703

The actin cytoskeleton of budding yeast contains an extensiveset of actin-associated proteins with conserved mammaliancounterparts. For more than 20 years, yeast has been used asa model organism to dissect the in vivo functions of thesefactors, revealing an intricate web of genetic interactions in thecell. Now, a surge of biochemical reports is defining thephysical interactions and activities of these proteins andproviding mechanistic insights into their cellular roles. Theemerging view is that most actin-associated proteins do notact alone but, rather, associate to form modular proteincomplexes that regulate actin assembly and organization.

Addresses*Rosenstiel Medical Center, Brandeis University, 415 South Street,Waltham, Massachusetts 02454, USA; e-mail: goode@brandeis.edu†Molecular and Cell Biology Department, University of CaliforniaBerkeley, 401 Barker Hall, Berkeley, California 94720-3202, USA; e-mail: arodal@uclink4.berkeley.edu

Current Opinion in Microbiology 2001, 4:703–712

1369-5274/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAAP actin-associated proteinABP actin-binding proteinARK actin-regulating kinaseARP actin-related proteinCAP cyclase-associated proteinCmd calmodulinEnd endocytosis Rvs reduced viability upon starvationSla synthetic lethal with Abp1Vrp very rich in prolineWASP Wiskott–Aldrich Syndrome proteinWIP WASP-interacting protein

IntroductionActin polymerization provides mechanical force used bycells to drive cell crawling, cell morphogenesis, endocytosisand Listeria-like intracellular ‘rocketing’ of vesicles andorganelles. Budding yeast has served for many years as anideal model organism to identify and study the componentsof the actin cytoskeleton, because of the unique opportunityto perform parallel genetic and biochemical analyses.Years of combined effort by many groups has led to theidentification of a long list of actin-associated proteins(AAPs), reviewed in [1]. Most of these AAPs are highlyconserved from yeast to mammals and, in many cases, havesimilar or indistinguishable activities (see Table 1). Untilrecently, the activities of most AAPs on actin have beeninvestigated in two-component in vitro assays, using purifiedactin and the protein of interest. However, it is now apparentthat many activities can not be detected in such assays andrequire interactions between two or more AAPs. Here, wereview the emerging concept that most AAPs physicallyinteract in complexes, and that the activities of the complexesare often not realized by studying single isolated components.

We discuss three modules that regulate actin nucleationmediated by the actin-related protein (Arp) 2/3 complex.

Genetic studies in yeast have been instrumental inestablishing the importance of AAPs in vivo and in placingthem into functional groups. A diagram of these geneticinteractions reveals an intricate web of functional interplayamong AAPs in the cell (reviewed in [1]). However, thephenotypes observed for most mutants of these factors (forexample, depolarized actin patches, loss of visible actincable staining and/or swollen cell morphologies) often donot provide mechanistic insights into their function, leavingtheir specific roles in the cell unclear. A next step that canbe taken is to purify the AAP and study its effects on actin,and these can provide insights into its functional capabilities.However, there is often no significant activity observed onactin. For example, the actin-related protein [Arp]2/3complex, Wiskott–Aldrich Syndrome protein [WASP] andactin-binding protein [Abp]1p alone each show little or noeffect on actin assembly [2•,3•], and Aip1p alone does notaffect the rate of actin disassembly [4]. How, then, do wedefine more precisely the cellular functions of an AAP? Newstudies reveal that many AAPs that were once viewed asfunctioning separately assemble into complexes with bothstable and dynamically associated components, and thesecomplexes have activities that involve interactions amongmultiple components.

Organization and function of the yeast actin cytoskeletonYeast cells contain three types of filamentous actin structures:actin cables, an actin–myosin contractile ring and corticalactin ‘patches’, all of which undergo extensive reorganizationthroughout the cell cycle [1]. The actin–myosin contractilering assembles transiently at the bud neck to facilitatecytokinesis and is reviewed by Tolliday, Bouquin and Li(pp 690–695 in this issue). Actin cables are oriented alongthe mother–daughter cell axis and are thought to serve aspolarized tracks for myosin-V-dependent delivery of cargoesinto the daughter cell. These cargoes include secretoryvesicles, mRNAs and organelles such as mitochondria, Golgi,nucleus and vacuole.

The ultrastructure of yeast actin cables has not yet beendetermined, because cables have been difficult to detectby electron microscopy within the ‘crowded’ yeast cytoplasm.It is likely that cables are comprised of bundled actinfilaments, given that they contain the known actin-bundling proteins, Sac6p/fimbrin [5] and Abp140p [6].However, it remains unclear whether the filaments incables are of uniform (parallel) or mixed (antiparallel)polarity and, if uniform, whether the barbed ends face themother or bud. Given that myosin V is a barbed-end-directedmotor, it seems likely that at least some actin filaments in

Modular complexes that regulate actin assembly in budding yeastBruce L Goode* and Avital A Rodal†

704 Growth and development

Table 1

Components of yeast actin assembly modules.

Yeast protein Putative mammalian Localization Known activities Physical interactions amongorthologue components of Figure 1

Abp1p mABP1 [75] A [42] Binds F-actin [3• ,75] and F-actin (P [3•• ,42])Arp2/3 complex [3•]

latS [8] Activates Arp2/3 complex [3•] Srv2p (L [49], BO [48], 2H [37•• ])Arp2/3 complex (P [3•])Rvs167p (BO [48], 2H [55])Ark1p/Prk1p (L, CoIP, 2H [46• ,47])

Ark1p/Prk1p GAK [46•] A [45• ,46•] Phosphorylates Pan1p Abp1p (L [46• ,47], CoIP [47], 2H [47])and Sla1p [45•]

Sla2p (2H [46•])

Arp2/3 complex Arp2/3 complex A [23•• ] Nucleates actin filament Abp1p (P [3•• ])assembly [2•]

B [12,20] Branches actin filaments(a) Las17p (CoIP [2• ,34•])latR [8] Myo3/5p (CoIP [16•• ,17•], 2H [17•], P [16•• ])

Cmd1p (2H [23•• ], CoIP [23•• ], P [72])

Cmd1p Calmodulin Multiple locations, Light chain for Myo3/5p [71] Myo3/5 (CoIP, 2H, BO [71])including cortical Binds directly to Arp2/3 Arp2/3 complex (2H [23•• ], CoIP [23•• ], patches [70] complex [23•• ,72] P [72])

End3p EH-domain-containing Unknown Inhibits phosphorylation Pan1p (2H [76], CoIP [76], L [76])proteins [65] of Pan1p by Prk1p [45•] Sla1p (2H [63•], CoIP [63•])

Las17p WASP/SCAR A [34•] Binds actin monomer Actin (2H [34• ,37•• ])B [2• ,26] Activates Arp2/3 complex [2•] Arp2/3 complex (2H [17•], CoIP [2•], L [2•])latR [34•] Rvs167p (2H [34• ,55])

Vrp1p (2H [34•,60], CoIP [16•• ,17•], CoM [31•])Sla1p (2H [37•• ], CoIP [25•]) Myo3/5p (CoIP [16•• ,17•])Novel SH3-domain proteins, includingLsb1-4p, Lsb7p (2H [34•])

Myo3/5p Myosin I B [38] Actin filament motor; F-actin via motor domain (CoIP [40•])latR [39] may activate Arp2/3 Arp2/3 complex (CoIP [16•• ,17•],

complex 2H [17•], P [16•• ])Vrp1p (CoIP [39], 2H [39])Las17p (CoIP [16•• ,17•], 2H [17•])Cmd1p (CoIP, 2H, BO [71])

Pan1p Eps15 Activates Arp2/3 complex [15•] End3p (2H [76], CoIP [76], L [76])Sla1p (2H [63•], CoIP [63•])

B [61] Endocytic machinery, includingyAP180p, Ent1/2p (2H [67], CoIP [67])

Rvs161p Amphiphysin Mostly diffuse Activities unknown Rvs167p (2H [55,77], coIP [78])cytoplasmic,some puncta [79]

Rvs167p Amphiphysin A [81] May deform lipid bilayers [56] Rvs161p (2H [54,77], coIP [78])latR [81] Abp1p (BO [48], 2H [55])

Las17p (2H [34• ,55])Srv2p, Sla2p, Sla1p, Lsb4p (2H [37•• ])Act1p (2H [37•• ,80])

Sla1p Intersectin?? A [36•] Activities unknown Las17p (2H [37•• ], CoIP [26])latR [8] End3p (2H [63•], CoIP [63•])

Rvs167p, Srv2p (2H [37•• ])Lsb4p (2H [37•• ])Sla2p (L [36•])

Sla2p HIP1R [82] B [84] Actin filament binding [83] F-actin (P [83])latR [8] HIP1R promotes clathrin Ark1p (2H [46•])

assembly and binds Rvs167p (2H [37•• ])lipids [68•] Sla1p (L [36•])

Srv2p CAP A [48,49] Actin monomer binding G-actin (coIP [51], P [51])B [50] and sequestration [51] Abp1p (L [49], BO [48], 2H [37•• ])

Rvs167p, Sla1p (2H [37•• ])

Vrp1p WIP B [35] WIP binds actin monomers Las17p (2H [34•,60], CoIP [16•• ,17•], CoM [31•])latR [35] and filaments [31•] Act1p (2H [35])

Myo3/5 (CoIP [60], 2H [60])

cables have their barbed ends oriented towards the bud.Another open issue is whether actin cables are composedof long individual filaments or short crosslinked filaments.One of the few clues here is the observation that actin cablesdisassemble rapidly (within about one minute) upon treatmentof cells with latrunculin-A, an actin-monomer-sequesteringagent [7], which is consistent with cable filaments beingshort. Resolving these and other important ultrastructureissues presents a technical challenge for the future.

The cellular functions of cortical actin patches are not aswell-defined as cables. Actin patches appear as cortical‘puncta’ by actin immunofluorescence and GFP–actinfluorescence and are comprised of actin filaments thatundergo rapid turnover [8]. Although actin is essential formaintaining cell polarity, cell wall integrity and endocytosisin yeast, it is not yet clear which, if any, of these functionsis performed by actin patches. The strongest functional linkfor actin patches is to endocytosis, given that many actin-associated proteins found at actin patches are required forendocytosis [1]. One speculation is that cortical patchesmay provide mechanical forces (from actin polymerizationand/or myosin action) that help drive vesicle internalizationand/or vesicle transport. Another possibility is that actinpatches serve as cortical ‘scaffolds’ that help assembleendocytic machinery [9]. However, the precise role of actinin endocytosis remains to be determined.

One intriguing aspect of cortical actin patches is that theyare highly motile, as revealed unambiguously in live cellsusing GFP-tagged actin and AAPs [10,11]. The importanceof actin patch motility in vivo is not yet understood, butthere is evidence that motility may be driven by actinpolymerization nucleated by the Arp2/3 complex [12]. Thishas led to speculation that actin patches may move by amechanism similar to that which drives Listeria monocytogenesmotility, leading edge protrusion during cell migration andactin-based rocketing of intracellular vesicles. An importantnext step is to define the precise molecular basis of actinpatch motility and to establish which, if any, physiologicalprocesses depend on patch motility.

General mechanisms that regulate actin assemblyActin polymerization in vivo occurs at free barbed ends offilaments, the availability of which is limited and regulatedby AAPs. There are three known mechanisms for gener-ating free barbed ends: severing of existing filaments,uncapping of filaments and de novo nucleation of newbarbed ends [13,14].

The Arp2/3 complex is composed of seven highly conservedsubunits, including two actin-related proteins (Arp2p andArp3p) and five unique proteins (Arc40p/p40, Arc35/p35,Arc19/p20, Arc18/p21 and Arc15/p15). In all species tested,the Arp2/3 complex has only weak actin nucleation activityalone and requires an ‘activator’ protein to stimulate rapidactin nucleation. In yeast, two strong activators have beenidentified, Las17p/WASP [2•] and Abp1p [3•], and oneweaker activator, the yeast eps15 homologue Pan1p [15•].In addition, genetic evidence suggests that the yeasttype I myosins (Myo3p and Myo5p) may activate theArp2/3 complex [16••,17•]. Consistent with this idea,Schizosaccharomyces pombe myosin I activates the Arp2/3complex [18], but this has not yet been demonstrated forSaccharomyces cerevisiae myosin I.

The Arp2/3 complex also generates branched actin filamentnetworks [14]. This is accomplished by binding of theArp2/3 complex to either the side or end of an existing actinfilament and, upon activation, nucleating polymerizationof a new (daughter) filament at a 70° angle to the motherfilament. No comparative analysis has yet been reported ofactin filament branching by the yeast Arp2/3 complex andits activators.

Genetic analyses of the yeast Arp2/3 complex havedemonstrated its essential role in multiple actin-dependentprocesses. Complete gene deletions of six of the sevensubunits of the complex (all but arc18) are lethal or causesevere defects in cell growth and actin organization [19•].This suggests that the Arp2/3 complex performs at leastone key, non-redundant function. Conditional alleles ofarp2, arp3 and arc35 exhibit defects in actin organization,actin patch motility, endocytosis, cell polarity, cell mor-phogenesis, organelle inheritance and nuclear export[12,20–22,23••,24•,25•]. Biochemical analyses of theArp2/3 complex isolated from these mutant strains shouldprovide mechanistic insights into cellular functions of theArp2/3 complex and may identify new regulators of itsactivities. Below, the evidence for three protein modulesin yeast that regulate Arp2/3-complex-dependent actinassembly (depicted in Figure 1) are summarized.

Module I: the Las17p complex WASP was the first cellular activator of the Arp2/3 complexto be identified [14]. However, prior to this discovery, theyeast homologue of WASP, Las17p (also known as Bee1p),had been isolated as a factor required for actin assembly ina permeabilized cell assay for polarized cortical actin-patch

Modular complexes regulating actin assembly in budding yeast Goode and Rodal 705

Table 1 legend

A, tight co-localization with cortical actin patches; B, partialco-localization with cortical actin patches (suggests ligands orfunctions associated with non-patch structures); BO, blot overlayassay; CAP, cyclase-associated protein; CoIP, co-immunoprecipitationor other affinity co-precipitation from extracts (for example, GSTpull-downs); CoM, co-migration in cell extracts using gel filtrationchromatography or sedimentation velocity through sucrosegradients; L, Localization to cortical patches depends on this

component; latR, localization to cortical patches retained after cellsare treated with latrunculin-A to disassemble visible actinstructures; latS, localization to cortical patches abolished afterlatrunculin-A treatment; Lsb, Las17p-binding protein; P, physicalinteraction of purified components; 2H, Two-hybrid assay; WASP,Wiskott–Aldrich Syndrome protein; WIP, WASP-interacting protein.(a) B Goode, unpublished data. Numbers in square brackets referto references.

assembly [26,27]. Subsequently, it was demonstrated that acarboxy-terminal fragment of Las17p (containing a proposedactin-monomer-binding domain and an acidic motif) issufficient to activate Arp2/3 complex in vitro [2•]. Theacidic motif in activators is postulated to bind to and helpactivate the Arp2/3 complex, whereas the actin-monomer-binding motif is thought to ‘present’ an actin monomer toArp2p and/or Arp3p, generating a pseudotrimer that seedsactin assembly. However, it is important to note that this‘monomer presentation’ model has not been tested directly,leaving open other possible mechanisms of activation. Forexample, a new proposed model is that activators stimulate

ATP exchange on the Arp2p subunit of the complex,promoting a conformation more favorable for nucleationand branching [28].

In order to activate the Arp2/3 complex, WASP itself mustfirst be activated. Structure/function studies have shownthat the amino terminus of mammalian WASP acts in anautoinhibitory fashion, preventing association of its carboxylterminus with Arp2/3 complex. Upon binding to stimulatorymolecules, such as Cdc42, Nck and PIP2, WASP unfolds,exposing its Arp2/3 complex activation domain [29,30].However, it is not clear if yeast WASP functions similarly.

706 Growth and development

Figure 1

SH3ADFH PPP

Abp1p

A A

Ark1p/Prk1pkinases

BAR

Rvs161p/167pamphiphysin

BAR SH3GPABAR SH3GPA

S/T kinasePP

Srv2p/CAPcomplex

PP PP

Arp3

Arp2

p35

Myosin I

Cmd1pSH3 AGPA

Motor

Vrp1p/WIP

PP

P

Las17/WASP

W APPPWH1

Sla2p/HIP1R

Sla1pSH3

SH3SH3

Pan1p/eps15

A

EH

EH

PP

EH

End3p

ENTH talin-likeENTH talin-like

Current Opinion in Microbiology

LSBs

SH3

Arp3Arp2

p35

Arp3Arp2

p35

(b) Abp1p module

(c) Las17p module

(c) Pan1p module

Phosphorylation

Activation of Arp2/3 complex

Suggested physical interaction

Targeted for phosphorylation

Actin monomer

On the basis of physical and genetic interactions, the yeastactin-associated proteins shown above have been organized intothree functional modules: (a) the Las17p module, (b) the Abp1pmodule, and (c) the Pan1p module. Each module regulatesArp2/3-complex-nucleated actin assembly and endocytosis.However, the precise roles of each module in these processes andthe functional interactions that occur between modules are unclear.

A, acidic motif required for Arp2/3-complex binding and activation;ADFH, actin-depolymerizing factor homology domain [73]; BAR,Bin1/amphiphysin/Rvs domain; EH, eps15 homology domain [65];ENTH, epsin amino-terminal homology domain [74] (which may bindto phosphoinositides); PP, polyproline motif; SH3, Src homology 3domain. The references supporting physical interactions are listed inTable 1.

Autoinhibition has not been addressed for Las17p, becausefull-length protein has not been isolated. In addition,although both Cdc42 and Las17p are required for actinassembly in the permeabilized cell assay, direct regulation ofLas17p by Cdc42p is not likely to occur. Las17p does notcontain a Cdc42p-interacting motif, suggesting thatCdc42 regulation, if any, is likely to occur through anintermediate factor [31•].

Another factor that may function with Las17p is Vrp1p, theyeast homologue of WASP-interacting protein (WIP).Mammalian WIP binds to the WH1 domain of WASP andto actin, and inhibits Cdc42 stimulation of actin assemblymediated by the Arp2/3 complex [32]. Vrp1p may functionsimilarly. First, deletion of the VRP1 gene is complementedby mammalian WIP, suggesting that Vrp1p/WIP function isconserved [33]. Second, Vrp1p binds to the WH1 domainof Las17p [16••,17•,34•,35], and Las17p and Vrp1pco-migrate in yeast extracts fractionated by gel filtration[31•]. These data suggest that Las17p and Vrp1p may forma stable complex (see Figure 1; Las17p module). Next, itwill be important to isolate full-length Vrp1p and Las17pproteins and study their activities together.

Another set of factors that bind to and may regulateLas17p function contain SH3 domains (Table 1). Theseinclude type I myosins [16••,17•], which themselves mayactivate the Arp2/3 complex (see below), Sla1p [26] andLas17p-binding proteins (LSBs) [34•]. Similar to the mech-anism by which Nck activates mammalian WASP [30], SH3domains in these yeast proteins may bind to the polyprolineregion of Las17p and cause release of autoinhibition,stimulating actin assembly mediated by the Arp2/3 complex.A tempting candidate for such an effect is the third SH3domain of Sla1p. Although its ligands have not been deter-mined, this SH3 domain is absolutely required for actinassembly in the permeabilized cell assay, whereas theother two SH3 domains of Sla1p are not [36•]. In addition,five novel SH3-domain-containing proteins (LSBs) havebeen implicated in Las17p interactions through the two-hybrid assay [34•]. Although the LSB proteins have not yetbeen characterized genetically and biochemically, they arepredicted to make interactions with key components ofthe actin cytoskeleton [37••], suggesting that they haveimportant roles in actin assembly.

A central component of the Las17p functional module istype I myosin. There are two type I myosins in yeast,Myo3p and Myo5p, which together perform an essentialin vivo function in endocytosis and actin organization [38]and are required for actin assembly in the permeabilizedcell assay [16••]. The SH3 domains of Myo3p and Myo5pwere found to bind to two components of the Las17pmodule, Vrp1p and Las17p, as determined by two-hybridassay and co-immunoprecipitation [16••,17•,39]. Furthermore,Myo3p and Myo5p contain acidic motifs similar to thatfound in Las17p, through which they interact with theArp2/3 complex [16••,17•]. These acidic motifs share a

genetically redundant function with the acidic motif ofLas17p, both in vivo and in the permeabilized cell assay.Taken together, these data suggest that type I myosinsmay activate the Arp2/3 complex in the same functionalpathway and physical complex as Las17p.

What remains poorly defined is the mechanism by whichtype I myosins activate the Arp2/3 complex. Two mechanismsfor activation have been established, one requiring theactivator to bind to actin monomers (the ‘monomer pre-sentation’ model; as for WASP above) and the otherrequiring binding to actin filaments (the ‘filament recruit-ment’ model; see Abp1p below). It is difficult to saywhether myosin I utilizes either mechanism. A fragment ofS. pombe type I myosin is sufficient to activate the Arp2/3complex in vitro [18]. However, this fragment does notbind actin monomers and binds only very weakly to actinfilaments (Kd = ~20 µM), leaving its mechanism of activationuncertain. Moreover, a similar fragment of S. cerevisiaetype I myosin fails to activate the Arp2/3 complex in vitro[31•]. This has led investigators to hypothesize mechanismsinvolving additional factors that function in trans. Evangelistaet al. [17•] have proposed that intermolecular interactionsbetween the actin-binding domain of Vrp1p and the acidicmotif of myosin I may activate the Arp2/3 complex.Consistent with such a model, Vrp1p associates with theSH3 domain of type I myosins [16••,17•,39]. Furthermore,Vrp1p is required for extract-dependent actin polymerizationnucleated by beads coated with a Myo5p fragment [40•].The next challenge is to test these hypotheses directlyusing purified full-length components (such as myosin I,Vrp1p and Las17p).

Although the primary focus of Las17p module research hasbeen its effects on activation of the Arp2/3 complex, geneticevidence suggests that there may be additional functionsfor this module. Deletion of the domains of Las17p andtype I myosins that are sufficient for activating the Arp2/3complex cause much less severe defects in vivo than a complete deletion of either gene [2•,16••,17•]. This suggeststhat Las17p and myosin I may each perform other functionsin addition to their redundant role in stimulating theArp2/3 complex. Of particular interest is the motor activityof type I myosin, which is required for actin assembly inpermeabilized cells [16••] but is not required in S. pombemyosin I for activation of the Arp2/3 complex in vitro [18].This suggests that the permeabilized cell assay may measureArp2/3-dependent and -independent functions of myosin I.

What, then, is the cellular function of the Las17p module?Interestingly, the localization of Las17p, myosin I andVrp1p to cortical patches is maintained in cells aftertreatment with latrunculin-A [34•,35,39]. This suggeststhat these components may function upstream of actinassembly, which is further supported by their requirementfor actin assembly in the permeabilized cell assay [16••].Taken together, the available data suggest that recruitmentof the Las17p module to the cell cortex may determine

Modular complexes regulating actin assembly in budding yeast Goode and Rodal 707

sites of actin patch assembly. This is further supported bystudies showing that myosin I and Vrp1p control thedirection and persistence of cortical actin patch movements,which, in turn, are required for maintaining a polarizeddistribution of actin patches [41•].

Module II: the Abp1p complexAbp1p was one of the first ABPs to be identified in yeast,and was shown to localize to cortical actin patches [42], butits activities remained unknown for many years. Recently,Abp1p was shown to bind strongly to and activate theArp2/3 complex [3•]. Together with the mammalian ABPcortactin [43,44], Abp1p defines a new class of activators,which stimulate the Arp2/3 complex by a mechanism distinctfrom the ‘monomer presentation’ model. Abp1p and cortactinboth require acidic motifs for activation, but show noactin monomer binding. Instead, these activators requiretheir actin-filament-binding domains for activation anddemonstrate the ability to ‘recruit’ the Arp2/3 complex tothe sides of actin filaments. This has led to the ‘filamentrecruitment’ model of Arp2/3 complex activation. In addition,cortactin stabilizes actin filament branches formed by theArp2/3 complex [44], in contrast to branches formed by theWASP–Arp2/3 complex. The effects of Abp1p on branchinghave not yet been determined.

Abp1p serves as the scaffold for its module, tethering tothe actin cytoskeleton two actin-regulating kinases(ARK) — Ark1p and Prk1p — and Srv2p/CAP. These ARKkinases localize to cortical actin patches, perform a geneticallyoverlapping function critical for cell growth, and phosphorylatePan1p and Sla1p [45•,46•]. Both Prk1p and Ark1p requireAbp1p for their normal localization to actin patches [46•]and bind via their proline-rich sequences to the SH3 domainof Abp1p [47]. Abp1p serves as the actin cytoskeleton‘docking’ site for these kinases, which phosphorylate andregulate components of the Pan1p module (see below). Ithas not yet been determined whether binding of the kinasesto Abp1p affects phosphorylation of Pan1p or the ability ofAbp1p to activate the Arp2/3 complex.

Srv2p, the yeast homologue of CAP (cyclase-associatedprotein) also interacts with the SH3 domain of Abp1p via aproline-rich motif [48,49]. The SH3 domain of Abp1p isrequired for normal localization of Srv2p/CAP to cortical actinpatches [48], similar to the Abp1p-dependent localizationof Ark1p and Prk1p mentioned above. Abp1p is one of themost abundant components of the yeast actin cytoskeleton(B Goode, unpublished data), possibly explaining how itsimultaneously links multiple factors to cortical actinpatches. Although Srv2p/CAP can associate with adenylatecyclase and participates in RAS signaling events, thisfunction is separate from its role in the actin cytoskeleton[50•]. Srv2p/CAP binds to actin monomers in vitro [51],shows genetic interactions with the actin-monomer-bindingprotein profilin [52], and has been suggested by geneticstudies in other organisms to promote actin assembly [53].However, the biochemical activities of Srv2p/CAP have

not been investigated thoroughly, and it remains uncertainhow this actin-monomer-binding protein may promoteactin assembly in cells. In this regard, it is important tonote that WASP was considered to be an actin-monomer-sequestering protein prior to its identification as anactivator of the Arp2/3 complex [54]. Similarly, it is possiblethat Srv2p/CAP has activities that depend on its interactionwith other factors in the cytoskeleton (Table 1).

Another factor that may be part of the Abp1p module isRvs167p, the yeast homologue of amphiphysin. The SH3domain of Rvs167p associates with the proline-rich regionof Abp1p in two-hybrid and blot-overlay assays [48,55].However, this interaction has not been established in vivo.Rvs167p may deform lipids in a manner similar to itsmammalian counterpart amphiphysin [56] and, thus, linkAbp1p and the Arp2/3 complex to membranes duringendocytosis. Another possibility is that, like studies onWASP regulation have shown, factors such as Rvs167p thatbind to the proline-rich region (and others that bind to theSH3 domain) of Abp1p may regulate its activity on Arp2/3complex. Thus, it will be interesting to test the effects ofpurified Srv2p, Ark1p/Prk1p and Rvs167p on Abp1p activity.

A key question that remains is: what is the cellular functionof this Abp1p module, and what is its relationship to theother two modules? Genetic studies have provided thebest clues, but also paint a complex picture. As mentionedabove, Abp1p is required for the recruitment of Ark1p,Prk1p and Srv2/CAP to cortical actin patches, and deletionof these genes (Ark1p and Prk1p or Srv2/CAP) causessevere defects in cell polarity, cell growth and actin organi-zation. However, deletion of ABP1 alone causes no obviouscellular defects. This suggests that the cortical localizationof Ark1p, Prk1p and Srv2p is not crucial to their cellularfunctions, and that these factors are able to function(although perhaps not optimally) even when they aremislocalized to the cytoplasm. What, then, is the cellularfunction of Abp1p? Genetic studies have shown that ABP1mutants are lethal in combination (synthetic lethal) withmutations in sla1 and sla2 [57], two components of thePan1p module. This suggests that the Abp1p modulelikely functions in endocytosis [58] and performs a taskthat may be similar to and genetically redundant with thePan1p module. On the other hand, the relationshipbetween the Abp1p and Las17p modules is less clear.Although both modules can activate the Arp2/3 complexand have roles in endocytosis, the limited genetic evidenceavailable suggests an antagonistic relationship betweenthese two modules (B Goode, unpublished data). Clearly,further genetic and biochemical analyses will be requiredto investigate this functional relationship.

Module III: the Pan1p complex The yeast actin cytoskeleton has had long-standingfunctional connections to endocytosis, but few direct linkshave been identified to explain this connection [1].Although components of the Las17p module are required

708 Growth and development

for endocytosis [38,59,60], its direct role in endocytosis isnot understood. More recently, Pan1p (the yeast homologueof endocytic adapter eps15) was identified as a directmolecular link between endocytosis and actin assemblymediated by the Arp2/3 complex [15••]. Mutants of pan1have defects in actin organization and endocytosis [61,62],and purified Pan1p activates the Arp2/3 complex in vitro[15••]. Furthermore, pan1 mutants exacerbate the phenotypeof arp2-1 mutants, suggesting that Pan1p–Arp2/3 interactionsmay be important in vivo. As with myosin I, the mechanismby which Pan1p activates the Arp2/3 complex is unclear,because Pan1p has not been demonstrated to bind to actinmonomers or filaments.

Pan1p may link the Arp2/3 complex and actin assembly toendocytosis through its multivalent interactions with severalcomponents of the endocytic machinery. These includeEnd3p and Sla1p, two proteins required for organizationof the actin cytoskeleton, and it has been suggested thatPan1p, Sla1p and End3p may form a complex [63•]. Afourth endocytic protein, Sla2p, requires Sla1p for itslocalization [36•] and, therefore, may bind to Sla1p andcontribute to the activity of the Pan1p module. Sla1p andSla2p remain associated with cortical patches after treatmentof cells with latrunculin-A [8], and are required for actinassembly in the permeabilized cell assay [64], suggestingthat they may function upstream of actin assembly.Taken together, these data indicate a role for the Pan1pmodule in linking Arp2/3-complex-dependent actin assemblyand endocytosis.

Interestingly, the Pan1p module has potential functionalinteractions with both the Las17p and Abp1p modules.First, Sla1p physically associates with Las17p by two-hybridassay and co-immunoprecipitation [26,37••••]. Thus, therequirement for Sla1p in the permeabilized cell assay mayinvolve its interactions with Las17p or Pan1 or both.Importantly, mutants of Pan1p abolishing its interactionswith Sla1p and the Arp2/3 complex have been generated[15••,63•], and could be used to test these possibilities.Another possibility is that Sla1p interactions with Las17pand Pan1p facilitate distinct, sequential steps in corticalactin patch assembly and/or endocytosis. This could beinvestigated by performing order-of-events experiments inthe permeabilized cell assay using mutants that disruptspecific interactions among the relevant components.

As mentioned above, Pan1p is regulated by a componentof the Abp1p module, the actin-regulating kinase (ARK)Prk1p and, possibly, its homologue, Ark1p [45•].Phosphorylation of Pan1p by Prk1p inhibits Pan1p functionin vivo [45•], suggesting that the Pan1p module may bedownregulated at cortical actin patches. What might thisfunction be? The Pan1p module contains components withthe potential to bring together membrane proteins targetedfor endocytosis, plasma-membrane lipids and actin. Pan1pand End3p contain eps15 homology (EH) domains, whichbind to NPF motifs in the cytoplasmic domains of proteins

targeted for internalization [65]. Furthermore, the Pan1p-associated proteins Sla2p and ENT1-4p contain epsinN-terminal homology (ENTH) domains, which may bind tolipids in the membrane [66]. Interactions between thesedomains and their ligands, coupled to Pan1p interactionswith the Arp2/3 complex and/or Sla2p interactions withactin, may promote actin-assembly-dependent membraneremodeling during the internalization step of endocytosis.

Alternatively, a clathrin-dependent mechanism forendocytosis could be coupled to the Pan1p module.Clathrin mutants in yeast have defects in endocytosis, andPan1p binds to the clathrin adaptor protein yAP180 [67].Furthermore, the mammalian homologue of Sla2p,HIP1R, binds to clathrin and promotes clathrin assembly,and Sla2p could have similar activities [68•]. Genetic evidence suggests that phosphorylation of the Pan1pmodule by Ark1p and Prk1p kinases may negatively regulatesuch activities, possibly disassembling the complex to allowthe next step of endocytosis [45•].

Other regulators of function of the Arp2/3 complex There are undoubtedly numerous other factors in the cellthat regulate activities of the Arp2/3 complex, some ofwhich will associate with one or more of the modulesabove, and others that will act independently. One factorthat can not yet be grouped into a specific module iscalmodulin. Yeast calmodulin (Cmd1p) performs at leastfour different genetically separable functions in vivo [69]and localizes to distinct structures (spindle pole bodies,bud tip, bud neck and cortical actin patches) that maycorrelate with these different functions [70]. At corticalactin patches, Cmd1p interacts with two different AAPs.First, Cmd1p binds to myosin I, serving as a light chain thatis required for myosin I function [71]. Second, overexpressionof CMD1 suppresses the effects of a conditional allele ofARC35 (arc35-1), the gene encoding the p35 subunit ofthe Arp2/3 complex [23]. Importantly, it has beendemonstrated that this genetic interaction with ARC35is independent of interactions between Cmd1p andmyosin I [25••]. Furthermore, there is now strong evidencethat Cmd1p and Arc35p interact directly. Cmd1p associateswith the Arp2/3 complex by both two-hybrid andco-immunoprecipitation assays [25••], and Cmd1p wasshown to bind directly to Arc35p in a high-throughputproteomic screen for protein interactions [72]. A betterunderstanding of this functional interaction should comefrom characterization of the activities of the Arp2/3 complexisolated from the arc35-1 mutant yeast strain, and fromtesting of the potential effects of purified calmodulin onthe Arp2/3 complex.

ConclusionsIt is important to note that Figure 1 is only a workingmodel of the mechanisms that regulate actin assembly inyeast. Undoubtedly, this view will undergo markedrefinement in the coming years. However, we hope that

Modular complexes regulating actin assembly in budding yeast Goode and Rodal 709

the model shown in Figure 1 will provide a conceptualframework to help inspire new experiments and investiga-tions into the cellular functions of AAPs. Ultimately, acomplete understanding of the mechanisms regulatingactin assembly will require overhauling of all of the partsinvolved, definition of their abundance in cells, determinationof the strengths of the physical interactions and activitiesinvolved, testing of functional relationships using geneticanalyses, and re-assembly of working actin machines frompurified components. The first phase of this long-term goalappears to be nearing completion, as we have identifiedmany, if not the majority, of yeast AAPs. More recently,the focus has shifted to definition of the interactions andactivities of these proteins and definition of geneticinteractions among components. This has allowed us tobegin to group factors into functional complexes or modules,such as those outlined above. The next major challengewill be to determine how the activities of these modulesare spatially and temporally regulated in cells, and how thesefactors link actin assembly to different cellular processes,such as endocytosis.

AcknowledgementsWe are grateful to members of the Goode laboratory for helpful discussionsregarding actin assembly complexes and, in particular, to Heath Balcer forhis helpful comments on the manuscript. This work was supported by aPew Scholars Award to BL Goode and a Howard Hughes Medical Institutepre-doctoral fellowship to AA Rodal.

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