A Conserved Sequence That Signifies F-Actin Binding in Functionally Diverse Proteins from Yeast to Mammals

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5679–5684, May 1997 Cell Biology

The I͞LWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals

RICHARD O. MCCANN*† AND SUSAN W. CRAIG*‡

Departments of *Biological Chemistry and ‡Pathology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Communicated by Thomas D. Pollard, Salk Institute for Biological Studies, La Jolla, Ca, March 27, 1996 (received for review January 21, 1997)

ABSTRACT Talin is an actin-binding protein involved in functions associated with individual talin domains are dis- integrin-mediated cell adhesion and spreading. The C- persed among proteins having cellular roles distinct from those terminal 197 amino acids of vertebrate talin are 45% similar of talin. These proteins appear to fall into three groups. The to the C-terminal residues of Sla2, a yeast protein implicated first of these is the erythrocyte band 4.1 superfamily. Talin is in polarized assembly of the yeast actin cytoskeleton. Talin is a member of this group based on similarity between the 47-kDa also homologous in this region to nematode talin, cellular fragment of talin and the N-terminal region of erythrocyte slime mold filopodin, and an Sla2 homolog from nematode. band 4.1 (1). The 4.1 superfamily also includes the related Analysis of the conserved C-terminal sequences of these five cytoskeletal proteins ezrin, moesin, and radixin, the tumor proteins with BLOCK MAKER reveals a series of four blocks, suppressor schwannomin (NF-2), and several protein tyrosine which we name the I͞LWEQ module after the conserved initial phosphatases (20). All of these proteins are related by a residues in each block. Experiments presented here show that homologous 4.1-like domain. All members of the 4.1 super- the conserved protein domain represented by the I͞LWEQ family are known, or potential, membrane-associated proteins. module competes quantitatively with native talin for binding A second group of proteins contains members that are to F-actin in vitro. Furthermore, the corresponding domain of similar to vertebrate talin at their N-terminal 4.1 region and at Sla2 binds to both yeast and vertebrate F-actin in vitro. their C termini. One such talin homolog has been identified in Mutation of one of the conserved residues in the fourth Dictyostelium (21). This 2,491-residue protein, filopodin, ac- conserved block abolishes the interaction of the Sla2 I͞LWEQ cumulates in response to chemoattractant at the leading edge module with F-actin. These results establish the location of an of motile cells, where F-actin is also enriched. Other than at F-actin binding domain in native talin, demonstrate that their N and C termini, talin and filopodin are not very similar. direct interaction of Sla2 with actin is a possible basis for its In contrast to filopodin, an apparently bona fide talin has been effect on the actin cytoskeleton in vivo, and define the I LWEQ ͞ identified in Caenorhabditis elegans (22). This 2,553-residue consensus as a new actin-binding motif. talin is 59% similar to mouse talin over its entire length, with greatest similarity at its N terminus, including the band 4.1 Talin is a modular protein (1, 2) found in focal adhesions (3), region, and at its C terminus. multiprotein assemblies mediating interactions between the A third group of proteins is homologous to talin only at the actin cytoskeleton of cultured cells and the extracellular C-terminal 200 residues. This group is represented by Sla2, a environment (4). Perturbation of talin in vivo indicates that 968-residue yeast protein that is required for polarized assem- talin functions in cell adhesion and spreading (5–7). Experi- bly of the actin cytoskeleton (23). SLA2 is allelic with END4, ments with purified proteins demonstrate that talin interacts whose product is required for endocytosis (24), and with with several focal adhesion components, including acidic phos- MOP2, which is necessary for the proper plasma membrane pholipids (8), actin (9–11), vinculin (12), ␤1 integrin (13), ␤3 ϩ integrin (14), and focal adhesion kinase (FAK) (15). Talin has localization of the H -ATPase encoded by PMA1 (25). Talin a calculated monomer mass of 269 kDa and is a dimer in its and Sla2 (End4͞Mop2) also share their similar C terminus actin-binding form (16, 17) and at protein concentrations with a putative Sla2 protein from C. elegans (26). greater than 1 ␮M (16). Calpain cleaves talin into two frag- Because Sla2 is required for nucleated assembly of actin in ments that have different activities in vitro. The 190-kDa, a permeabilized yeast cell model (27), and because talin both C-terminal fragment binds to G and F-actin (2, 9) and to nucleates actin polymerization and binds to F-actin in vitro (9, vinculin (18), and nucleates actin polymerization (17). Recent 10, 17), we hypothesized that Sla2 might interact directly with studies with glutathione S-transferase (GST) fusion proteins actin and that the actin remodeling activities of talin and Sla2 provide evidence for two, nonoverlapping F-actin binding reside in their conserved C-terminal domain. To test this idea, sequences in the 190-kDa talin fragment (19), and one F-actin we constructed two GST-fusion proteins incorporating the binding sequence from the N-terminal, 47-kDa region. In C-terminal 197 residues of mouse talin and the homologous other studies, the 47-kDa proteolytic fragment did not bind to residues of yeast Sla2 and evaluated their potential to interact F-actin or nucleate actin polymerization (2, 17). The 47-kDa with vertebrate and yeast actin. fragment contains all of the acidic phospholipid binding activity of intact talin (2), which has led to the hypothesis that MATERIALS AND METHODS the N terminus of talin mediates talin-membrane interactions in vivo. Protein Preparation. Rabbit skeletal muscle actin was pu- Several proteins have been identified that are similar to one rified as described (28), with an additional gel filtration step or more limited regions of talin, suggesting that the specific (29). Pyrene-labeled actin was produced by labeling F-actin with pyrene iodoacetamide (30, 31). Yeast actin (32) and The publication costs of this article were defrayed in part by page charge chicken gizzard talin (33) were purified as described. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Sequence Analysis. The C-terminal sequences of accordance with 18 U.S.C. §1734 solely to indicate this fact. talin and its putative homologs were aligned and blocks 1–4 of

Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424͞97͞945679-6$2.00͞0 Abbreviation: GST, glutathione S-transferase. PNAS is available online at http:͞͞www.pnas.org. †To whom reprint requests should be addressed.

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the I͞LWEQ module identified using BLOCK MAKER (34) Airfuge) for 20 min and the supernatant and pellet were [www.blocks.fhcrc.org͞]. separated for subsequent analysis on SDS͞PAGE. GST Fusion Proteins. The mouse talin GST fusion protein F-Actin Bundling. Actin (3 ␮M) was polymerized in the construct (GST-Tn.2345–2541) was prepared by amplifying presence of GST, GST-Tn.2345–2541, or GST-Sla2.771–968 (3 the coding region extending from I2345 to the stop codon, using ␮M each). The resulting F-actin preparations were adsorbed to a mouse talin cDNA as the template, and subcloning this PCR grids, negatively stained with uranyl formate, and visualized on fragment into pCR 2.1 (Invitrogen). The following primers a Zeiss model 10A electron microscope. were used: 5Ј-ATCCTAGAAGCTGCC-3Ј (7192–7206; Nucleation of Actin Polymerization. G-actin (6 ␮M, 54% pyrene-labeled) was polymerized as outlined above in the StartϭI2345); 5Ј-TTAGTGCTCGTCTCG-3Ј [7785–7771; numbering corresponds to that in Rees et al. (1)]. The EcoRI presence of talin, GST-Tn, GST-Sla2, or GST (2 ␮M each), insert-containing fragment from pCR2.1 was then subcloned except for the addition of 30 mM NaCl and a final Tris into EcoRI-digested pGEX-2T (Pharmacia). The Sla2 GST concentration of 7 mM. Polymerization was monitored by the fusion protein construct (GST-Sla2.771–968) was prepared increase in pyrene fluorescence (Perkin–Elmer model LS 50 B; similarly, except that yeast genomic DNA (strain SM1060) was excitation: 365 nm; emission 407 nm). Gelsolin (2 nM) was used as a positive control for nucleation. used as the template. The following primers were used for the Sla2 construct: 5Ј-CCATTGTTGTCATTGGC-3Ј (Chr XIV: 190360–190376; StartϭP771); 5Ј-GATCAATCATCATC- RESULTS CTGG-3Ј (Chr XIV: 190958–190944; numbering from the At the start of this study, BLOCK MAKER (34) was used to aid Saccharomyces Genome Database). The fusion proteins were in identification of the most conserved patterns of amino acids purified using glutathione-agarose as previously described in the conserved C termini of the four proteins then identified (35). as talin homologs (1, 21, 23, 26). BLOCK MAKER uses automated F-Actin Cosedimentation. Cosedimentation of mouse talin, versions of MOTIFS (36) and GIBBS (37) in conjunction with GST-Tn.2345–2541, and GST-Sla2.772–968 with F-actin was MOTOMAT (38) for identification of the best set of conserved measured essentially as described in Schmidt et al. (11) by amino acid patterns (motifs) in homologous protein sequences. mixing G-actin (in buffer G: 2 mM Tris, pH 7.5, 9490.2 mM The MOTIFS algorithm with the parameters s, r, and d (36) CaCl2͞0.2 mM ATP͞0.2 mM dithiothreitol) with talin or one automatically fixed at 4, 0, and 17, respectively, found blocks of the GST fusion proteins and then initiating actin polymer- 1–4 (Fig. 1) when queried with all four sequences. Recently, ization by adding MgCl2 to 2 mM and KCl to 50 mM (50 ␮l the sequence of C. elegans talin was reported (22), and when total volume). After polymerization was complete (60 min, it was included in the BLOCK MAKER analysis, MOTIFS no longer 22ЊC) the mixture was centrifuged at 150,000 g (Beckman found block 1 because the values for s, r, and d automatically

FIG.1. I͞LWEQ module sequence alignment. Alignment of the conserved C-terminal domain common to mouse (Mm) and C. elegans (Ce) talin, the Dictyostelium (Dd) talin homolog filopodin, the putative C. elegans Sla2 homolog, and S. cerevisiae (Sc) Sla2. The four conserved blocks, originally identified using the BLOCK MAKER program (34), are boxed; identities between all five sequences are highlighted by shading in the column, conservative substitutions are indicated by ϩ. The most conserved positions (at least four out of five residues identical) are marked by a solid circle above the column. Block 1 contains 17% identities and 48% similarites; the values for blocks 2–4 are 63%͞80%, 57%͞83%, and 46%͞66%, respectively. Downloaded by guest on September 25, 2021 Cell Biology: McCann and Craig Proc. Natl. Acad. Sci. USA 94 (1997) 5681

change to 5, 0, and 17. This means that in the block 1 region there is no longer an amino acid triplet pattern of the form aa1-d1-aa2-d2-aa3 in which the amino acids at positions 1, 2, and 3 are identical across the five sequences, as specified by the value for s. In Fig. 1 the columns of amino acids that are identical between the five aligned proteins are shaded. Posi- tions that are identical in at least four out of the five sequences are indicated by a dot above the column. The GIBBS algorithm consistently found blocks 2 and 4. Based on the patterns found in the initial four proteins we designed fusion proteins starting at the beginning of block 1. We have named the conserved C-terminal set of motifs the I͞LWEQ module, after the conserved initial residues of blocks 1–4 (Fig. 1). To test the hypothesis that the I͞LWEQ module interacts with actin, we constructed GST-Tn.2345–2541 (GST- Tn), which begins with the initial residue of block 1 of mouse talin and extends to the C terminus, and GST-Sla2.771–968 (GST-Sla2), which begins with Pro-771, immediately N- terminal to block 1 of yeast Sla2, and extends to the C terminus. Thus both GST-fusions include all four conserved blocks from their respective proteins. We then examined these fusion proteins for interaction with vertebrate and yeast actin in vitro. Binding of talin, GST-Tn and GST-Sla2 to F-actin was assayed by measuring cosedimention with vertebrate F-actin (Fig. 2). With increasing actin concentration, native talin, GST-Tn, and GST-Sla2 are depleted from the residual supernatant following ultracentrifugation, and this depletion is accompanied by an increase of talin or each fusion protein in the corresponding pellet fractions (Fig. 2A). Cosedimentation of GST-Tn and GST-Sla2 with F-actin could not be measured directly by densitometry of the fusion protein pellet fractions because the large amount of sedimented actin obscures the fusion proteins; therefore we used supernatant depletion of the fusion proteins as a measure of cosedimentation. Immunoblots of the fusion protein pellet fractions with anti-GST showed that depletion of GST-Tn and GST-Sla2 from their respective supernatants was accompanied by enrichment in their respective F-actin pellet fractions (Fig. 2A). With native talin and GST, where it was possible to measure directly the amount of protein in each supernatant and pellet sample from densitometry of Coomassie blue stained gels, protein depleted from each supernatant fraction could be accounted for quantitatively in the corresponding pellet. GST did not bind to F-actin, and the GST-fusion proteins did not sediment in the absence of F-actin (Fig. 2A). A quantitative analysis of the gels illustrated in Fig. 2A is shown in Fig. 2B. Both GST-fusion proteins bound to F-actin to a greater extent than native talin, with GST-Sla2 binding to a greater extent than GST-Tn. The actin concentration required for half-maximal binding of fusion protein was Ϸ3 ␮M for GST-Tn and GST-Sla2 and Ϸ5 ␮M for native talin. In this particular experiment, Ϸ45% of the talin bound to F-actin at the saturating actin concentration (30 ␮M), which is within the range we have found in several other experiments using different talin and actin preparations. Other investigators have also observed that only a portion of purified native talin cosediments with actin. In fact, in the earliest studies (3, 12) none of the purified chicken gizzard talin cosedimented with actin. Subsequently other investiga- FIG. 2. F-actin cosedimentation: vertebrate actin. (A) SDS͞PAGE. tors reported that talin does bind actin (9–11). Evidence Rabbit skeletal muscle actin (0–30 ␮M) was mixed with either native suggests that the amount of active talin depends on the mouse talin (2.5 ␮M), GST-Tn (2.0 ␮M), GST-Sla2 (2.0 ␮M), or GST (2.0 purification protocol (9) and on the conditions of ionic ␮M), and F-actin cosedimentation analyzed. All of the supernatant panels strength and pH used in the binding assay (11). Our prepara- and the talin and GST pellet panels were visualized with Coomassie blue; tions contain 45–60% active talin that was not significantly the fusion protein pellet fractions were detected by immunoblotting (see affected by pH (in the range between 6.6–7.4), or length of text). (B) Quantitative analysis. Supernatant depletion, as a measure of cosedimentation, of talin (E), GST-Tn (F), GST-Sla2 (É), and GST (ç) incubation with actin (up to 24 hr) in the cosedimentation was determined densitometrically using the gels shown in A.(C) Quanti- assay. All talin molecules in our preparation have an intact C tative analysis: GST-Sla2 mutant. Supernatant depletion was performed as terminus as assessed by quantitative immunoprecipitation with in A on GST-Sla2 (E), the mutant GST-Sla2R958G(É), and GST (F). The an antibody raised to the C-terminal 21 amino acids of murine shift in electrophoretic mobility of the Sla2 mutant is shown (Inset). Downloaded by guest on September 25, 2021 5682 Cell Biology: McCann and Craig Proc. Natl. Acad. Sci. USA 94 (1997)

talin (data not shown). Thus the molecular basis for functional heterogeneity in our talin preparations is unknown. One of the GST-Sla2 clones generated by PCR yielded a fusion protein that failed to bind to F-actin (Fig. 2C). The DNA sequence of this clone showed a single base change resulting in an R to G substitution at position 958 in Sla2. This mutant had a slightly retarded migration on SDS gels com- pared with the wild-type fusion protein (Fig. 2C Inset), suggesting that the mutation affects a structural fold on which the amino acids contacting actin are arrayed. This function- blocking mutation at a conserved residue of the I͞LWEQ domain provides preliminary support for the hypothesis that the conserved residues of the I͞LWEQ module are responsible for the actin-binding activity of the module. Yeast and vertebrate actin are 87% identical and 94% similar, which is substantially less homologous than the six vertebrate actins are to each other. To test the assumption of functional homology, the I͞LWEQ modules of mouse talin and yeast Sla2 were tested for binding to yeast F-actin. The results show that mouse talin, GST-Tn and GST-Sla2 also interact with yeast F-actin (Fig. 3). Once again both GST-Tn FIG. 4. Competition of GST-Tn.2345–2541 for talin F-actin binding activity. Rabbit actin (10 ␮M) and talin (1 ␮M) were mixed in the and GST-Sla2 bound to F-actin to a greater extent than native presence of increasing amounts of GST-Tn (0–20 ␮M), and actin talin, with GST-Sla2 binding to the greatest extent. GST alone polymerization, centrifugation, SDS͞PAGE, and densitometry were did not bind to yeast F-actin. Therefore, the I͞LWEQ module performed as previously described (Fig. 2). Values reported are from both a vertebrate and a yeast protein is able to mediate averages of three separate experiments. GST (0–20 ␮M) had no effect an interaction in vitro with F-actin from either source. on talin binding to F-actin (not shown). To evaluate the extent to which the I͞LWEQ module accounts for the interaction between native talin and F-actin, may be conformationally regulated, as recently demonstrated GST-Tn was tested for its ability to compete with talin for for vinculin (39). binding to F-actin (Fig. 4). In this experiment, Ϸ55% of native Talin nucleates actin polymerization at the relatively high talin bound to vertebrate F-actin at a 10:1 molar ratio of actin molar ratio of 1:3 (talin͞actin) (2, 10). When tested for similar to talin. Increasing the GST-Tn concentration from zero to 20 activity, neither GST-Tn nor GST-Sla2 nucleated actin poly- ␮M completely displaced the talin from F-actin (GST alone merization. Native talin at a ratio of 1:3 (talin͞actin) did had no effect; data not shown). This result indicates that the shorten the lag phase associated with actin polymerization I͞LWEQ module is responsible for all of talin’s F-actin binding (Fig. 5). By contrast, gelsolin is a much more efficient nucle- capacity, as measured in vitro by cosedimentation. This finding suggests that the additional F-actin binding sites recently identified in talin using GST-fusion proteins (19) may be inaccessible in the purified molecule and further, raises the interesting possibility that the full actin-binding activity of talin

FIG. 3. F-actin cosedimentation: yeast actin. Cosedimentation of FIG. 5. F-actin bundling. Actin (3 ␮M) was polymerized in the talin (E), GST-Tn (F), GST-Sla2 (É), and GST (ç) with yeast F-actin presence of (A) talin, (B) GST, (C) GST-Tn, or (D) GST-Sla2 (3 ␮M was measured as described in Fig. 2 except for the following changes each), and the resulting F-actin preparations were negatively stained in protein concentrations: yeast actin (0–10 ␮M); talin, GST-Tn, with uranyl formate and visualized by electron microscopy. (Bar ϭ 10 GST-Sla2, and GST (2.0 ␮M each). ␮M). Downloaded by guest on September 25, 2021 Cell Biology: McCann and Craig Proc. Natl. Acad. Sci. USA 94 (1997) 5683

musculus (vertebrate) and C. elegans (invertebrate) talin plus D. discoideum filopodin and another composed of Saccharo- myces cerevisiae Sla2 and its putative C. elegans homolog. Filopodin is clearly related to talin by virtue of its band 4.1 region and I͞LWEQ module, but it is not otherwise talin. Thus, filopodin may represent a third class of I͞LWEQ- containing proteins, with its ultimate classification depending on further molecular, cellular, and genetic characterization.

DISCUSSION We have shown that the conserved C-terminal domains of yeast Sla2 and mammalian talin represent a novel actin binding element. This conserved sequence element, which we call the I͞LWEQ module, binds to F-actin and bundles actin filaments. The I͞LWEQ module is unrelated to the F-actin binding motif found in the ␣-actinin family that includes ABP 120, fimbrin, dystrophin, filamin, and spectrin (44). Conservation of sequence and F-actin binding function of the I͞LWEQ domain between yeast and vertebrate proteins allows some new predictions. Although the direct interaction of talin with actin in vitro is well established, such information on the other I͞LWEQ proteins is lacking. Filopodin was identified in a screen using a panel of monoclonal antibodies FIG. 6. Nucleation of F-actin polymerization. Actin (6 ␮M, 54% raised against Dictyostelium proteins that had been enriched on pyrene-labeled) was polymerized alone (E) or in the presence of talin (2 ␮M, É), GST-Tn, GST-Sla2, or GST (2 ␮M each (F), or gelsolin (2 an F-actin affinity matrix, and the protein accumulates at the nM, ç) and polymerization was monitored by the increase in pyrene tips of filopodia, cell surface extensions that contain a core of fluorescence. bundled F-actin filaments (21). Although it is not known whether filopodin interacts directly with F-actin, the conserved ator of actin polymerization (Fig. 5). function of the I͞LWEQ module from mouse talin and yeast Native talin induces the assembly of thick bundles of actin Sla2 strongly implies that the I͞LWEQ module of filopodin filaments (Fig. 6A), which are similar to those observed in mediates a filopodin͞F-actin interaction in Dictyostelium. The other studies (40), whereas GST-Tn and GST-Sla2 aggregate finding that GST-Tn. 2345–2541 and GST-Sla2.771–968 bun- actin filaments into small bundles (Fig. 6 C and D). GST alone dle F-actin filaments in vitro suggests that filopodin, through its had no discernible effect on filament morphology (Fig. 6B). I͞LWEQ module, bundles actin filaments in Dictyostelium Talin is a homodimer when it binds to actin (16), GST is a filopodia. dimer (41, 42), and GST and our fusion proteins migrate as SLA2 was identified in a synthetic lethal screen as being dimers by gel filtration analysis in physiological saline (data not required along with ABP1 for polarized assembly of the actin shown). Thus differences in the properties of talin and the cytoskeleton (23). SLA2 is also necessary for polarized nucle- I͞LWEQ domain fusion proteins do not reflect differences in ation of cortical actin filament assembly in permeabilized yeast monomer vs. dimer configurations. cells (27). Although the I͞LWEQ module of Sla2 mediates an The five proteins that possess an I͞LWEQ module are interaction with vertebrate and yeast F-actin, GST-Sla2.771– members of a talin superfamily that includes the band 4.1 968 did not nucleate (vertebrate) actin polymerization in vitro. superfamily and the Sla2 family. Representative members of However, this negative result may not reflect the properties of these families are diagrammed to relative scale in Fig. 7. The intact Sla2 because the in vitro nucleation activity of talin was I͞LWEQ actin-binding module is indicated by the four shaded not reproduced by GST-Tn.2345–2541. C-terminal blocks. The presence of the I͞LWEQ module Our data show that the I͞LWEQ module competes quan- groups the talins and Sla2 proteins. Although the I͞LWEQ titatively for the interaction of talin with F-actin in cosedi- module is not found in the ezrin, moesin, and radixin family, mentation assays, but does not reproduce all of the effects of some ezrin, moesin, and radixin proteins do have a C-terminal native talin on actin assembly and structure. Reasons for the actin binding domain (43). The presence of an N-terminal band differences between intact talin and the I͞LWEQ module 4.1 region groups talins, filopodin, and members of the ezrin, remain to be defined, but probably reflect the additional moesin, and radixin family (represented by ezrin). The con- N-terminal actin-binding sequences in talin (19), or a require- served N-terminal blocks in yeast Sla2 and its C. elegans ment for a particular arrangement (e.g., antiparallel) of the homolog are distinct from those of the band 4.1 superfamily. I͞LWEQ domains in the GST fusion protein dimer. Because Fig. 7 also shows that the I͞LWEQ-containing proteins can all of the F-actin binding activity of native talin is competed by be grouped into at least two classes; one consisting of Mus the I͞LWEQ domain, we hypothesize that the additional

FIG.7. I͞LWEQ actin-binding module superfamily. The five proteins containing the I͞LWEQ module (mouse and C. elegans talin are represented together) are diagrammed to relative scale. (u) Conserved sequence blocks identified using BLOCK MAKER (34). See text for discussion. Downloaded by guest on September 25, 2021 5684 Cell Biology: McCann and Craig Proc. Natl. Acad. Sci. USA 94 (1997)

actin-binding sequences that have been described by fusion 13. Horwitz, A., Duggan, K., Buck, C., Beckerle, M. C. & Burridge, protein analysis (19) are cryptic in native talin and require K. (1986) Nature (London) 320, 531–533. some type of regulated exposure for function. 14. Knezevic, I., Leisner, I. & Lam, S. C-T. (1995) J. Biol. Chem. 271, The observation that yeast genes END4 (24) and MOP2 (25), 16416–16421. are allelic with SLA2 (Saccharomyces Genome Database) 15. Chen, H. C., Appeddu, P. A., Parsons, J. T., Hildebrand, J. D., Schaller, M. D. & Guan, J.-L. (1995) J. Biol. Chem. 270, 16995– raises some interesting possibilities. Several lines of evidence 16999. indicate that intracellular vesicle trafficking in yeast is polar- 16. Goldmann, W. H., Bremer, A., Haner, M., Aebi, U. & Iserberg, ized and associated with the actin cytoskeleton (45, 46). G. (1994) J. Struct. Biol. 112, 3–10. Identification of the Sla2(End4) I͞LWEQ module as an 17. 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