Afi1p Functions as an Arf3p Polarization-specific Docking Factor for Development of Polarity

ADP-ribosylation factors (Arfs) are highly conserved small GTPases and are critical components of vesicle trafficking. Yeast Arf3p, despite its similarity to mammalian Arf6, is not required for endocytosis but is involved in polarity development. In this study, we identified an Arf3p interacting protein 1 (Afi1p), which, through its N-terminal conserved region, specifically interacts with GTP-bound Arf3p. Afi1p is distributed asymmetrically at the plasma membrane and is required for polarized distribution of Arf3p but not of an Arf3p guanine nucleotide-exchange factor, Yel1p. However, Afi1p is not required for targeting of Arf3p or Yel1p to the plasma membrane. Like arf3 mutant yeast, afi1 mutant yeast exhibited an abnormal budding pattern and partially delayed actin patch polarization. An Afi1p, 38KLGP4A-Afi1p, mutated at the Arf3p-binding region, loses its ability to interact with Arf3p and maintain the polarized distribution of Arf3p. Although 38KLGP4A-Afi1p still possessed a proper polarized distribution, it lost its ability to rescue actin patch polarization in afi1 mutant cells. Our findings demonstrate that Afi1p functions as an Arf3p polarization-specific adapter and participates in development of polarity. ADP-ribosylation factors (Arfs) are highly conserved small GTPases and are critical components of vesicle trafficking. Yeast Arf3p, despite its similarity to mammalian Arf6, is not required for endocytosis but is involved in polarity development. In this study, we identified an Arf3p interacting protein 1 (Afi1p), which, through its N-terminal conserved region, specifically interacts with GTP-bound Arf3p. Afi1p is distributed asymmetrically at the plasma membrane and is required for polarized distribution of Arf3p but not of an Arf3p guanine nucleotide-exchange factor, Yel1p. However, Afi1p is not required for targeting of Arf3p or Yel1p to the plasma membrane. Like arf3 mutant yeast, afi1 mutant yeast exhibited an abnormal budding pattern and partially delayed actin patch polarization. An Afi1p, 38KLGP4A-Afi1p, mutated at the Arf3p-binding region, loses its ability to interact with Arf3p and maintain the polarized distribution of Arf3p. Although 38KLGP4A-Afi1p still possessed a proper polarized distribution, it lost its ability to rescue actin patch polarization in afi1 mutant cells. Our findings demonstrate that Afi1p functions as an Arf3p polarization-specific adapter and participates in development of polarity. Arfs are guanine nucleotide-binding proteins, which belong to the Ras superfamily. They are critical for vesicular trafficking and organization of the actin cytoskeleton (1D'Souza-Schorey C. Chavrier P. Nat. Rev. Mol. Cell Biol. 2006; 7: 347-358Crossref PubMed Scopus (1041) Google Scholar). Arfs are activated by guanine nucleotide-exchange factors (GEFs), 3The abbreviations used are: GEF, guanine nucleotide-exchange factor; GFP, green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; RFP, red fluorescent protein; mRFP, mono-RFP. 3The abbreviations used are: GEF, guanine nucleotide-exchange factor; GFP, green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; RFP, red fluorescent protein; mRFP, mono-RFP. which facilitate replacement of GDP with GTP, and are inactivated by GTPase-activating proteins, which stimulate GTP hydrolysis to GDP (2Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Arfs are active in the GTP-bound state and competent to interact with downstream effectors such as coatomer, GTPase-activating protein, phospholipase D, and phosphatidylinositol 4/5-kinase to initiate vesicle formation or actin cytoskeleton reorganization (3Donaldson J.G. J. Biol. Chem. 2003; 278: 41573-41576Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 4Nie Z. Hirsch D.S. Randazzo P.A. Curr. Opin. Cell Biol. 2003; 15: 396-404Crossref PubMed Scopus (188) Google Scholar, 5Hernandez-Deviez D.J. Roth M.G. Casanova J.E. Wilson J.M. Mol. Biol. Cell. 2004; 15: 111-120Crossref PubMed Scopus (135) Google Scholar). The best known Arf proteins are Arf1 and Arf6; the former is required for intra-Golgi and Golgi-endoplasmic reticulum trafficking (6Balch W.E. Kahn R.A. Schwaninger R. J. Biol. Chem. 1992; 267: 13053-13061Abstract Full Text PDF PubMed Google Scholar), and the latter is required for membrane trafficking and actin cytoskeleton regulation at the plasma membrane (3Donaldson J.G. J. Biol. Chem. 2003; 278: 41573-41576Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 7Turner C.E. Brown M.C. Curr. Biol. 2001; 11: R875-R877Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Arf6 is involved in many cellular events, including constitutive and stimulated endocytosis, membrane ruffling, membrane protrusion, secretory granule exocytosis, and cytokinesis (8Altschuler Y. Liu S. Katz L. Tang K. Hardy S. Brodsky F. Apodaca G. Mostov K. J. Cell Biol. 1999; 147: 7-12Crossref PubMed Scopus (119) Google Scholar). Although Arf3p is the yeast homologue of mammalian Arf6, the functions of these two proteins are not the same (9Lee F.J. Stevens L.A. Kao Y.L. Moss J. Vaughan M. J. Biol. Chem. 1994; 269: 20931-20937Abstract Full Text PDF PubMed Google Scholar). In our previous study, we found that Arf3p was not required for fluid phase Lucifer yellow or FM4-64 endocytosis or for mating factor receptor internalization. Furthermore, Arf3p was not directly required for reorganization of the yeast actin cytoskeleton (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar). In contrast, the cellular location of Arf3p changes during the cell cycle. In the G1 phase, Arf3p is present at the plasma membrane and in the cytosol. In the S phase, however, it is concentrated at the plasma membrane of the emerging bud. Because of its polarized localization and its critical function in the normal budding pattern of yeast, Arf3p was hypothesized to be a regulator of vesicle trafficking, which is important for polarized growth. Recently, many studies provide evidence to support the involvement of Arf3p in cell polarity and the organization of the actin cytoskeleton (11Costa R. Warren D.T. Ayscough K.R. Biochem. J. 2005; 387: 649-658Crossref PubMed Scopus (23) Google Scholar). It was found that ARF3 could be a genetic suppressor for pfy1, las17, and vrp1 mutant cells, and Bni1p overexpression could rescue the budding pattern abnormality of an arf3 mutant. In addition, Arf3p shows a genetic interaction with Bud6p and is required for the proper location of GGA-like protein Lsb5p (12Lambert A.A. Perron M.P. Lavoie E. Pallotta D. FEMS Yeast Res. 2007; 7: 782-795Crossref PubMed Scopus (23) Google Scholar). However, the molecular mechanism of how Arf3p participates in polarity establishment and actin organization is still unclear. To identify regulators and effectors of Arf3p, we used Arf3Q71L-d17N, a constitutively active and N-terminally truncated Arf3p that removes the amphipathic helix, as bait to identify interacting proteins by yeast two-hybrid screening. A hypothetical open reading frame, YOR129c, was identified and named Arf3-interacting protein 1 (AFI1). The predicted amino acid sequence does not contain any recognized structural or functional motifs. We show here that one function of Afi1p is to localize Arf3p at the polarized plasma membrane. We identified a specific conserved sequence at the N terminus of Afi1p, which is important for its interaction with Arf3p. An Afi1p mutated at the Arf3p-binding region did not affect its proper polarized distribution, but lost its ability to maintain the polarization of Arf3p and to rescue the defect of actin patch polarization in afi1 mutant cells. We infer that Afi1p acts a polarization-specific adapter for Arf3p and participates in development of polarity. Strains, Media, and Plasmids—Table 1 lists the yeast strains used in this study. Yeast culture media were prepared as described by Sherman et al. (13Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Scholar). and yeast amino and and for strains were at the (13Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Scholar). Yeast strains were by the (13Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Scholar). were to J. A Laboratory Cold Spring Harbor Laboratory Press, Cold Spring Scholar). was as described 1999; 15: PubMed Scopus Google Scholar). The used for are in and the used in this are in The was by Y. P.A. S. PubMed Scopus Google strains used in this in a used in this in a used in this or acid A ARF3 ARF3 ARF3 in in a was and was and were in our Yeast two-hybrid was the Methods Mol. Biol. Google Scholar), in which the bait was with the protein The yeast was the The the yeast to proteins as to an Yeast the was with In the were and in the were yeast and for of the were and N-terminal of the the and a to the Afi1p N terminus a D.J. P. 14: PubMed Scopus Google Scholar). The were with the and and the in interaction of Arf3p and Afi1p, and were and with in and a and at for to the was with and with were by to the of Arf3p. the interaction Arf3p and Afi1p, 1 of or was with 1 of or in 1 of 1 1 of at the was with and the proteins were by were in or and prepared for as described (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar, F.J. C.F. Lin M.C. Moss J. Vaughan M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was with a and were a were at a of and were with and with as described R.A. Methods Cell Biol. PubMed Scopus Google Scholar). were with with and with for as described (13Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Scholar). were in and with a and at a of in were and to as described Y.W. Lee Lee F.J. J. Cell 2006; PubMed Scopus Google Scholar). the was to for at to the and The was membrane and by for 1 of were to Arf3p with the N of identify proteins that interact with Arf3p, we used a constitutively active Arf3p with N-terminal as bait in a yeast two-hybrid and the were N-terminal of a with which was named protein 1 (AFI1). In yeast two-hybrid but not or with the N terminus of Afi1p that the interaction of Arf3p and Afi1p is and A and Although could not interact with the N-terminally truncated could interact with in the yeast two-hybrid However, this interaction be to the similarity and Arf3p and does not in a protein of amino with to any known or sequence or of similarity to protein However, predicted of were found in the and In addition, to a predicted protein was The function of the and K. not In the of actin at polarization by the of a protein, C.L. K. Mol. Biol. Cell. 2004; 15: PubMed Google Scholar). the interaction region of Afi1p with Arf3p, of the open reading were and for interaction with in yeast two-hybrid shows that the N-terminal of could interact with that the interacting sequence be distributed this region or that the N terminus is required for proper that is to interact with Arf3p. To the in interaction of Arf3p and Afi1p, we Afi1p and Arf3p or in arf3 mutant of these were the of was with and the of proteins was by and to a but not that Arf3p interacts with Afi1p in a We and and small proteins to interaction in but not or could interact with support the of a specific and interaction Arf3p and The of Afi1p we to an in our the of the was to for the of Afi1p, were the of by of cell that of and phase Afi1p and phase show a Afi1p Afi1p was in in phase and that of Afi1p, of Arf3p, is in in in the pattern is to the by et al. M. 2003; PubMed Scopus Google Scholar). Afi1p for a budding to or in an pattern and in a pattern J. J. Cell Biol. PubMed Scopus Google Scholar). Our previous that an ARF3 an abnormal budding pattern (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar). To Afi1p a in yeast polarized we that an ARF3 or or with the yeast were by and by not is a and with the of and ARF3 are and exhibited defect at that afi1 is not a with A and shows that of afi1 exhibited a or budding pattern as did arf3 cells, of which show a with a of abnormal budding with cells, was in the which that Afi1p and Arf3p function in the same to maintain the proper budding pattern in Afi1p in previous that an arf3 actin patch polarization in the emerging at (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar). To Afi1p is involved in the development of actin patch arf3 afi1 and mutant were at or and was used to actin and actin patch polarization were normal in strains at However, at and the exhibited in actin patch polarization and to actin patch in yeast with small The of abnormal in these mutant strains is not actin polarization defect that Afi1p and Arf3p in the same which is involved in actin patch of Afi1p the the localization of Afi1p, we to with but was we used a a however, we still could not any with not Because the Afi1p is to be by a was by of the and the in of the The shows that was at the plasma membrane and in the In cells, its at the was that it be involved in in the or the in to the plasma membrane. membrane and of are lost The yeast cell was to Arf3p, membrane and in were identified by Although of the Afi1p Arf3p, in the membrane of the Afi1p was found in the that the membrane of Afi1p is that of Arf3p. that and are for the localization of the ARF3 but not the polarized localization the plasma membrane (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar), that overexpression the polarization To Afi1p is asymmetrically distributed the plasma many were including of the of the and the yeast in to its However, these by the of its a we polarized distribution of in with a Although in the of could the at the plasma membrane of and be this the that Afi1p a polarized distribution, and this be required for the polarization of Arf3p. The in the of Afi1p the of the or its is of Afi1p in the Arf3p that constitutively active Arf3p was at the plasma membrane (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar). of to the plasma membrane the that Afi1p interacts with active Arf3p at the plasma membrane. To the functional of the we Arf3p is involved in the localization of Afi1p or The localization of was not in the arf3 mutant that Arf3p is not a of Afi1p We Afi1p is required for the plasma membrane targeting of Arf3p. In cells, by a the of its was at the plasma membrane and concentrated at the emerging In afi1 cells, however, lost its polarization distribution In addition, constitutively active Arf3p lost its distribution in afi1 mutant that Afi1p be a for Arf3p targeting to the polarized membrane. Afi1p of Yel1p was to be a guanine factor for Arf3p and was with at the plasma to the and the emerging S. 2007; PubMed Scopus Google Scholar). We the of Afi1p is required for the polarization of Yel1p. We the of the in and afi1 was to the plasma membrane at the and the in the afi1 mutant that Afi1p does not affect polarized localization of Yel1p. We the localization of Afi1p in mutant cells. The polarized distribution of Afi1p was not in the of Yel1p that Afi1p is not required for polarized location of Yel1p and Yel1p for of Arf3p and in to be required for the polarized localization of Arf3p to the plasma membrane S. 2007; PubMed Scopus Google Scholar). To this we in or afi1 mutant was at the and in the but lost its location in afi1 and mutant cells. with previous and plasma membrane localization in mutant of the Arf3p in and afi1 mutant that the of but not the of the plasma membrane of We the distribution of Arf3p was in the and afi1 were two plasma and and by The of Arf3p in the and in the but not is in the that Afi1p and Yel1p function in in the localization of Arf3p to the plasma membrane. To Arf3p, is involved in actin patch the or were at and with to actin polarization. The arf3 and cells, but not cells, delayed or actin patch polarization in small The of actin patch in is not arf3 and mutant Yel1p and Arf3p in the same which is involved in actin cytoskeleton A at the N of Afi1p for and the of Afi1p for with Afi1p homologue with known the that the N terminus is highly conserved and be for its function we mutant of Afi1p as a of N-terminal amino that are not conserved in Afi1p a of amino of the conserved and an mutant in the conserved to the of the Afi1p N terminus in its localization and interaction with Arf3p. yeast two-hybrid we that the amino of Afi1p are not required for the interaction Afi1p and Arf3p, and proper localization of Afi1p and However, the of the N-terminal amino of Afi1p could not interact with Arf3p and lost its ability to with the plasma membrane. The mutant of Afi1p could not interact with Arf3p but did not its polarized with the plasma membrane. these of Afi1p and that the N-terminal amino of Afi1p are important for its and the conserved amino of Afi1p are critical for interaction with Arf3p. with Afi1p for and of the specific interaction Afi1p and Arf3p functional we or mutant of with its afi1 cells. strains were with localization of in was in afi1 Afi1p and However, lost its polarized localization in afi1 that interaction with Afi1p is important for polarized localization of Arf3p. We could rescue the defect of actin patch polarization in afi1 cells. The afi1 with and were at with and by and but not could rescue the defect in actin patch polarization in afi1 mutant of actin patch in the was and is in that conserved Afi1p and its are and for its In addition, the interaction of Arf3p and Afi1p is critical for the of Afi1p Arf3p location and for actin patch polarization. In this study, we show that a protein Afi1p functions as a polarization-specific adapter for Arf3p. Afi1p and Arf3p are distributed the polarized plasma membrane and in normal budding pattern and actin patch polarization. of evidence support the that Afi1p acts as a polarization-specific adapter for Arf3p and participates in development of polarity as Arf3p loses its polarized distribution, but not plasma membrane in the of a conserved region at Afi1p, critical for its interaction with Arf3p, is required for the polarized distribution of and an Afi1p mutated at the Arf3p-binding region its proper polarized distribution but loses its ability to rescue actin patch polarization in afi1 mutant cells. Furthermore, Afi1p is not required for polarized distribution of Yel1p and we infer that Arf3p is activated by Yel1p and is Afi1p to the polarized plasma Arf3p development of polarity. Arf3p with Afi1p in a yeast two-hybrid we that the N-terminal amino are and to interact with active of Arf3p and of the N-terminal amino could not interact with Arf3p, that the N-terminal amino are to a proper to interact with Arf3p. In to Arf3p, the truncated active of interact with the localization of and the plasma localization of it is that Afi1p interacts with Arf3p in with the interaction of and Afi1p in yeast two-hybrid the similarity and Arf3p. In the interaction of Arf3p with effectors of was in our such as and with the in and in interaction it is that Afi1p interacts with Arf3p directly and specifically at the plasma membrane. of Arf3p and Afi1p at the the localization of Afi1p, was the of the and was to localize at the plasma membrane and with the of et al. M. 2003; PubMed Scopus Google in which a protein was Although et al. 2003; PubMed Scopus Google that in the this be an of the protein of Afi1p, which is to we found that of with which be one of the that an could not the of its We to with however, we could not the Afi1p location a or did not show polarized localization the plasma membrane (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar). in of or could these proteins be at the plasma membrane of cells, that overexpression the polarization and Afi1p or Arf3p the of the lost for polarized Arf3p could still partially localize to the we were to show and in the that Arf3p lost its polarized localization in afi1 mutant and cells, and S. we that the pattern of Arf3p proper polarized distribution of It that are of involved in proteins in a polarized distribution J.E. C.L. D.J. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). a polarized interacts with the protein of its such as a of a of a such as the the of distribution a with constitutively polarized and endocytosis, of which the actin such as protein receptor The of Afi1p polarized distribution and the of actin for the localization (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Arf3p in the of polarized However, Afi1p is polarized proteins and is not an membrane protein as Afi1p a of polarized In addition, the mechanism by which Afi1p its distribution is Afi1p and Arf3p in the to of and and ARF3 are strains and but budding pattern abnormality and actin patch to these that are involved in the same polarity Although it that Afi1p a in mating F. J. J. E. Wilson R.A. 2000; PubMed Scopus Google Scholar), we with a previous C.L. K. Mol. Biol. Cell. 2004; 15: PubMed Google Scholar), that afi1 in the formation of mating or in mating and S. Although it that Afi1p interact with a protein M. 2003; PubMed Scopus Google Scholar), this was to demonstrate the cellular function of Afi1p in the Afi1p was in the It is that Afi1p the and plasma to factors such as D. J. Cell 2000; PubMed Google Scholar), and and of polarity of in an abnormal budding pattern to that in cells, and S. that the proper protein of Afi1p is important for its function in polarity development. are required for polarity and an for polarization J. Rev. Cell Biol. 1999; 15: PubMed Scopus Google Scholar). the of polarity establishment many cellular such as actin cytoskeleton vesicle trafficking, and cell with the budding pattern abnormality of such as K. M. Rev. PubMed Scopus Google Scholar), the abnormal budding pattern ARF3 or is which that Arf3p and Afi1p are involved in polarity development polarity In yeast, actin is actin and actin are and are of plasma membrane actin are that as for vesicle to actin by proteins E. Perron M. Pallotta D. 2003; PubMed Google Scholar). at the cell and to the that is for polarized cell growth. Like the arf3 the afi1 mutant shows actin patch polarization to the emerging at a and the of ARF3 and these that Afi1p and Arf3p in the same to actin patch polarization. Recently, Arf3p was found to be a suppressor of the of actin patch polarization to the emerging and actin formation and ARF3 this K. M. Rev. PubMed Scopus Google Scholar). is an actin protein, in the of actin of Arf3p in rescue the actin patch polarization but not the actin formation K. M. Rev. PubMed Scopus Google Scholar). is with our that Arf3p and Afi1p are required for actin patch polarization but not actin In addition, it was that ARF3 genetic interaction with the polarity (12Lambert A.A. Perron M.P. Lavoie E. Pallotta D. FEMS Yeast Res. 2007; 7: 782-795Crossref PubMed Scopus (23) Google Scholar). studies support the cellular function of Arf3p in cell polarity and actin Furthermore, we show an Arf3p guanine factor, is involved in actin patch polarization. our provide a Arf3p and actin Arf3p and Afi1p actin patch polarization by proteins or by with Afi1p for and of the studies an N-terminal of Afi1p, we show that the N-terminal region of Afi1p a sequence that is not important for its localization but for interacting with Arf3p. We that the sequence of Afi1p is important for interacting with Arf3p, Arf3p, and actin patch polarization. Because or could not interact with Arf3p, the sequence of Afi1p be to interact with Arf3p. of this sequence proper the N-terminal region which is to interact with Arf3p. However, of by this mutant did not to affect the polarized localization of that or of Afi1p required for interacting with Arf3p and to its receptor or be our previous that the N-terminal of Arf3p is for its plasma membrane localization (10Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; 14: 3834-3847Crossref PubMed Google Scholar), this that of activated Arf3p with the plasma membrane is a for its interaction with It is that at the polarized plasma membrane with Afi1p in this of Afi1p and Yel1p in of the we show that membrane of Afi1p is that of Arf3p that of Arf3p with the plasma membrane is that of Furthermore, in an afi1 Arf3p did not the plasma membrane and was still distributed the plasma membrane in and polarized cells. that activated Arf3p or specific membrane for Arf3p In addition, of Arf3p with the membrane be in and polarized cells. It is that of Afi1p with specific the polarized plasma membrane is a for its interaction with activated Arf3p. Because Afi1p is Arf3p and of Afi1p at phase we that Afi1p is a polarization-specific factor that activated Arf3p to the polarized membrane and a specific in polarized cells. It is to that Arf3p a in cells, of the of Arf3p in the The by and S. 2007; PubMed Scopus Google with our here that in mutant Arf3p was to the and distributed the plasma membrane. They that the of in mutant an of Arf3p, which is with the membrane. However, our that the constitutively of Arf3p, is in the is that be for Arf3p and S. 2007; PubMed Scopus Google that localization of Yel1p was to the and that of Arf3p, and our that Yel1p was to the They that Arf3p its of and the membrane. However, our is that be by Afi1p the of which is by Yel1p. that the localization of the activated Arf3p, but not the Arf3p is required for the proper of downstream However, we that Arf3p localize to two of the plasma membrane. In this study, Afi1p was identified as an protein that as a polarization-specific factor of Arf3p. the an in which Afi1p and Yel1p in the of Arf3p to the polarized plasma membrane by interacting directly with of these In of Yel1p to the polarized plasma membrane at the of to in be by Afi1p to the polarized plasma membrane Afi1p as a protein to maintain proper location of Arf3p and proteins that are for the function of Arf3p actin patch polarization. of interacting of Arf3p and Afi1p the of Arf3p in polarity development. We Y. for the We and for critical of this with