facilitates temporal stability and native protein distribution in
transient and stable expression studies. Plant J., in press.
Grefen, C., Lalonde, S., and Obrdlik, P. (2007). Split-ubiquitin system
for identifying protein-protein interactions in membrane and full-length
proteins. Curr. Protoc. Neurosci. 5.27.1–5.27.41.
Grefen, C., Obrdlik, P., and Harter, K. (2009). The determination of
protein-protein interactions by the mating-based split-ubiquitin sys-
tem (mbSUS). Methods Mol. Biol. 479: 217–233.
Groves, J.D., and Tanner, M.J.A. (1992). Glycophorin A facilitates the
expression of human band 3-mediated anion transport in Xenopus
oocytes. J. Biol. Chem. 267: 22163–22170.
Heese, A., Ludwig, A.A., and Jones, J.D.G. (2005). Rapid phosphor-
ylation of a syntaxin during the Avr9/Cf-9-race-specific signaling
pathway. Plant Physiol. 138: 2406–2416.
Honsbein, A., Sokolovski, S., Grefen, C., Campanoni, P., Pratelli, R.,
Paneque, M., Chen, Z., Johansson, I., and Blatt, M.R. (2009). A
tripartite SNARE-K+ channel complex mediates in channel-depen-
dent K+ nutrition in Arabidopsis. Plant Cell 21: 2859–2877.
Hu, C., Ahmed, M., Melia, T.J., Sollner, T.H., Mayer, T., and Rothman,
SNARE-K+ Channel Interaction Motif 3091
J.E. (2003). Fusion of cells by flipped SNAREs. Science 300: 1745–
1749.
Jahn, R., Lang, T., and Sudhof, T.C. (2003). Membrane fusion. Cell
112: 519–533.
Kato, T., Morita, M.T., Fukaki, H., Yamauchi, Y., Uehara, M.,
Niihama, M., and Tasaka, M. (2002). SGR2, a phospholipase-like
protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravi-
tropism of Arabidopsis. Plant Cell 14: 33–46.
Leung, Y.M., Kwan, E.P., Ng, B., Kang, Y., and Gaisano, H.Y. (2007).
SNAREing voltage-gated K+ and ATP-sensitive K+ channels: Tuning
beta-cell excitability with syntaxin-1A and other exocytotic proteins.
Endocr. Rev. 28: 653–663.
Leyman, B., Geelen, D., Quintero, F.J., and Blatt, M.R. (1999). A
tobacco syntaxin with a role in hormonal control of guard cell ion
channels. Science 283: 537–540.
Li, J.F., Park, E., von Arnim, A.G., and Nebenfuhr, A. (2009). The FAST
technique: A simplified Agrobacterium-based transformation method
for transient gene expression analysis in seedlings of Arabidopsis and
other plant species. Plant Methods 5: 6.
Li, L.G., Kim, B.G., Cheong, Y.H., Pandey, G.K., and Luan, S. (2006).
A Ca(2)+ signaling pathway regulates a K(+) channel for low-K re-
sponse in Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 12625–12630.
Lipka, V., Kwon, C., and Panstruga, R. (2007). SNARE-ware: The role
of SNARE-domain proteins in plant biology. Annu. Rev. Cell Dev. Biol.
23: 147–174.
Marquardt, D. (1963). An algorithm for fleast-squaires estimation of
nonlinear parameters. J. Soc. Ind. Appl. Math. 11: 431–441.
Meharg, A.A., Maurousset, L., and Blatt, M.R. (1994). Cable correc-
tion of membrane currents recorded from root hairs of Arabidopsis
thaliana L. J. Exp. Bot. 45: 1–6.
Michaelevski, I., Chikvashvili, D., Tsuk, S., Singer-Lahat, D., Kang,
Y.H., Linial, M., Gaisano, H.Y., Fili, O., and Lotan, I. (2003). Direct
interaction of target SNAREs with the Kv2.1 channel. Modal regulation
of channel activation and inactivation gating. J. Biol. Chem. 278:
34320–34330.
Naren, A.P., Nelson, D.J., Xie, W.W., Jovov, B., Pevsner, J., Bennett,
M.K., Benos, D.J., Quick, M.W., and Kirk, K.L. (1997). Regulation of
CFTR chloride channels by syntaxin and Munc18 isoforms. Nature
390: 302–305.
Nuhse, T.S., Boller, T., and Peck, S.C. (2003). A plasma membrane
syntaxin is phosphorylated in response to the bacterial elicitor flagel-
lin. J. Biol. Chem. 278: 45248–45254.
Obrdlik, P., et al. (2004). K+ channel interactions detected by a genetic
system optimized for systematic studies of membrane protein inter-
actions. Proc. Natl. Acad. Sci. USA 101: 12242–12247.
Pajonk, S., Kwon, C., Clemens, N., Panstruga, R., and Schulze-
Lefert, P. (2008). Activity determinants and functional specialization of
Arabidopsis PEN1 syntaxin in innate immunity. J. Biol. Chem. 283:
26974–26984.
Parlati, F., Weber, T., McNew, J.A., Westermann, B., Sollner, T.H.,
and Rothman, J.E. (1999). Rapid and efficient fusion of phospholipid
vesicles by the alpha-helical core of a SNARE complex in the absence
of an N-terminal regulatory domain. Proc. Natl. Acad. Sci. USA 96:
12565–12570.
Press, W., Flannerly, B., Teukolsky, S., and Vetterling, W. (1992).
Numerical Recipes: The Art of Scientific Computing. (Cambridge, UK:
Cambridge University Press).
Qi, D., and Scholthof, K.B.G. (2008). A one-step PCR-based method
for rapid and efficient site-directed fragment deletion, insertion, and
substitution mutagenesis. J. Virol. Methods 149: 85–90.
Robatzek, S., Chinchilla, D., and Boller, T. (2006). Ligand-induced
endocytosis of the pattern recognition receptor FLS2 in Arabidopsis.
Genes Dev. 20: 537–542.
Saito, C., Morita, M.T., Kato, T., and Tasaka, M. (2005). Amyloplasts
and vacuolar membrane dynamics in the living graviperceptive cell of
the Arabidopsis inflorescence stem. Plant Cell 17: 548–558.
Sokolovski, S., Hills, A., Gay, R.A., and Blatt, M.R. (2008). Functional
interaction of the SNARE protein NtSyp121 in Ca2+ channel gating,
Ca2+ transients and ABA signalling of stomatal guard cells. Mol. Plant
1: 347–358.
Sottocornola, B., Visconti, S., Orsi, S., Gazzarrini, S., Giacometti, S.,
Olivari, C., Camoni, L., Aducci, P., Marra, M., Abenavoli, A., Thiel,
G., and Moroni, A. (2006). The potassium channel KAT1 is activated by
plant and animal 14-3-3 proteins. J. Biol. Chem. 281: 35735–35741.
Sudhof, T.C., and Rothman, J.E. (2009). Membrane fusion: Grappling
with SNARE and SM proteins. Science 323: 474–477.
Surpin, M., Zheng, H.J., Morita, M.T., Saito, C., Avila, E., Blakeslee,
J.J., Bandyopadhyay, A., Kovaleva, V., Carter, D., Murphy, A.,
Tasaka, M., and Raikhel, N. (2003). The VTI family of SNARE proteins
is necessary for plant viability and mediates different protein transport
pathways. Plant Cell 15: 2885–2899.
Sutter, J.U., Campanoni, P., Tyrrell, M., and Blatt, M.R. (2006). Selec-
tive mobility and sensitivity to SNAREs is exhibited by the Arabidopsis
KAT1 K+ channel at the plasma membrane. Plant Cell 18: 935–954.
Sutter, J.U., Sieben, C., Hartel, A., Eisenach, C., Thiel, G., and Blatt,
M.R. (2007). Abscisic acid triggers the endocytosis of the Arabidopsis
KAT1 K+ channel and its recycling to the plasma membrane. Curr.
Biol. 17: 1396–1402.
Tyrrell, M., Campanoni, P., Sutter, J.-U., Pratelli, R., Paneque, M.,
Sokolovski, S., and Blatt, M.R. (2007). Selective targeting of plasma
membrane and tonoplast traffic by inhibitory (dominant-negative)
SNARE fragments. Plant J. 51: 1099–1115.
Uemura, T., Ueda, T., Ohniwa, R.L., Nakano, A., Takeyasu, K., and
Sato, M.H. (2004). Systematic analysis of SNARE molecules in
Arabidopsis: Dissection of the post-Golgi network in plant cells. Cell
Struct. Funct. 29: 49–65.
Ungar, D., and Hughson, F.M. (2003). SNARE protein structure and
function. Annu. Rev. Cell Dev. Biol. 19: 493–517.
Very, A.A., and Sentenac, H. (2003). Molecular mechanisms and
regulation of K+ transport in higher plants. Annu. Rev. Plant Biol. 54:
575–603.
Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K.,
Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C.,
Harter, K., and Kudla, J. (2004). Visualization of protein interactions
in living plant cells using bimolecular fluorescence complementation.
Plant J. 40: 428–438.
Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl,
M., Parlati, F., Sollner, T.H., and Rothman, J.E. (1998). SNAREpins:
Minimal machinery for membrane fusion. Cell 92: 759–772.
White, P.J., and Lemtiri-Chlieh, F. (1995). Potassium currents across
the plasma membrane of protoplasts derived from rye roots: A patch
clamp study. J. Exp. Bot. 46: 497–511.
Wiser, O., Bennett, M.K., and Atlas, D. (1996). Functional interaction of
syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+
channels. EMBO J. 15: 4100–4110.
Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L., and Wu, W.H.
(2006). A protein kinase, interacting with two calcineurin B-like proteins,
regulates K+ transporter AKT1 in Arabidopsis. Cell 125: 1347–1360.
Yano, D., Sato, M., Saito, C., Sato, M.H., Morita, M.T., and Tasaka,
M. (2003). A SNARE complex containing SGR3/AtVAM3 and ZIG/
VTI11 in gravity-sensing cells is important for Arabidopsis shoot
gravitropism. Proc. Natl. Acad. Sci. USA 100: 8589–8594.
Zhang, Z.G., Feechan, A., Pedersen, C., Newman, M.A., Qiu, J.L.,
Olesen, K.L., and Thordal-Christensen, H. (2007). A SNARE-protein
has opposing functions in penetration resistance and defence signal-
ling pathways. Plant J. 49: 302–312.
3092 The Plant Cell
DOI 10.1105/tpc.110.077768; originally published online September 30, 2010; 2010;22;3076-3092Plant Cell
BlattChristopher Grefen, Zhonghua Chen, Annegret Honsbein, Naomi Donald, Adrian Hills and Michael R.
Arabidopsis Channel KC1 and Channel Gating in +A Novel Motif Essential for SNARE Interaction with the K
This information is current as of February 5, 2021
Supplemental Data /content/suppl/2010/09/13/tpc.110.077768.DC1.html
References /content/22/9/3076.full.html#ref-list-1
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