Arf-like GTPases: not so Arf-like after all

Arf-like GTPases: not so Arf-like after allChristopher G. Burd, Todd I. Strochlic and Subba R. Gangi Setty

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA

ADP-ribosylation factor (Arf) GTP-binding proteins are

among the best-characterized members of the Ras

superfamily of GTPases, with well-established functions

in membrane-trafficking pathways. A recent watershed

of genomic and structural information has identified a

family of conserved related proteins: the Arf-like (Arl)

GTPases. The best-characterized Arl protein, Arl2,

regulates the folding of b tubulin, and recent data

suggest that Arl1 and Arf-related protein 1 (ARFRP1) are

localized to the trans-Golgi network (TGN), where they

function, in part, to regulate the tethering of endosome-

derived transport vesicles. Other Arl proteins are

localized to the cytosol, nucleus, cytoskeleton and

mitochondria, which indicates that Arl proteins have

diverse roles that are distinct from the known functions

of traditional Arf GTPases.

ADP-ribosylation factor (Arf) GTPases are crucial regula-tors of secretion, endocytosis, phagocytosis and signaltransduction, and much has been learned about how theirnucleotide-dependent conformational changes are har-nessed to maintain and modulate these cellular activities.The best-characterized members of the Arf family – Arf1,Arf6 and Sar1 – regulate the composition of secretory andendocytic organelles by recruiting vesicle coat proteins(COPI, COPII, clathrin, clathrin adapters and GGAproteins) that mediate the sorting and transport ofproteins and lipids between compartments, and byactivating lipid kinases and lipases [1,2]. Early studiesto identify genes encoding Arf GTPases in evolutionarilydivergent organisms led to the discovery of relatedproteins that have been termed Arf-like (Arl) GTPases[3,4]. Collectively, the Arf, Sar and Arl proteins constitutethe Arf family of GTPases.

More than ten genes encoding Arl proteins have beenidentified in the human genome, and their products havebeen classified into related groups on the basis ofdistinguishing primary sequence features [5,6] (Table 1).A unifying aspect of most Arf family GTPases is thestructural mechanism by which GDP and GTP binding isharnessed for signaling [5] (Box 1). Many Arl GTPases arehighly conserved throughout eukaryotic evolution [6],indicating that they have important roles; however, theroles of most Arl GTPases are completely unknown. In thisreview, we discuss recently published work that has begunto elucidate the structures and functions of several ArlGTPases.

Corresponding author: Christopher G. Burd ([email protected]).

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Arl GTPases: diversity in the Arf family

The three-dimensional structures of Arl1, Arl2 and Arl3conform to the Ras archetype of a six-stranded b-sheetsurrounded by five a-helices [7–9]. A comparison of thestructures of Arl1–GDP [7] and Arl1–GTP [10,11] showsthat the conserved structural features of the Arf familyinterswitch toggle mechanism are also used to link GTPbinding and membrane targeting in Arl1 (Box 1), as wouldbe expected from its amino acid sequence, which is closelyrelated to that of Arf1 (human Arl1 shares 56% identityand 76% similarity with Arf1).

Pasqualato et al. [5] carried out a comparative analysisof Arl protein sequences in the context of the availablestructures of Arf family GTPases and concluded that thereare significant structural differences between some Arland Arf proteins in the regions surrounding the nucleo-tide-binding pocket and in the short amino (N)- andcarboxy (C)-terminal regions that extend from the GTPasedomain (Table 1). These differences suggest that aspects ofthe enzymatic nucleotide cycles, including the require-ment of a guanine-nucleotide-exchange factor (GEF) forbinding to GTP, the conformational changes that accom-pany GTP binding, and the intracellular targetingmechanisms and functions of some Arl GTPases, will befound to differ substantially from the Arf prototype [5].

A diagnostic feature of Arf family GTPases is a segmentof 10–25 amino acids containing a myristoylated, amphi-pathic a-helix that extends from the N terminus of theGTPase domain (Box 1). In Arf1, this segment interactswith membrane lipids – an interaction that helps toanchor the GTPase to the cytoplasmic leaflet of organellemembranes and facilitates GTP binding, which alsorequires a GEF [12–14]. At position C2 of most Arf familyGTPases is a glycine that is myristoylated; however, thesequences of Arl8A, Arl8B and Arf-related protein 1(ARFRP1, also called ARP) do not contain myristoylationsites and Arl2 has been reported to not be myristoylated,despite the presence of a myristoylation motif [15]. Thisfinding suggests that the intracellular targeting andactivation mechanisms of these non-myristoylated Arlproteins differ from those of the myristoylated Arfproteins. Recent evidence indicates that ARFRP1 islocalized to Golgi membranes even though it is notmyristoylated, and interesting details have emergedabout its targeting mechanism.

ARFRP1 and its orthologs contain a tyrosine orphenylalanine at position C2, and recent work hasshown that this residue is crucial for acetylation ofthe N-terminal initiator methionine [16,17]. Acety-lation of N-terminal residues is a common modificationof cellular proteins, and thus ARFRP1 is not unusual in

Review TRENDS in Cell Biology Vol.14 No.12 December 2004

. doi:10.1016/j.tcb.2004.10.004

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Table 1. Arf-like GTPasesa,b

Arlc Organisms Distinguishing

features

Localization Function Interacting proteins Refsd

Arl1 Hs, Mm, Dr,

Ce, Dm, At,

Sc

Arl protein most

similar to Arf1

TGN Endosome-to-

Golgi trafficking;

TGN protein

sorting; ion

homeostasis

(yeast)

Golgin-97, Golgin-245,

GCC88, GCC185, Imh1,

VFT/GARP, SCOCO,

POR1/Arfaptin2,

Pericentrin, HRG4,

MKLP1, PDEd

[10,11,19,20,

30–32,43,48]

Arl2

(ScCin4p,

SpAlp41p,

AtTTN5/HAL,

CeEVL-20)

Hs, Mm, Dr,

Ce, Dm, At,

Sc

Not myristoylated Cytosol,

microtubules,

mitochondria

Microtubule

biogenesis;

mitochondrial

function?

BART, PDEd, cofactor D,

PP2A HRG4

[8,15,27,48,63]

Arl3 Hs, Mm, Dr,

Ce, Dm, At

Related to Arl2 Cytosol,

microtubules

Possible role in

microtubule

biogenesis

RP2, BART, PDEd, HRG4,

Golgin-245

[29,48,64]

Arl4 Hs, Mm, Dr,

Dm

Spontaneous GTP

binding; insertion in

interswitch region;

NLS

Cytosol, nucleus

or nucleoli

Possible role in

spermatogenesis

Importin-a [54,55]

Arl4B Hs, Mm, Dr Unknown Unknown None known [6]

Arl7 Hs, Mm, Dr,

Dm

Spontaneous GTP

binding; insertion in

interswitch region;

candidate NLS;

related to Arl4

Cytosol Possible role in

cholesterol export

None known [5,54,65]

Arl9 Hs, Mm, Dr Related to Arl4 Unknown Unknown None known [6]

Arl5 Hs, Mm, Dr,

Ce, Dm, At

NLS Nucleus or

nucleoli

Unknown HP1a [56]

Arl5B Hs Unknown Unknown None known [6]

Arl6 Hs, Mm, Ce,

Dm

Gly/Ser

substitution in

switch II region;

candidate NLS

Cytosol Might be mutated

in Bardet–Biedl

syndrome

Sec61b [5,54,66,67]

Arl8 Mm, Dr, Ce,

Dm, At

No myristoylation

signal

Unknown Unknown None known [5,6]

Arl8A Hs, Mm No myristoylation

signal


Arl8B Hs, Mm, Dr No myristoylation

signal


Arl10 Hs, Mm Unknown Unknown None known [6]

Arl11 Hs, Mm Unknown Unknown None known [6]

ARFRP1 or

yeast Arl3

Hs, Mm, Dr,

Ce, Dm, At,

Sc

Insertion preceding

switch I;

spontaneous

GTPase activity

in vitro; not

myristoylated;

N-terminal acetylation

and Sys1 required

for Golgi localization

TGN Required to target

Arl1 to the Golgi

Slo1p (yeast), Sys1 [16,17,19,43]

aAbbreviations: Arf, ADP-ribosylation factor; Arl, Arf-like GTPase; At, Arabidopsis thaliana; BART, Binder of Arl2; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster;

Dr, Danio rerio; EVL, Ena/VASP-like; HRG, Heregulin; Hs, Homo sapiens; MKLP, mitotic kinase-like protein; Mm, Mus musculus; NLS, nuclear localization signal; PDE,

phosphodiesterase; PP, protein phosphatase; Sc, Saccharomyces cerevisiae; TGN, trans-Golgi network.bSolidi (‘/’) denote different names for the same protein.cNomenclature is based on Ref. [6].dRefs refer to interacting proteins unless none is known, in which case they refer to information in the ‘Distinguishing features’ and/or ‘Function’ columns.


this respect. ScArl3, the Saccharomyces cerevisiae ortho-log of ARFRP1 (which should not be confused withmetazoan Arl3), is mislocalized to the cytosol in mutantcells that lack the NatC N-terminal acetyltransferaserequired for its acetylation, and downstream signaling byScArl3 is abrogated [16,17]. Targeting of ScArl3 to theGolgi also requires a conserved integral membraneprotein, Sys1, that can be co-purified with ScArl3 in thepresence of a chemical crosslinking reagent, whichsuggests that Sys1 and ScArl3 are associated in a complex[16,17] (Figure 1).

Elegant experiments by Behnia et al. [16] imply thatSys1 directly recognizes the N-terminal helical segment of

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ScArl3, including the acetylated methionine. By redirect-ing Sys1 to the endoplasmic reticulum (ER) from the Golgi(by placing a Lys-Lys-x-x signal for ER localization on itsC terminus), it has been shown that a chimeric GTPase,comprising the N-terminal segment of ARFRP1 fused tothe GTPase domain of Arf1, also localizes to the ER. Itseems that the N-terminal segments of ARFRP1 andScArl3 (S.R. Gangi Setty and C. Burd, unpublished) areSys1-dependent targeting signals, at least when appendedto another GTPase of the Arf family.

The targeting of another non-myristoylated Arf familyGTPase, Sar1, depends in part on an interaction betweenits N-terminal helix and an integral membrane protein,


Box 1. Nucleotide-dependent structural changes in the Arf

family interswitch toggle mechanism

A comparison of the recently described structures of yeast GDP-

bound ADP-ribosylation factor (Arf)-like GTPase (Arl)1 [7] and

mammalian Arl1–GTP [10,11] reveals the structural changes that

occur during the interswitch toggle mechanism that unifies the Arf,

Sar and Arl GTPases (Figure I), which was originally proposed on the

basis of the GDP-bound and GTP-bound structures of several Arf

family GTPases [8,59,60]. Arl1 is localized to the cytosol in its GDP-

bound state and translocates to membranes of the trans-Golgi

network on GTP binding [30]. Themembrane association of Arl1, like

that of other myristoylated members of the Arf family, is dependent

on the myristoylated, amphipathic, N-terminal helical domain

(Figure I, blue). In the GDP-bound state, this helix packs into a

hydrophobic groove on the opposite side of the molecule to the

nucleotide-binding site. Amino acids of the switch I (yellow) and

switch II (green) segments, in addition to the interswitch segment

(red) that connects them, participate in nucleotide binding, and the

conformations of these regions undergo marked changes on GTP

binding that eject the helix from its binding site. The structure of the

N-terminal helix was not determined in the Arl1–GTP structures, but

has been modeled here on the basis of results showing that Arf1

interacts with membrane lipids [12,13].

In the Arl1–GDP structure, switch I forms a b-strand that is

characteristic of Arf family GTPases (it is not observed in most

other Ras-related GTPases), and this conformation prevents spon-

taneous GTP binding. Thus, Arf family GTPases require a guanine-

nucleotide-exchange factor (GEF) to facilitate the conformational

changes required for GTP binding [61,62]. Note that in Arl1–GDP, the

switch I b-strand makes b-sheet contacts with b2 of the interswitch

segment, but these interactions are eliminated by the conformation-

al change that occurs on GTP binding. The register of the b2–b3

strands (the interswitch) shifts by two residues (almost 7 A) from the

nucleotide-binding pocket, thereby ejecting the N-terminal helix. In

the Arl1–GTP structure, the switch I segment has swung outward

and the b-strand is not observed. The orientation of switch II has

changed to accommodate the binding of GTP (in the GDP-bound

conformation, part of switch II is not ordered and is indicated as a

dotted line). Signaling by most Arf family GTPases is thought to be

terminated by GTPase-activating proteins (GAPs) that stimulate the

rate of GTP hydrolysis.

TRENDS in Cell Biology

Arl1–GTPArl1–GDP

GEF

GAP

Figure I. Nucleotide-dependent structural changes in ADP-ribosylation factor

(Arf)-like GTPase (Arl)1 demonstrate the Arf family interswitch toggle

mechanism.

Review TRENDS in Cell Biology Vol.14 No.12 December 2004 689

Sec12, which functions as a GEF for Sar1 [18]. Might Sys1function as a GEF for ScArl3? The fact that a constitu-tively activated mutant form of ScArl3, which shouldbypass the requirement for GEF-mediated activation, stillinteracts with Sys1 suggests that Sys1 is not functioningas a GEF for ScAr13, although this remains to be tested


directly [17]. Overall, the results suggest that Sys1functions to localize ScArl3 constitutively to Golgi mem-branes, and it might be that the GTP cycle of ScArl3regulates an activity of Sys1 that remains to be identified.The targeting of ScArl3 represents a model mechanism inwhich an integral membrane protein is important for theintracellular targeting of an Arf family GTPase, and it islikely that other integral membrane proteins will bediscovered that function as receptors for membrane-associated Arl and Arf proteins.

Distinguishing Arl from Arf

The downstream functions of Arf family GTPases aremediated by effector proteins that specifically bind to theGTP-bound form of the enzyme. Given the high degree ofstructural similarity between Arf and Arl proteins, it isnot obvious how effectors might specifically distinguishamong these proteins to achieve specificity for Arlsignaling pathways. This is particularly instructive forArl1, the Arl GTPase that is most closely related to Arf1,because crystal structures of Arl1–GTP in a complex witha GRIP [Golgin-97, RanBP2a, Imh1p and p230 (also calledGolgin-245 or tGolgin-1)] domain from Golgin-245 havebeen recently determined [10,11]. The GRIP domain is amodule of about 50 amino acids that binds specifically toArl1–GTP, but not to Arf1–GTP [10,19,20].

In general, hydrophobic interactions predominate atthe interface between Arl1 and the GRIP domain, whichincludes contacts between residues of the switch I,interswitch and switch II regions of Arl1. Specific bindingis conferred largely by interactions involving amino acidsof the switch II region with an a-helix (a1) of the GRIPdomain [10,11]. In the structures of Arf1 in a complex withseveral different effectors, contacts are also observedbetween switch II and an a-helix of the effector; however,small differences in the interacting surfaces of Arl1 andArf1 with their effectors seem to account for the specificityof these interactions [10,11].

For example, both Arl1 and Arf1 contain a hydrophobicpocket in the switch II region that is occupied by a sidechain from a bound effector, but small differences in theshape of this pocket lead to steric constraints such thatonly the appropriate effector can bind. In addition, aconserved cysteine residue (Cys80) in switch II is uniqueto Arl1 and seems to be specifically recognized by theGRIP domain. Despite the overall similarity in theinteracting surfaces of Arl1 and Arf1 with their effectors,it seems that relatively subtle differences on the surfacesof Arl proteins are sufficient to confer specific recognition.The identification and characterization of other ArlGTPase effectors will aid our understanding of thespecificity of Arl GTPase signaling pathways and willobviously provide important clues to the functions of Arlproteins.

Functions of Arl GTPases

The cellular functions of most Arl GTPases are unknown,and speculation regarding their functions has been drivenlargely by knowledge of their intracellular localization andthe identification of Arl-binding proteins that are putativeeffectors. In contrast to the Arf GTPases, only two Arl


GEF?

GRIP

Arl1 GDP

GTP

Sys1

GTP

ARFRP1

NatC

Sys1

GDP

GEF?

Endosome

Golgin-97

ARFRP1

ARFRP1 ARFRP1Arl1

Transport vesicles

trans-Golgi network


Figure 1. Targeting and functions of Golgi-localized ADP-ribosylation factor (Arf)-like GTPases (Arls). Golgi targeting of Arf-related protein 1 [ARFRP1 (termed ScArl3 in

Saccharomyces cerevisiae)] requires acetylation of its N-terminal methionine (black circle), which in yeast requires the NatC N-terminal acetyltransferase complex [16,17].

The Golgi-localized integral membrane protein Sys1 is also required for Golgi localization of ARFRP1. It is thought to recognize the N-terminal helical region of ARFRP1,

including the acetylated methionine [16,17]. Inactive GDP-bound Arl1 is localized to the cytosol; its localization to Golgi membranes requires activated ARFRP1 [19,30]. The

components of this signaling pathway that potentially link ARFRP1 signaling to activation of Arl1, including the guanine-nucleotide-exchange factors (GEFs) that activate

ARFRP1 and Arl1, have not been identified. Targeting of Arl1 to the Golgi also requires myristoylation of Arl1 (jagged black line) [30], although ARFRP1 is not myristoylated.

Proteins containing GRIP (Golgin-97, RanBP2a, Imh1p and p230) domains, such as Golgin-97, are recruited to the trans-Golgi network (TGN) by GTP-bound Arl1 [10,19,20,40].

Inhibition of Arl1 or Golgin-97 function perturbs vesicle-mediated trafficking between endosomes and the TGN in a manner consistent with the idea that Golgin-97 functions

as a tether for endosome-derived transport vesicles [32]. Only a single molecule of Golgin-97 is shown for simplicity, although Golgin-97 and all GRIP domain proteins are

likely to be dimers.


proteins, Arl1 and ARFRP1, have been shown to be localizedto intracellular membranes of secretory and endocyticorganelles; the others have been localized to the cytosol,nucleus, mitochondria and the cytoskeleton (Table 1).

Regulation of microtubule biogenesis by Arl2 and Arl3

A remarkable convergence of biochemical and geneticevidence in diverse organisms indicates that Arl2 andpossibly Arl3 regulate microtubule biogenesis. Geneticscreens for mutants with deficiencies in microtubule-dependent processes such as chromosome transmissionand cytokinesis have led to the identification of Arl2orthologs in S. cerevisiae [21], fission yeast (Schizosacchar-omyces pombe) [22], plant (Arabidopsis thaliana) [23,24]and nematode (Caenorhabditis elegans) [25]. In multi-cellular organisms, mutations in Arl2 lead to loss of thecortical microtubules required for cytokinesis, and inC. elegans Arl2 (also known as EVL-20) localizes to thesemicrotubules [24,25].

An important clue to the molecular function of Arl2 hascome from the discovery that Arl2 associates with thetubulin folding chaperone cofactor D, which binds tob tubulin and assists in assembling the tubulinab heterodimer [26,27] (Figure 2). Cofactor D bindsreversibly to b tubulin in a cycle that involves interactionswith other tubulin folding cofactors and leads to theformation of an assembled ab heterodimer. In vitrobiochemical assays have shown that Arl2–GDP can inhibit


this cycle and can also inhibit the ability of cofactor D tomediate the destruction of microtubules when the cofactoris overexpressed in cultured cells [26].

Because most G-protein effectors bind to theGTP-bound conformation of the enzyme, the observationthat only Arl2–GDP is active in associating with andregulating cofactor D provides an interesting twist tointeractions between G proteins and their effectors.Moreover, these results leave open the possibility thatArl2–GTP might have functions that are distinct from theregulation of tubulin folding. In fact, a small proportion ofArl2 (less than 10%) has been localized to the innermembrane space of mitochondria, and an Arl2–GTPeffector, Binder of Arl2 (BART), can bind to the adeninenucleotide transporter (ANT) thereby forming anArl2–GTP–BART–ANT complex [15]. Further work willbe needed to determine whether Arl2 regulates mitochon-drial functions, in addition to microtubule dynamics, andwhether or not Arl3 regulates tubulin folding.

The sequence and structure of Arl3 are highly related tothose of Arl2, and the sequence of a putative humanArl3–GTP effector, retinitis pigmentosa 2 (RP2) protein, issimilar to that of tubulin folding cofactor C, raising thepossibility that Arl3 and RP2 might also regulate tubulinbiogenesis [28,29]. The discovery that Arl2 regulates thebiogenesis of tubulin sets a clear precedent for functionsof Arl GTPases that are distinct from the membrane-trafficking roles of Arf and Sar GTPases. Recent work



co-D

co-D

Arl2Arl2

α

βco-D

co-Eco-C

α

βGTP GDP

α

β

co-D

α

β

co-D

co-E

co-C

GDPGDP

Arl2

Arl2

(a)

(b)(i)

(ii)

Figure 2.ADP-ribosylation factor (Arf)-like GTPase (Arl)2 regulates cofactor (co)-D, a

b-tubulin chaperone. (a) GDP-bound Arl2 binds to co-D. (b) Arl2 has been shown to

regulate formation of the tubulin ab heterodimer by two biochemical activities [26].

(i) In the final step of assembling an ab-tubulin heterodimer, three chaperones

(co-C, co-D and co-E) are associated with the ab-tubulin heterodimer and promote

GTP hydrolysis by functioning as a GTPase-activating protein (GAP) for b tubulin.

GTP hydrolysis by b tubulin releases the assembled heterodimer, which is then

available for incorporation into microtubules. By directly binding to co-D, Arl2

inhibits the GAP activity of the supercomplex containing ab tubulin and co-C, co-D

and co-E, thereby preventing the release of native ab tubulin (a). (b)(ii) When co-D is

overproduced in cells (e.g. by transfection of its gene), it causes the destabilization

of microtubules by sequestering b tubulin, which, in turn, destroys the ab-tubulin

heterodimer; Arl2 has been shown to inhibit this process. These results indicate that

GDP-bound Arl2 inhibits the function of co-D in tubulin biogenesis.


on two other Arl GTPases, Arl1 and ARFRP1, suggests,however, that these Arl GTPases do regulate membranetrafficking.

Golgi-localized Arl GTPases

The trans-Golgi network (TGN) is the face of the Golgi atwhich proteins are sorted into different types of transportvesicle for export from the Golgi. In addition, the TGNreceives material from endosomes through retrogradevesicle-mediated transport. Both Arl1 and ARFRP1 arelocalized to the TGN, and functional depletion of eitherprotein in cultured mammalian cells results in the dis-sociation of a subset of TGN peripheral membrane proteins,a slowing in the export rate of secretory cargo, and a defect inendosome-to-Golgi transport [16,17,20,30–32]. In yeast,arl1 and arl3 null mutants are viable and have minordefects in protein sorting in the TGN, indicating that Arl1and ScArl3 are not required for constitutive proteinsecretion or for sorting to the lysosome-like vacuole[33–36]. The plasma membrane of yeast arl1 null mutantcells is hyperpolarized, leading to defects in ion homeo-stasis, suggesting that Arl1 regulates the localization oractivities of ion transporters [37–39]. Recent evidencefrom experiments in yeast and mammalian cells has


identified a family of Arl1 effectors, and at least one ofthese proteins seems to regulate vesicle-mediated endo-some-to-TGN transport.

Proteins containing GRIP domains have beenrecently identified as a family of Arl1-specific effectors[10,19,20,40] (Table 1 and Figure 3). As mentioned above,the GRIP domain binds specifically to Arl1–GTP, and theGRIP domains of several proteins have been shown tobe both necessary and sufficient for their localization tothe Golgi [41–43]. In addition to their C-terminal GRIPdomain, all of these peripheral membrane proteins arelarge (from w900 to more than 2000 amino acids) andcontain a preponderance of heptad repeats that areindicative of dimeric rod-shaped structures. For thesereasons, GRIP domain proteins are considered to bemembers of the loosely defined Golgin family that hasfunctions in vesicle-mediated trafficking, stacking of Golgicisternae and Golgi positioning in the cell [44,45]. Morespecifically, GRIP domain proteins have been proposed tofunction as vesicle tethers that promote transport vesicletargeting by mediating long-range contacts betweenopposing membranes [44–46].

Although most known tethering proteins are effectorsof Rab GTPases, recent evidence supports the idea thatthe Arl1 effector Golgin-97, which binds to Arl1 throughits GRIP domain, regulates endosome-to-Golgi transportvia tethering of endosome-derived vesicles to the TGN.Perturbing the function of Arl1 or Golgin-97 blocks thedelivery of endocytosed Shiga toxin B, which piggybacksthrough the cell on a glycolipid, to the TGN in a processthat precedes the requirement for Syntaxin 16, a TGNSNARE [32]. These results suggest that Golgin-97 func-tions upstream of the SNARE-dependent fusion of endo-some-derived vesicles with the TGN, as would be expectedfor a vesicle tether. Glycolipids en route from endosomes tothe TGN are also misrouted in cells depleted of the GRIPdomain protein Golgin-245, although it is not clearwhether this is because Golgin-245 has a direct role inretrograde transport (M. Marks, pers. commun.).

Among the effects of depleting Arl1 (by RNA inter-ference) or Arl1–GTP (by high-level expression of a GRIPdomain) is a skewing to endosomal compartments of thesteady-state localization of proteins such as TGN46 thatcycle between the TGN and endosomes [20,31]. In a yeastmutant that lacks the GRIP domain protein Imh1,retrieval of Kex2 (a furin-related protease) from endo-somes back to the Golgi seems to be defective [47]. Anotheryeast Arl1 effector, the GARP–VFT complex, is also aputative vesicle tether that has been implicated inendosome-to-Golgi trafficking [40]. Taken together, theseresults suggest that Arl1 regulates retrograde trafficking(transport from later compartments to earlier compart-ments) by recruiting vesicle tethers. Notably, one of theprimary functions of Arf1 is also to regulate retrogradetrafficking; however, Arf1 achieves this by regulating therecruitment of vesicle coat proteins that are required toform transport vesicles that carry cargo between Golgicompartments and from the Golgi to the ER.

A subset of Arf1 effectors, Arfaptin2 (also termedPOR1) and MKLP1, have the ability to bind to Arl1in vitro, and expression of a mutant, the GTP-restricted


Wild type

arl1∆

Wild type

ScArl3∆

(a) (b)

(c) (d)

Figure 3. GRIP (Golgin-97, RanBP2a, Imh1p and p230) domains are recruited to the

Golgi by ADP-ribosylation factor (Arf)-like GTPase (Arl)1. Localization studies in

yeast cells show that a GRIP domain that binds specifically to Arl1–GTP localizes to

the Golgi in an Arl1-dependent manner. (a) Visualization for Arl1 fused to green

fluorescent protein (GFP) shows that Arl1 is localized to approximately ten discrete

puncta that correspond to late Golgi compartments [19,40]. (b) Expression of the

GRIP domain of the yeast Imh1 protein fused to red fluorescent protein (RFP) in the

same cells shows colocalization of Arl1–GFP and RFP–GRIP (yellow signals).

(c) GFP–GRIP localizes to the cytosol in mutant cells lacking the gene encoding Arl1

(arl1D), indicating that Arl1 is required for recruitment of the GRIP domain to the

Golgi. (d) Arl1–GFP is localized to the cytosol in mutant cells lacking ScArl3 [the

yeast ortholog of Arf-related protein 1 (ARFRP1)]. These experiments suggest an Arl

GTPase signaling pathway in which ARFRP1 functions upstream of Arl1.


form of Arl1, in cultured mammalian cells leads to anincrease in the amounts of Arf1-regulated vesicle coatproteins, COPI and AP-1, that are associated with theGolgi [30,48]. These results imply that Arl1 sharesoverlapping roles with Arf1, in addition to its functionsthat are carried out by unique effectors such as GRIPdomain proteins. However, the physiological significanceof these interactions is not yet clear because in cellsexpressing high levels of GTP-restricted Arl1, the amountof Arf1 observed on Golgi membranes was also increased,so it was not possible to distinguish whether Arl1recruited Arf1 effectors directly [30]. These experimentsraise the possibility that Arl1 can influence the activationand recruitment of Arf1 to the Golgi, and recent studieshave shown that the functions of Arl1 and ARFRP1 arealso intertwined.

In both yeast and cultured mammalian cells, inhibitionof signaling by ScArl3 and ARFRP1, respectively, leads toa loss of Arl1 and its effectors from the TGN [16,19,40](Figure 3). One way to explain these observations is thatARFRP1 and Arl1 are activated sequentially, akin to thecascade of yeast Golgi Rab GTPases in the exocyticpathway [49,50] (Figure 1). For example, the GEF forArl1 might be an effector of ARFRP1. Intriguingly, theArf1 GEF mSec7 (also known as Cytohesin) has beenshown to bind to GTP-restricted ARFRP1 [51], althoughthe GEF domain of mSec7 does not stimulate binding ofGTP-gS to Arl1 in in vitro GEF assays [48].

The identification of additional effectors and regulatorsof ARFRP1 and Arl1 will be required to dissect themolecular linkage between them and to examine more


closely the possibility that signaling by Arl1 is linked toactivation of Arf1 in the TGN. The sequential action ofARFRP1 and Arl1 could ensure the ordered assembly ofprotein and/or lipid complexes that might be required forvectoral transport to, through or out of the Golgi.Alternatively, this arrangement could result in a localizedpool of activated Arl1 that might serve to define asubdomain of the TGN. In support of the latter hypothesis,some GRIP domain proteins seem to be localized todistinct subdomains of the TGN [52,53].

Less characterized Arl GTPases

Arl1, Arl2 and ARFRP1 are the best-characterized ArlGTPases and it is not yet possible to assign functions toany of the others. The analysis of these other proteins is atan early stage, and most studies have so far focused onidentifying their tissue expression patterns, intracellularlocalization and putative effectors (Table 1).

The Arl4 and Arl5 proteins are localized to the nucleusand nucleoli [54–56], and mouse Arl4 has been implicatedin testis development [55,57]. The genes encoding mouseARFRP1 and fruitfly ARL1 are essential for earlydevelopment, but their specific functions are not yetknown [3,58]. Genetic analysis of the genes encoding Arlproteins in experimental organisms in which developmentand the physiological functions of organs can be monitoredshould lead the way in elucidating the roles of the lesscharacterized Arl GTPases.

Concluding remarks

For more than a decade, the existence of Arl GTPases hasbeen appreciated, yet their biochemical characterizationand the knowledge of their functions have lagged farbehind those of the traditional Arf proteins. Recentbiochemical and structural analyses of Arl1, Arl2 andArl3 indicate that they operate by the same interswitchtoggle mechanism that has been described for Arf and Sarproteins. However, the observations that Arl GTPases arelocalized to various organelles, coupled with the discoverythat the role of the integral membrane protein Sys1 isinvolved in Golgi targeting of non-myristoylated ARFRP1,suggest that their mechanisms of intracellular targetingand activation can differ considerably from those of thetraditional Arf family GTPases. Moreover, the identifi-cation of diverse regulatory functions such as the foldingof tubulin by Arl2 and the tethering of endosome-derivedvesicles to the Golgi by Arl1 and Golgin-97 suggests thatthe roles of Arl GTPases vary considerably.

Far less is known about the functions of other ArlGTPases, although the application of genetic and proteo-mic technologies should aid the identification ofArl-specific effectors and regulators that will provideclues to their functions. Other important issues to addressin the future include the identification of GEF activatorsand GTPase-activating proteins that terminate Arl sig-naling, and the elucidation of mechanisms by which ArlGTPase signaling is integrated with other signalingpathways, as has been suggested by the discovery of thelinkage between ARFRP1, Arl1 and possibly Arf1. Withthe study of Arl GTPases poised to expand the cell biologyof Arf family GTPases into exciting new areas, we



anticipate that, as more is learned about them, theseproteins will turn out to be not so Arf-like after all.

AcknowledgementsWe apologize to those colleagues whose work we have cited indirectlythrough review articles owing to space limitations. We thank MickeyMarks, John Murray, Phong Tran and Jonathan Raper for helpfuldiscussions; Kartik Narayan for help with the figures; the anonymousreviewers for their suggestions; and Mickey Marks for suggesting thetitle. Research in our laboratory is supported by a grant from the NationalInstitutes of Health.

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Arf-like GTPases: not so Arf-like after all