De-ubiquitination: a new player in Golgi to ER retrograde transport

1Mickaël Cohen, 2Françoise Stutz and 1Catherine Dargemont*

1Nucleocytoplasmic transport group. Institut Jacques Monod. Unité Mixte de Recherche

7592, CNRS, Universités Paris VI and VII, 2 Place Jussieu. Tour 43. 75251 Paris Cedex 05.

France.

2Dept. of Cell Biology. Sciences III. 30 Quai E. Ansermet. 1211 Genève 4. Switzerland.

Running title: De-ubiquitination of β’COP by the Ubp3p/Bre5p complex

*To whom correspondence should be addressed: Institut Jacques Monod. Unité Mixte de

Recherche 7592, CNRS, Universités Paris VI and VII, 2 Place Jussieu. Tour 43. 75251 Paris

Cedex 05. France.

Tel/Fax: 0033 1 44276956

E-mail : dargemont@ijm.jussieu.fr

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 30, 2003 as Manuscript C300451200 by guest on Septem

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Abstract

Modification by ubiquitin plays a major role in a broad array of cellular functions.

Although reversal of this process, deubiquitination, likely represents an important regulatory

step contributing to cellular homeostasis, functions of deubiquitination enzymes still remain

poorly characterized. We have previously shown that the ubiquitin protease Ubp3p requires a

co-factor, Bre5p, to specifically deubiquitinate the COPII subunit Sec23p, which is involved

in anterograde transport between ER and Golgi compartiments. In the present report, we show

that disruption of BRE5 gene also led to a defect in the retrograde transport from the Golgi to

the Endoplasmic reticulum. Further analysis indicate that the COPI subunit β'-COP represents

another substrate of the Ubp3p/Bre5p complex. All together, our results indicate that the

Ubp3p/Bre5p de-ubiquitination complex co-regulates anterograde and retrograde transports

between ER and Golgi compartments.

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Introduction

Modification of target proteins by ubiquitin participates in a wide array of biological

functions. Proteins destined for degradation or processing via the 26S proteasome are coupled

to multiple copies of ubiquitin. However, attachment of ubiquitin or ubiquitin-related

molecules may also result in changes in subcellular distribution or modification of protein

activity (1,2).

Understanding of mechanisms and regulation of ubiquitin conjugation considerably

improved over the past ten years and recent studies indicate that reversal of this modification,

namely deubiquitination, represents an additional level of regulation. The de-ubiquitination

process is catalyzed by proteases called deubiquitinating enzymes (DUB) which fall into four

distinct families (3), ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific processing

proteases (USPs or UBPs), OTU-domain ubiquitin-aldehyde-binding proteins (Otubains) and

Jab1/Pad1/MPN-domain-containing metallo-enzymes (JAMMs). Among these four families,

UBPs represent the most widespread and represented DUBs across evolution. In particular,

the Saccharomyces cerevisiae genome encodes for 16 UBPs and only one UCH but none of

these enzymes is essential for yeast viability. UBPs tend to release ubiquitin from a

conjugated protein. They display similar catalytic domains containing conserved Cys and His

boxes but divergent N-terminal and occasionally C-terminal extensions (4,5) which are

thought to function in substrate recognition, subcellular localization and protein-protein

interactions. Molecular basis for substrate recognition by UBP has been poorly described.

Indeed human HAUSP has been shown to be necessary and sufficient to de-ubiquitinate its

specific substrate, the p53 tumor suppressor (6) but no additional substrate for this Ubp has

been identified so far. The tumour suppressor CYLD has been shown to negatively regulate

NF-κB signalling by deubiquitinating NEMO and TRAF2 (7,8,9).

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We recently reported that the yeast Ubp3p forms a complex with Bre5p which

specifically de-ubiquitinates Sec23p, a component of the COPII complex essential for

anterograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus (10).

Ubp3p is the only yeast Ubp able to catalyze Sec23p deubiquitination indicating that Ubps

can exert their activity on specific substrates and are probably not redundant. Ubp3p directly

interacts with Sec23p and its catalytic cysteine residue is essential for the deubiquitinating

activity. In contrast Bre5p does not participate to Sec23p recognition nor complements a

catalytically inactive Ubp3p but rather acts as an essential positive regulator of Ubp3p. Bre5p

displays an N-terminal domain related to the Nuclear Transport Factor 2 (NTF2) and

responsible for the interaction with Ubp3p and and a C-terminal domain presenting putative

RNA-binding sites. Mammalian cells display two proteins homologous to Bre5p, G3BP1 and

G3BP2. Interestingly, G3BP1 has been shown to interact with USP10 (11), a human ubiquitin

protease sharing 46% similarity and 27% identity with Ubp3p. The interaction between

USP10 and G3BP1 suggests that the Ubp3p/Bre5p complex might have been conserved

during evolution. However there is no clue whether these yeast and human complexes are

implicated in similar functions.

In the present report, we analyzed whether the yeast Ubp3p/Bre5p complex could have

other substrates than Sec23p and whether the human USP10/G3BP1 complex could recognize

these substrates.

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Experimental procedures

Yeast strains and antibodies

The S. cerevisiae strains and plasmids used in this study are listed in the Supplementary Table

. When indicated, the BRE5 and UBP3 genes were disrupted as described (12). Rabbit

polyclonal antibodies to yeast Gcs1p (1:1000 dilution) and Glo3p (1:1000) were kindly

provided by G. Johnston, αCOP (1:800), β'COP (1:200) and δCOP (1:500) by F. Letourneur

and antibodies to βCOP#9381 (1:600), β'COP#9562 (1:1000), γCOP (1:1000), and εCOP

(1:800) were generous gifts from R. Duden.

ER retrieval defect assay of dilysine-tagged Ste2p

The in vivo assay to monitor ER retrieval of dilysine-tagged Ste2p in yeast was performed

essentially as previously described (13). Briefly, MATa cells (wt, ret1.1 and bre5∆) deleted of

the endogenous STE2 gene and expressing the Ste2-WBP1 fusion protein, were mated with

MATα cells (RH311-3D). Resulting diploids were selected on growth medium lacking uracil

and histidine.

Yeast extracts and GST Pull-Down Assay

Cells grown in YPD were collected during the exponential growth phase (A600 2). Total

protein extracts were prepared by the NaOH-TCA lysis method (14). Alternatively cells were

lysed at 4°C with glass beads in IP buffer (50 mM Hepes pH7.5, 150 mM NaCl, 1 mM DTT,

0,5% Triton, 10% Glycerol, supplemented with a mixture of protease inhibitors, 10µg/ml

aprotinine, pepstatine, leupeptine, 1 mM PMSF). The resulting lysate was centrifuged for 30

min at 13000 g. 3µg of GST-WBP1 or GST-WBP1-SS fusion proteins expressed from

plasmids generously provided by F. Letourneur. were incubated with lysates of BRE5 or

bre5∆ cells for 1h at 4°C and an additional hour at 4°C in the presence of glutathione-

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Sepharose beads. Beads were washed in IP buffer and heated in sample buffer before SDS-

PAGE and immunoblotting.

Two hybrid assay

A bait fusion protein between USP10 and the LexA DNA binding domain was expressed

from the pBTM116 plasmid (11). COPI bovine subunits fused to the Gal4 activation domain

were expressed from the pAD-Gal4-2.1 and pACTII plasmids (15). The two-hybrid assay was

performed as previously described (10).

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Results and Discussion

BRE5 genetically interacts with the GLO3 gene. Screening for mutations that induce

synthetic lethality upon disruption of the BRE5 gene recently led to the identification of a

genetic interaction between BRE5 and SFB3, a gene encoding for a Sec24-like component of

the COPII complex. Indeed Bre5p forms a complex with the ubiquitin protease Ubp3p that

de-ubiquitinates the COPII protein Sec23p (10). In order to identify additional substrates of

the Ubp3p/Bre5p complex, we characterized another mutant strain from this screen and found

that the viability of these cells could be rescued by the GLO3 gene. (Figure 1A). Both BRE5

and a truncated version of BRE5 encoding a protein deleted of its RNA-binding domain

(BRE5 (1-400)) could complement the bre5∆-glo3 mutant. In contrast, deletion of the Nuclear

Transport Factor 2 (NTF2)-like domain (BRE5 (146-515)) responsible for the interaction with

Ubp3p abolished the ability of Bre5p to rescue the viability of bre5∆-glo3 mutant (Figure

1B).

ER retrieval defect in cells disrupted for BRE5. Glo3p is one of the GTPase Activating

Proteins (GAP) for the small GTPase ADP-ribosylation factor 1 (Arf1p). Arf1p and the

heptameric coatomer complex (α, β, β’, γ, δ, ε, ζ-COP) form the Coat Protein Complex I

(COPI) involved in retrograde transport within the Golgi apparatus (16) and from the Golgi to

the ER (13,17). The GDP-bound form of Arf1p is likely recruited on Golgi membranes by the

transmembrane protein p23 prior nucleotide exchange. Arf1p-GTP as well as p23/p24

proteins then triggers coatomer binding and coat polymerization (18,19,20). Following

budding, Arf1p-dependent GTP hydrolysis initiates vesicle uncoating (21,22), a prerequisite

for vesicle docking to the acceptor compartment. The three yeast Arf1-GAPs share overall

similarity but appear specialized in specific transport routes. Gcs1p and Age2p have been

shown to be implicated in transport from the Trans Golgi Network (23), whereas Glo3p is the

major GAP involved in ER retrieval (24).

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The genetic interaction between GLO3 and BRE5 led us to analyze whether deletion of

BRE5 could induce defects in retrograde transport from the Golgi to the ER. For this purpose,

we used a previously described in vivo assay to monitor ER retrieval of dilysine-tagged

proteins in yeast (13). Indeed, it has been clearly shown that dilysine signals confer a COPI-

mediated localization to the ER (13). This assay is based on the use of MATa cells deleted

from the endogenous α-factor receptor STE2 gene but expressing a fusion protein between

Ste2p and WBP1. The dilysine retrieval motif of WBP1 confers an ER localization to the

chimeric protein resulting in the inability of these ste2∆ Ste2-WBP1 (WT) cells to mate

(Figure 2A). However, when retrograde transport from Golgi to ER is defective, Ste2-WBP1

is mislocalized to the cell surface, allowing mating to occur. In particular, a ts mutation in the

α subunit of the coatomer (ret1.1) was able to restore mating (Figure 2A) as previously

described (13). Deletion of BRE5 led to a partial rescue of the ability to mate indicating that

lack of Bre5p causes a defect in ER retrieval (Figure 2A).

Mutations affecting ER retrieval of dilysine-tagged proteins fall into two classes

distinguished by their ability to alter, or not, binding of coatomer to dilysine motifs in vitro

(13). To better characterize the effects of BRE5 deletion on retrograde transport, lysates from

BRE5 and bre5∆ cells were incubated with purified recombinant fusion proteins between

GST and WBP1 peptides presenting dilysine (GST-WBP1) or diserine motifs (GST-WBP1-

SS) and pulled-down with glutathione beads as previously described (17). Bound proteins

were revealed by immunoblotting with antibodies to β’-COP and δ−COP, two subunits of

COPI coatomer (Figure 2B). Neither β’-COP nor δ−COP were retained on GST-WBP1-SS

beads. Although both subunits were bound to GST-WBP1 beads, the efficiency of recruitment

was affected upon BRE5 deletion. A precise quantification performed on three independent

experiments indicated that the amount of bound β’-COP and δ−COP from bre5∆ cells

extracts corresponded to 50 and 65% respectively of material bound using wt extracts. This

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result suggests that coatomer assembly is likely altered in bre5∆ cells thus inducing an ER

retrieval defect. Consistently, cells disrupted for BRE5 encoding gene have been shown to be

hypersensitive to Brefeldine A (25), an inhibitor of nucleotide exchange on ARF which

blocks coatomer binding to membranes (26).

Bre5p/Ubp3p complex de-ubiquitinates the COPI subunit β’-COP/Sec27p. To analyze

the molecular basis of the ER retrieval defect in bre5∆ cells, expression levels of the different

COPI components were analyzed in wild-type, bre5∆ and ubp3∆ cells. No significant

difference could be observed in the steady-state level of the Arf-GAPs Gcs1p or Glo3p nor

coatomer subunits α−COP, β−COP, δ−COP, γ−COP or ε−COP in the various strains (Figure

3A). In contrast, a modified form of β’-COP with slower mobility than the β’-COP main

signal was reproducibly detected in bre5∆ and ubp3∆ cells but not in wild type cells (Figure

3A). The modified form of β’-COP not only displayed a molecular mass of ~10kD greater

than unmodified β’-COP and accumulated in the absence of the Ubp3p/Bre5p de-

ubiquitination complex but β’-COP has also been shown to be ubiquitinated (27). Thus the

modified form of β’-COP most likely corresponds to mono-ubiquitinated β’-COP that could

not be incorporated into the COPI complex and therefore affects coatomer assembly and ER

retrieval.

We recently reported that absence of the Bre5p/Ubp3p complex induces an

accumulation of mono-ubiquitinated Sec23p that facilitates its subsequent poly-ubiquitination

and degradation by the 26S proteasome(10). To address a possible role of the Bre5p/Ubp3p

complex in β’-COP turn-over, we first measured β’-COP stability in the presence of

cycloheximide and found that the half-life of this protein is shorter in bre5∆ cells, than in WT

cells (data not shown). Since polyubiquitination rather than modification with a single

ubiquitin triggers proteins for recognition and degradation by the 26S proteasome,

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modification of β’-COP was analyzed in cim3-1 thermosensitive mutants defective for the

proteasome activity (28). A two hours shift from 23 to 37°C in the presence of cycloheximide,

led to a rapid degradation of β’-COP in wild-type cells (CIM3) and bre5∆ cells whereas it was

stabilized in cim3-1 strains (Figure 3B) indicating that degradation of β’-COP depends on the

proteasome activity. Interestingly, deletion of BRE5 in cim3-1 cells led to an accumulation at

37°C of both unmodified and high molecular weight forms of β’-COP likely corresponding to

polyubiquitinated species of β’-COP (Figure 3B). These results indicate that absence of a

functional Ubp3p/Bre5p complex led to a faster polyubiquitination and degradation of β’-

COP by the proteasome.

Mammalian β’-COP interacts with USP10. G3BP1, one of the two human proteins similar

to Bre5p, has been previously shown to interact with USP10 (11), a human ubiquitin protease

sharing 46% similarity and 27% identity with Ubp3p mainly concentrated in the catalytic

domain (Figure 4A). Using a 2-hybrid assay, we found that USP10 not only interacts with

G3BP1 but also with G3BP2, the second human homologue of Bre5p (data not shown).

Moreover, similarly to the Ubp3p/Bre5p complex, the interaction between USP10 and G3BP1

or G3BP2 is mediated by the NTF2-like domain of G3BPs (data not shown).

Yeast β’-COP is very similar to bovine β’-COP (43% identity, 64% similarity). To test

whether Ubp3p/Bre5p and USP10/G3BP complexes could exert their de-ubiquitination

activity on similar substrates, we tested whether human USP10 could interact with COPI

subunits. For this purpose, we used a two hybrid approach where the LexA DNA binding

domain alone or fused to USP10 was co-expressed with bovine COPI coatamer subunits fused

to the Gal4 activating domain (Figure 4B). We found that Gal4-β’-COP strongly interacted

with LexA-USP10 whereas the other fusion proteins did not display any significant

interaction using this assay. This result suggests that the yeast Ubp3p/Bre5p and the

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mammalian USP10/G3BP complexes could be functionally homologous and could regulate

Golgi to ER transport by acting on the function and stability of β’-COP substrate.

In this study, we show that inactivation of the Ubp3p/Bre5p deubiquitination complex

induces an ER retrieval defect likely due to a defect of COPI coat assembly. This partial

defect led to cell lethality when combined with mutation on the ArfGAP Glo3p. Further

analysis indicates that the COPI subunit β’-COP is a substrate of the Ubp3p/Bre5p complex.

Indeed, inactivation of the deubiquitination complex leads to an accumulation of ubiquitinated

β’-COP and a faster proteasome-dependent degradation of the protein. The ability of USP10

to interact with mammalian β’-COP suggest that regulation of Golgi to ER transport by

ubiquitination could also occur in mammals. All together, these data therefore indicate that

the Ubp3p/Bre5p complex is able to regulate retrograde transport from the Golgi apparatus to

the ER by acting on the essential COPI subunit β’-COP. We have previously shown that the

Ubp3p/Bre5p complex can act on anterograde transport between ER and Golgi compartments

by deubiquitinating the COPII protein Sec23p (10). Therefore, the Ubp3p/Bre5p complex

appears to co-regulate anterograde and retrograde transports between ER and Golgi

compartments by deubiquitinating and controlling expression levels of COPII Sec23p and

COPI β’-COP respectively.

Interestingly, Ubp3p has been implicated in pheromone signaling (29), G3BP1 in the

Ras signaling pathway (30) and Bre5p as well as G3BP1 can be phosphorylated (31,32). It is

therefore tempting to speculate that Bre5p/G3BP phosphorylation is regulated by signaling

cascades and controls the activity of the ubiquitination complex. Ubp3/Bre5 complex might

therefore contribute to coordinate and adapt anterograde and retrograde transport routes

between ER and Golgi compartments to the cellular environment and physiological

conditions.

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Acknowledgments

We thank members of the lab as well as N. Belgareh and R. Tsapis-Haguenauer for

stimulatory discussions and critical reading of the manuscript. We are grateful to F.

Letourneur, R. Duden, G. Johnston, F. Wieland and G. Draetta for their generous gift of

antibodies, strains and expression plasmids. This study was funded by grants from the

Association de Recherche contre le Cancer and the Ligue contre le Cancer. M.C. is a fellow

from the Association de Recherche contre le Cancer.

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Figure legends

Figure 1: Genetic interaction between BRE5 and GLO3 involves the NTF2-like domain of

Bre5p. bre5∆-glo3 strain transformed with the pCH1122-BRE5 plasmid was transformed A,

with pLAC111 empty vector (Vector), pBRE5 (BRE5) or pGLO3 (GLO3) B, with p415-ADH

empty vector (Vector), with pBRE5 (BRE5), pBRE5-1.400 (BRE5(1-400)), or pBRE5-

146.515 (BRE5 (146-515)). Transformants were subsequently streaked on 5-FOA medium to

induce loss of pCH1122-BRE5.

Figure 2: Bre5p regulates retrograde transport between ER and Golgi compartments. A,

Cells disrupted for BRE5 display an ER retrieval defect. MATa WT (FLY264), ret 1.1

(FLY306) or bre5∆ (FLY264 bre5∆) cells were mated with ΜΑΤα RH311-3D cells and

diploids were selected on appropriate minimal media. B, Purified recombinant fusion proteins

between GST and WBP1 peptides presenting dilysine (GST-WBP1) or diserine motifs (GST-

WBP1-SS) were incubated with lysates from wild type cells (BRE5) or cells disrupted for

BRE5 (bre5∆) and pulled-down with glutathione beads. Bound proteins were revealed by

immunoblotting with antibodies to β’-COP and δ−COP. Loading was controlled using a non

specific band (*).

Figure 3: β’-COP is a substrate of the Ubp3p/Bre5p complex. A, Yeast extracts were

prepared from wild-type cells, bre5∆ and ubp3∆ deletion mutants, and analyzed by SDS-

PAGE and by Western blotting using specific antibodies against the indicated proteins. B, β’-

COP degradation is proteasome-dependent. Wild type CIM3 (YPH 499), cim3-1 (CMY 763),

bre5∆ and cim3-1 bre5∆ strains were grown in YPD at 23°C (A600 0.5) and then shifted to

37°C for 120 minutes (+) in the presence of 100µg /ml cycloheximide. Yeast extracts were

prepared and analyzed by SDS-PAGE and immunoblotting using an anti-β’-COP antibody.

Loading was controlled using a non specific band (not shown). (arrow) non specific. (*)

mono-ubiquitinated β’-COP.

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Figure 4: USP10 interacts with mammalian β’-COP. A, Alignment of S. cerevisiae Ubp3p

and human USP10. Identical or similar amino acids are shown in black or grey background

respectively B, Two hybrid analysis of LexA-USP10 bait versus Gal4-COPI subunit preys.

Preys consisting of Gal4 activation domain fused to bovine α, β, β’,δ , ε and γ-COP subunits

were tested for interaction with USP10 fused to LexA DNA binding domain (LexA-USP10)

or with LexA DNA binding domain (LexA) used as a negative control. Interactions between

proteins were measured by cell growth on media without histidine or by activation of the

LacZ reporter gene in β-galactosidase activity assay (data not shown).

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BRE5 GLO3

Vector

bre5∆-glo3

A

B

BRE5

Vector BRE5 (1-400)

BRE5 (146-515)

bre5∆-glo3

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WT

ret1.1

bre5∆

BRE5bre

5∆BRE5

bre5∆

GST-KK GST-SS

β'-COP

δ-COP

GST

*

A

B

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BRE5bre

5∆

UBP3ubp3∆

Gcs1p

Glo3p

β'-COP*β'-COP

ε-COP

α-COP

δ-COP

γ-COP

β-COP

CIM3 cim3-1 bre5∆bre5∆cim3-1/

β'-COP

β'-COP-Ub

shift at 37°C +- +- +- +-

-82

-175

A

B

*

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80 PQNNINGGSTTNNNNINKKKWHSNGITNNNGSSGNQGANSSGSGMSYNKSHTYHHNYSNNHIPMMASPNSGSNAGMKKQT 72 PRTPSYSISSTLN--PQAPEFILGCTASKITPDGITKEASYGSIDCQYPGSALALDGSSN-VEAEVLENDGVSGGLGQRE

160 NSSNGNG SPSYSSYNSSS YSSAT QYDL KFDVTKLKNLKENSSNLIQLPLFINTTEAEFAAASVQRYELNMKALNLNSESL 149 RKKKKKR----PPGYYSYLKDG----G--DDSISTEALVNGHANSAVPNSVSAEDAEFMGDMPPPLTP--RTCNSP----

240 ENSSVEKSSAHHHTKSHSIPKHNEEVKTETHGEEEDAHDKKPHASKDAHELKKKTEVKKEDAKQDRNEKVIQEPQATVLP 213 QNSTDSVSDIVPDSPFPGALGSDTRTAGQPEGGPGADFGQSCFPAEAGRDTLSRTAGAQPCVGTDTTENLG-VANGQILE

320 VVDKKEPEESVEENTSKTSSPSPSPPAAKSWSAIASDAIKSRQASNKTVSGSMVTKTPISGTTAGVSSTNMAAATIGKSS 292 SSGEGTATNGVELHTTESIDLDPTKPESASPPADG----------TGSASGTLPVSQPKSWASLFHDSK-------PSSS

400 SPLLSKQPQKKDKKYVPPSTKGIEPLGSIALRMCFDPDFISYVLRNKDVENKIPVHSIIPRGIINRANICFMSSVLQVLL 355 SPVAYVETKYSPPAISPLVSEKQVEVKEGLVPVSEDPVAIKIAELLENVTLIHKPVSLQPRGLINKGNWCYINATLQALV

480 YCKPFIDVINVLSTRNTNSRVGTSSCKLLDACLTMYKQFDKETYEKKFLE KKSNADDAEKTTESDA SKSKSFQHCATADA 435 ACPPMYHLMKFIPLYSKVQRP-CTSTPMIDSFVRLMNEFTNMPVPPKPRQ-------------ALG--DKIVRDIRPGAA

560 VKPDEFYKTLSTI KFKDLQWGHQEDAEEFLTHLLDQLHEELISAIDGLTD FIRNLSRYNEIQNMLQSINDEQLKVF GKA 499 FEPTYIYRLLTVN-KSSLSEKGRQEDAEEYLGFILNGLHEEMLNLKKLLSP------------------SNEKLTIS---

640 E

P

EFIKNASPRLK LIEKYGVINDDSTEENGWHEVSGSSKRGKKTKTAAKRTVEIVPSPISKLFGGQFRSVLDIPNNKESQS 557 -----------N-GPKNHSVNEEEQEEQGEGSEDEWEQVGPRNKTSVTRQADFVQTPITGIFGGHIRSVVYQQSSKES--

720 ITLDPFQTIQLDISDAGVNDLETAFKKFSEYELLP-FKSSSGNDVEAKKQTFIDKLPQVLLIQFKRFSFIN MNVNKDNA T 623 ATLQPFFTLQLDIQSDKIRTVQDALESLVARESVQGYTTKTKQEVEISRRVTLEKLPPVLVLHLKRFVYEK---------

YPKTSSPPPPTPTNMQIPIYQAPLQMYGYTQAPYLYPTQIPAYSFN-MVNQNQPIYHQSGSPHHLPPQYIFGDFSPDEFNQFFVTPRSSVELPPYSGTVLCGTQAVDKLPDGQEYQRIEFGVDEVIEPSDTL

Ubp3 1 MNMQDANKEESYSMUSP10 1 ---------MALHS

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

Ubp3 USP10

799 YN NAYNGRIEKIRKKIKYGHELIIPEESMSSITLKNNTS DDRRYKLTGVIYHHGVSSDGGHYTADVYHSEHNKWYRID 694 -T----GGCQKLIKNIEYPVDLEISKELLSPGVKNKNFK--CHRTYRLFAVVYHHGNSATGGHYTTDVFQIGLNGWLRID

879 DVNITELEDDDVLKG SDSRTAYILMYQKRN-- 767 DQTVKVINQYQVVKP----TAERTAYLLYYRRVD

GI

GEEALL

β'-COP

ε-COP

α-COP

δ-COP

γ-COP

β-COP

LexA-USP10 LexA

Gal4-

B

A

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Strain Genotype Reference

L40FY1679-10AFY10305B (bre5∆)BY10000BY16148 (ubp3∆)RH311-3DFLY264 (wt)

FLY264 (bre5∆)

FLY306 (Ret 1.1)

CH1305CH1462CH1305-bre5∆CH1462-bre5∆bre5∆↑glo3YPH 499CMY 763Cim wt bre5∆Cim 3-1 bre5∆

MATaade2 his3 leu2 trp1 LYS::lexA-HIS3 URA3::lexA-lacZ MATα ura3 trp1 his3 MATα ura3 trp1 his3 YNR051c::kanMX4 MATα ura3 leu2 his3 lys2 MATα ura3 leu2 his3 lys2 YER151c::kanMX4 MATα ura3 leu2 trp1MATa ura3 leu2 his3 ade2/3 trp2 ste2::LEU2 STE2-WBP1::URA3MATa ura3 leu2 his3 ade2/3 trp2 ste2::LEU2 STE2-WBP1::URA3 YNR051c::kanMX6MATa ura3 leu2 his3 ade2/3 trp2 ste2::LEU2 STE2-WBP1::URA3 ret1.1MATa ade2 ade3 leu2 ura3 lys2 can1 MATα ade2 ade3 leu2 ura3 his3 MATa ade2 ade3 leu2 ura3 lys2 can1 YNR051c::kanMX4 MATα ade2 ade3 leu2 ura3 his3 YNR051c::kanMX4 MATα ade2 ade3 leu2 ura3 his3 YNR051c::kanMX4 glo3MATa ura3 leu2 his3 trp1 lys2 ade2 MATα ura3 leu2 his3 cim3-1 MATa ura3 leu2 his3 trp1 lys2 ade2 YNR051c::kanMX6MATα ura3 leu2 his3 cim3-1 YNR051c::kanMX6

Gift from J. CamonisEurofanEurofanEuroscarfEuroscarfGift from F. Letourneur(24)

This study

(24)

(33)(33)(10)(10)This study(28)(28)(10)(10)

Plasmid

pBTM-116pLexA-USP10pAD-Gal4-2.1pGal4-αCOPpGal4-βCOPpGal4-βCOPpGal4-δCOPpGal4-εCOPpGal4-γCOPpCH1122-BRE5YCpLAC111pGLO3p415-ADHpBRE5pBRE5-146.515pBRE5-1.400

2µm TRP1 ADH-LEXA (DNA Binding Domain)2µm TRP1 ADH-LEXA-USP102µm LEU2 ADH-GAL4 (Activation Domain)2µm LEU2 ADH-GAL4-αCOP2µm LEU2 ADH-GAL4-βCOP2µm LEU2 ADH-GAL4-βCOP2µm LEU2 ADH-GAL4-δCOP2µm LEU2 ADH-GAL4-εCOP2µm LEU2 ADH-GAL4-γCOPCEN URA3 ADE3 BRE5(0.5kb 5 and 3flanking sequences)CEN LEU2CEN LEU2 GLO3 (+0.5kb 5 and 3 flanking sequences)CEN LEU2 ADH promCEN LEU2 ADH-BRE5 (codon 1 to 515)CEN LEU2 ADH-BRE5 (codon 146 to 515)CEN LEU2 ADH-BRE5 (codon 1 to 400)

(34)(11)Stratagene(15)(15)(15)(15)(15)(15)(10)(35)This study(36)(10)(10)(10)

Supplementary table: Strains and plasmids used in this study

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Mickael Cohen, Francoise Stutz and Catherine DargemontDe-ubiquitination: A new player in Golgi to ER retrograde transport

published online October 30, 2003J. Biol. Chem. 

  10.1074/jbc.C300451200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2003/11/12/C300451200.DC1

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