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Rvs161p and Rvs167p, the Two Yeast Amphiphysin Homologs, Function Together in Vivo

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.m008735200

1083-351X

Ruben Lombardi, Howard Riezman,

Photosynthetic Processes and Mechanisms

Mutations in RVS161 andRVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis. Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant. Here we demonstrate that the two proteins function together in vivo. We find that the steady state level of Rvs167p is strongly reduced in an rvs161Δ strain. Similarly, the level of Rvs161p is strongly reduced in an rvs167Δ strain. We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein. Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in anabp1Δ strain suggesting that Abp1p is not essential for this interaction. Mutations in RVS161 andRVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis. Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant. Here we demonstrate that the two proteins function together in vivo. We find that the steady state level of Rvs167p is strongly reduced in an rvs161Δ strain. Similarly, the level of Rvs161p is strongly reduced in an rvs167Δ strain. We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein. Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in anabp1Δ strain suggesting that Abp1p is not essential for this interaction. open reading frame polyacrylamide gel electrophoresis wild type 4-morpholineethanesulfonic acid tetramethylrhodamine B isothiocyanate 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside The yeast Rvs161p and Rvs167p, together with mammalian amphiphysin I and II, nematode, and fission yeast isoforms, constitute a family of conserved proteins (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The N termini of the different proteins share the highest homology, and this common domain was called the BAR domain (BIN/Amphiphysin/RVS domain (2Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (308) Google Scholar)). Rvs161p consists only of the BAR domain (Fig. 1), whereas the other members of the family have an SH3 domain at their C termini and a central domain varying among the different proteins. In the case of Rvs167p, the central domain is rich in glycine, proline, and alanine and therefore is called the GPA domain (Fig. 1).The mammalian homolog, amphiphysin I, was first identified as a brain protein enriched at presynaptic regions (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar). The identification of dynamin, synaptojanin, the αc-subunit of AP2-adaptin, and clathrin as amphiphysin I-interacting proteins further implicated amphiphysin I in endocytosis (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). A more broadly expressed isoform, amphiphysin II, has been identified by several groups and was found to interact with the same proteins that interact with amphiphysin I (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Interestingly, the two isoforms can be coimmunoprecipitated from brain extracts suggesting that they act in concert (4Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (208) Google Scholar). Indeed, two studies (4Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (208) Google Scholar, 31Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) found that the two amphiphysin isoforms colocalize in brain. However, a third study identified a different subcellular localization of the two isoforms (5Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (210) Google Scholar). Amphiphysin I was shown to be concentrated in the cortical cytoplasm of nerve terminals, whereas amphiphysin II was concentrated in axon initial segments and nodes of Ranvier (5Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (210) Google Scholar).The two yeast members of the amphiphysin family, encoded byRVS161 and RVS167, were first identified in a screen for mutations causing reduced viability upon nutrient starvation (6Crouzet M. Urdaci M. Dulau L. Aigle M. Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). RVS161 was also identified in a screen for endocytosis mutants (8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar). Mutations in rvs161 orrvs167 exhibit the same phenotypes except for a defect in cell fusion only found in the rvs161 mutant (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar). The mutant phenotypes include defects in endocytosis, cell polarization, bud site selection in diploid cells, and a depolarized actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar,10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Rvs161p and Rvs167p have been shown to interact through the BAR domain in the two-hybrid system (11Navarro P. Durrens P. Aigle M. Biochim. Biophys. Acta. 1997; 1343: 187-192Crossref PubMed Scopus (49) Google Scholar). However, their localization in the cell seems to be different, raising the question whether the Rvs161p-Rvs167p interaction is relevant in vivo. Rvs161p was shown to be mainly cytosolic in unbudded cells, and upon bud emergence it localizes mainly to the mother-bud neck region (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar). In contrast, in unbudded cells Rvs167p is localized mainly in small cortical patches throughout the cell which polarize at the bud emergence site and in small buds (12Balguerie A. Sivadon P. Bonneu M. Aigle M. J. Cell Sci. 1999; 112: 2529-2537Crossref PubMed Google Scholar). By using the two-hybrid system the BAR domain of Rvs167 has also been shown to mediate homodimerization in one study (13Colwill K. Field D. Moore L. Friesen J. Andrews B. Genetics. 1999; 152: 881-893Crossref PubMed Google Scholar), and another study failed to detect any Rvs167p-Rvs167p interaction (11Navarro P. Durrens P. Aigle M. Biochim. Biophys. Acta. 1997; 1343: 187-192Crossref PubMed Scopus (49) Google Scholar).Mutations in rvs161 and rvs167 affect the actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Interestingly, the SH3 domain of Rvs167p has been shown to interact with actin in the two-hybrid system (14Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar). The finding that Rvs167p interacts with the actin-binding protein Abp1p through its GPA/SH3 domains (15Lila T. Drubin D.G. Mol. Biol. Cell. 1997; 8: 367-385Crossref PubMed Scopus (145) Google Scholar) led to the hypothesis that Abp1p could mediate the interaction between Rvs167p and actin.In this study we investigated whether Rvs161p and Rvs167p indeed function together in vivo. We find that the steady state level of Rvs161p is strongly reduced in an rvs167Δ strain. Similarly, the level of Rvs167p is strongly reduced in anrvs161Δ strain. We demonstrate that these reduced protein levels at steady state are caused by a decreased stability of either Rvs protein in a strain mutated in the other rvs gene. Furthermore, we provide evidence that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we find that Abp1p is not required to mediate the interaction of Rvs167p with actin.DISCUSSIONIn this study we provide direct evidence that Rvs161p and Rvs167p function together in vivo. We find that the steady state levels of the two proteins are interdependent. This effect is caused by a dramatically decreased stability of either Rvs protein in the absence of its partner. These data provide evidence that the interaction of Rvs161p and Rvs167p is physiologically relevant and required for the stability of both proteins. Furthermore, these findings might explain the almost identical phenotypes that are seen upon mutation of the two genes individually. The mutant phenotypes detected in either mutant strain might be a combination of loss of both Rvs proteins. Nevertheless, the function of Rvs161p in cell fusion (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar) does not seem to require Rvs protein-protein interaction. Apparently, the highly reduced levels of Rvs161p in the rvs167Δ mutant are sufficient for this function. As seen in Fig. 3 B, Rvs161p is more sensitive to Rvs167p levels than vice versa. Since Rvs167p has two additional domains when compared with Rvs161p, this difference might reflect a partial stabilization of Rvs167p in the absence of Rvs161p via interactions mediated by these domains with other proteins (e.g. interaction with actin).As mentioned in the Introduction, two previous studies have investigated the cellular localization of Rvs161p (mostly cytoplasmic, at mother-bud neck region in small budded cells (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar)) and Rvs167p (small cortical patches that polarize upon bud emergence (12Balguerie A. Sivadon P. Bonneu M. Aigle M. J. Cell Sci. 1999; 112: 2529-2537Crossref PubMed Google Scholar)). The two proteins apparently do not colocalize in the cell; however, in this study we have provided evidence that these two proteins do function together in vivo. This discrepancy might be explained if one considers that Rvs161p localization is mainly cytosolic. Therefore, it might also be localized to the Rvs167p-containing patches but is not concentrated there. In the case of Rvs167p, the bright fluorescence caused by its high concentration in cortical patches might make it difficult to detect an additional weak diffuse cytosolic staining. Therefore, it is possible that a certain amount of Rvs167p is localized to the cytosol and interacts with Rvs161p.Interestingly, overexpression of either Rvs161p or Rvs167p alone in a WT strain has no major effect on the internalization step of endocytosis. However, co-overexpression of both proteins reduced the α-factor internalization rate to two-thirds of WT levels. Also, overexpression of Rvs161p in an rvs167Δ strain, as well as overexpression of Rvs167p in an rvs161Δ strain, exacerbated the endocytic defect found in the mutated strains. Since the amount of protein upon overexpression is similar in all the strains (WT, rvs161Δ, and rvs167Δ, data not shown), we conclude that both the amount and the ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. We have tested the strains used for the endocytosis assays for an actin and a growth phenotype. None of the strains overexpressing the Rvs proteins exhibited an obvious change in their actin cytoskeleton when compared with the strains with the empty vectors. Also the growth rates were almost identical with the exception of a mild defect detected upon overexpression of Rvs167p in the rvs161Δ strain. The endocytic defect we detect in some of the strains overexpressing the Rvs proteins might be explained in two ways. One possibility would be that overexpression of the Rvs proteins causes a very subtle actin defect not detectable by immunofluorescence but affecting endocytosis. Another possibility would be that by overexpressing the Rvs proteins another protein required for endocytosis is titrated out and therefore endocytosis is affected.By using the two-hybrid system, we detected an interaction of Rvs167p with itself mediated via the BAR domain as previously described (13Colwill K. Field D. Moore L. Friesen J. Andrews B. Genetics. 1999; 152: 881-893Crossref PubMed Google Scholar). Since the same domain also mediates the interaction with Rvs161p, we wanted to determine whether the Rvs167p-Rvs167p interaction is direct or mediated via Rvs161p. Two-hybrid analysis in an rvs161Δstrain demonstrated that Rvs161p is not required for this interaction and, therefore, Rvs167p either interacts directly with itself or the interaction is mediated via a protein other than Rvs161p. Interestingly, amphiphysin I has also been shown to interact with itself in mammalian cells (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar).Mutations in rvs161 and rvs167 have been shown to affect the actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). A previous study has shown that Rvs167p interacts with actin via its SH3 domain (14Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar), and it was suggested that the actin-binding protein Abp1p could mediate this Rvs167p-actin interaction (15Lila T. Drubin D.G. Mol. Biol. Cell. 1997; 8: 367-385Crossref PubMed Scopus (145) Google Scholar). Here we show that in the two-hybrid system Rvs167p interacts with actin in an abp1Δ strain showing that Abp1p is not required for this interaction. There are several possible explanations for this finding. First, it could be that in the absence of Abp1p another protein takes over its function. Second, there could be more than one protein involved in mediating this interaction. Third, Rvs167p could interact directly with actin. Another potential candidate to mediate the Rvs167p-actin interaction could be Las17p, since it was shown to interact with the GPA/SH3-domains of Rvs167p in the two-hybrid system (32Madania A. Dumoulin P. Grava S. Kitamoto H. Schärer-Brodbec C. Soulard A. Moreau V. Winsor B. Mol. Biol. Cell. 1999; 10: 3521-3538Crossref PubMed Scopus (138) Google Scholar).rvs mutant cells show pleiotropic phenotypes including sensitivity to nonoptimal growth conditions, defects in the organization of the actin cytoskeleton, defects in bud-site selection, and endocytosis (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Interestingly, Rvs167p was identified as a target of the Pho85 cyclin-dependent kinase and thus might be involved in the regulation of the actin cytoskeleton early in cell cycle (28Lee J. Colwill K. Aneliunas V. Tennyson C. Moore L. Ho Y. Andrews B. Curr. Biol. 1998; 8: 1310-1321Abstract Full Text Full Text PDF PubMed Google Scholar). Also its mammalian homolog, amphiphysin, has been shown to be regulated by phosphorylation (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar, 29Bauerfeind R. Takei K. De Camilli P. J. Biol. Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In addition, amphiphysin I has been implicated in the autoimmune Stiff-Man syndrome disorder associated with breast cancer, and amphiphysin II has been shown to interact with the MYC oncoprotein implicating it in cell cycle control (2Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (308) Google Scholar, 30David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar). Taken together these data suggest that some general functions and regulation of these proteins have been conserved through evolution. In addition, these findings implicate the members of this protein family not only as important players in intracellular membrane trafficking but also in mediating signals during the cell cycle.In summary, we demonstrate for the first time that the yeast amphiphysin homologs Rvs161p and Rvs167p function together in vivo. The interaction between the proteins seems to be crucial for their stability. Furthermore, we show that the amount and ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. The yeast Rvs161p and Rvs167p, together with mammalian amphiphysin I and II, nematode, and fission yeast isoforms, constitute a family of conserved proteins (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The N termini of the different proteins share the highest homology, and this common domain was called the BAR domain (BIN/Amphiphysin/RVS domain (2Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (308) Google Scholar)). Rvs161p consists only of the BAR domain (Fig. 1), whereas the other members of the family have an SH3 domain at their C termini and a central domain varying among the different proteins. In the case of Rvs167p, the central domain is rich in glycine, proline, and alanine and therefore is called the GPA domain (Fig. 1). The mammalian homolog, amphiphysin I, was first identified as a brain protein enriched at presynaptic regions (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar). The identification of dynamin, synaptojanin, the αc-subunit of AP2-adaptin, and clathrin as amphiphysin I-interacting proteins further implicated amphiphysin I in endocytosis (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). A more broadly expressed isoform, amphiphysin II, has been identified by several groups and was found to interact with the same proteins that interact with amphiphysin I (1Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Interestingly, the two isoforms can be coimmunoprecipitated from brain extracts suggesting that they act in concert (4Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (208) Google Scholar). Indeed, two studies (4Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (208) Google Scholar, 31Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) found that the two amphiphysin isoforms colocalize in brain. However, a third study identified a different subcellular localization of the two isoforms (5Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (210) Google Scholar). Amphiphysin I was shown to be concentrated in the cortical cytoplasm of nerve terminals, whereas amphiphysin II was concentrated in axon initial segments and nodes of Ranvier (5Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (210) Google Scholar). The two yeast members of the amphiphysin family, encoded byRVS161 and RVS167, were first identified in a screen for mutations causing reduced viability upon nutrient starvation (6Crouzet M. Urdaci M. Dulau L. Aigle M. Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). RVS161 was also identified in a screen for endocytosis mutants (8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar). Mutations in rvs161 orrvs167 exhibit the same phenotypes except for a defect in cell fusion only found in the rvs161 mutant (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar). The mutant phenotypes include defects in endocytosis, cell polarization, bud site selection in diploid cells, and a depolarized actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar,10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Rvs161p and Rvs167p have been shown to interact through the BAR domain in the two-hybrid system (11Navarro P. Durrens P. Aigle M. Biochim. Biophys. Acta. 1997; 1343: 187-192Crossref PubMed Scopus (49) Google Scholar). However, their localization in the cell seems to be different, raising the question whether the Rvs161p-Rvs167p interaction is relevant in vivo. Rvs161p was shown to be mainly cytosolic in unbudded cells, and upon bud emergence it localizes mainly to the mother-bud neck region (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar). In contrast, in unbudded cells Rvs167p is localized mainly in small cortical patches throughout the cell which polarize at the bud emergence site and in small buds (12Balguerie A. Sivadon P. Bonneu M. Aigle M. J. Cell Sci. 1999; 112: 2529-2537Crossref PubMed Google Scholar). By using the two-hybrid system the BAR domain of Rvs167 has also been shown to mediate homodimerization in one study (13Colwill K. Field D. Moore L. Friesen J. Andrews B. Genetics. 1999; 152: 881-893Crossref PubMed Google Scholar), and another study failed to detect any Rvs167p-Rvs167p interaction (11Navarro P. Durrens P. Aigle M. Biochim. Biophys. Acta. 1997; 1343: 187-192Crossref PubMed Scopus (49) Google Scholar). Mutations in rvs161 and rvs167 affect the actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Interestingly, the SH3 domain of Rvs167p has been shown to interact with actin in the two-hybrid system (14Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar). The finding that Rvs167p interacts with the actin-binding protein Abp1p through its GPA/SH3 domains (15Lila T. Drubin D.G. Mol. Biol. Cell. 1997; 8: 367-385Crossref PubMed Scopus (145) Google Scholar) led to the hypothesis that Abp1p could mediate the interaction between Rvs167p and actin. In this study we investigated whether Rvs161p and Rvs167p indeed function together in vivo. We find that the steady state level of Rvs161p is strongly reduced in an rvs167Δ strain. Similarly, the level of Rvs167p is strongly reduced in anrvs161Δ strain. We demonstrate that these reduced protein levels at steady state are caused by a decreased stability of either Rvs protein in a strain mutated in the other rvs gene. Furthermore, we provide evidence that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we find that Abp1p is not required to mediate the interaction of Rvs167p with actin. DISCUSSIONIn this study we provide direct evidence that Rvs161p and Rvs167p function together in vivo. We find that the steady state levels of the two proteins are interdependent. This effect is caused by a dramatically decreased stability of either Rvs protein in the absence of its partner. These data provide evidence that the interaction of Rvs161p and Rvs167p is physiologically relevant and required for the stability of both proteins. Furthermore, these findings might explain the almost identical phenotypes that are seen upon mutation of the two genes individually. The mutant phenotypes detected in either mutant strain might be a combination of loss of both Rvs proteins. Nevertheless, the function of Rvs161p in cell fusion (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar) does not seem to require Rvs protein-protein interaction. Apparently, the highly reduced levels of Rvs161p in the rvs167Δ mutant are sufficient for this function. As seen in Fig. 3 B, Rvs161p is more sensitive to Rvs167p levels than vice versa. Since Rvs167p has two additional domains when compared with Rvs161p, this difference might reflect a partial stabilization of Rvs167p in the absence of Rvs161p via interactions mediated by these domains with other proteins (e.g. interaction with actin).As mentioned in the Introduction, two previous studies have investigated the cellular localization of Rvs161p (mostly cytoplasmic, at mother-bud neck region in small budded cells (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar)) and Rvs167p (small cortical patches that polarize upon bud emergence (12Balguerie A. Sivadon P. Bonneu M. Aigle M. J. Cell Sci. 1999; 112: 2529-2537Crossref PubMed Google Scholar)). The two proteins apparently do not colocalize in the cell; however, in this study we have provided evidence that these two proteins do function together in vivo. This discrepancy might be explained if one considers that Rvs161p localization is mainly cytosolic. Therefore, it might also be localized to the Rvs167p-containing patches but is not concentrated there. In the case of Rvs167p, the bright fluorescence caused by its high concentration in cortical patches might make it difficult to detect an additional weak diffuse cytosolic staining. Therefore, it is possible that a certain amount of Rvs167p is localized to the cytosol and interacts with Rvs161p.Interestingly, overexpression of either Rvs161p or Rvs167p alone in a WT strain has no major effect on the internalization step of endocytosis. However, co-overexpression of both proteins reduced the α-factor internalization rate to two-thirds of WT levels. Also, overexpression of Rvs161p in an rvs167Δ strain, as well as overexpression of Rvs167p in an rvs161Δ strain, exacerbated the endocytic defect found in the mutated strains. Since the amount of protein upon overexpression is similar in all the strains (WT, rvs161Δ, and rvs167Δ, data not shown), we conclude that both the amount and the ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. We have tested the strains used for the endocytosis assays for an actin and a growth phenotype. None of the strains overexpressing the Rvs proteins exhibited an obvious change in their actin cytoskeleton when compared with the strains with the empty vectors. Also the growth rates were almost identical with the exception of a mild defect detected upon overexpression of Rvs167p in the rvs161Δ strain. The endocytic defect we detect in some of the strains overexpressing the Rvs proteins might be explained in two ways. One possibility would be that overexpression of the Rvs proteins causes a very subtle actin defect not detectable by immunofluorescence but affecting endocytosis. Another possibility would be that by overexpressing the Rvs proteins another protein required for endocytosis is titrated out and therefore endocytosis is affected.By using the two-hybrid system, we detected an interaction of Rvs167p with itself mediated via the BAR domain as previously described (13Colwill K. Field D. Moore L. Friesen J. Andrews B. Genetics. 1999; 152: 881-893Crossref PubMed Google Scholar). Since the same domain also mediates the interaction with Rvs161p, we wanted to determine whether the Rvs167p-Rvs167p interaction is direct or mediated via Rvs161p. Two-hybrid analysis in an rvs161Δstrain demonstrated that Rvs161p is not required for this interaction and, therefore, Rvs167p either interacts directly with itself or the interaction is mediated via a protein other than Rvs161p. Interestingly, amphiphysin I has also been shown to interact with itself in mammalian cells (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar).Mutations in rvs161 and rvs167 have been shown to affect the actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). A previous study has shown that Rvs167p interacts with actin via its SH3 domain (14Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar), and it was suggested that the actin-binding protein Abp1p could mediate this Rvs167p-actin interaction (15Lila T. Drubin D.G. Mol. Biol. Cell. 1997; 8: 367-385Crossref PubMed Scopus (145) Google Scholar). Here we show that in the two-hybrid system Rvs167p interacts with actin in an abp1Δ strain showing that Abp1p is not required for this interaction. There are several possible explanations for this finding. First, it could be that in the absence of Abp1p another protein takes over its function. Second, there could be more than one protein involved in mediating this interaction. Third, Rvs167p could interact directly with actin. Another potential candidate to mediate the Rvs167p-actin interaction could be Las17p, since it was shown to interact with the GPA/SH3-domains of Rvs167p in the two-hybrid system (32Madania A. Dumoulin P. Grava S. Kitamoto H. Schärer-Brodbec C. Soulard A. Moreau V. Winsor B. Mol. Biol. Cell. 1999; 10: 3521-3538Crossref PubMed Scopus (138) Google Scholar).rvs mutant cells show pleiotropic phenotypes including sensitivity to nonoptimal growth conditions, defects in the organization of the actin cytoskeleton, defects in bud-site selection, and endocytosis (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Interestingly, Rvs167p was identified as a target of the Pho85 cyclin-dependent kinase and thus might be involved in the regulation of the actin cytoskeleton early in cell cycle (28Lee J. Colwill K. Aneliunas V. Tennyson C. Moore L. Ho Y. Andrews B. Curr. Biol. 1998; 8: 1310-1321Abstract Full Text Full Text PDF PubMed Google Scholar). Also its mammalian homolog, amphiphysin, has been shown to be regulated by phosphorylation (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar, 29Bauerfeind R. Takei K. De Camilli P. J. Biol. Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In addition, amphiphysin I has been implicated in the autoimmune Stiff-Man syndrome disorder associated with breast cancer, and amphiphysin II has been shown to interact with the MYC oncoprotein implicating it in cell cycle control (2Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (308) Google Scholar, 30David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar). Taken together these data suggest that some general functions and regulation of these proteins have been conserved through evolution. In addition, these findings implicate the members of this protein family not only as important players in intracellular membrane trafficking but also in mediating signals during the cell cycle.In summary, we demonstrate for the first time that the yeast amphiphysin homologs Rvs161p and Rvs167p function together in vivo. The interaction between the proteins seems to be crucial for their stability. Furthermore, we show that the amount and ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. In this study we provide direct evidence that Rvs161p and Rvs167p function together in vivo. We find that the steady state levels of the two proteins are interdependent. This effect is caused by a dramatically decreased stability of either Rvs protein in the absence of its partner. These data provide evidence that the interaction of Rvs161p and Rvs167p is physiologically relevant and required for the stability of both proteins. Furthermore, these findings might explain the almost identical phenotypes that are seen upon mutation of the two genes individually. The mutant phenotypes detected in either mutant strain might be a combination of loss of both Rvs proteins. Nevertheless, the function of Rvs161p in cell fusion (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar) does not seem to require Rvs protein-protein interaction. Apparently, the highly reduced levels of Rvs161p in the rvs167Δ mutant are sufficient for this function. As seen in Fig. 3 B, Rvs161p is more sensitive to Rvs167p levels than vice versa. Since Rvs167p has two additional domains when compared with Rvs161p, this difference might reflect a partial stabilization of Rvs167p in the absence of Rvs161p via interactions mediated by these domains with other proteins (e.g. interaction with actin). As mentioned in the Introduction, two previous studies have investigated the cellular localization of Rvs161p (mostly cytoplasmic, at mother-bud neck region in small budded cells (9Brizzio V. Gammie A.E. Rose M.D. J. Cell Biol. 1998; 141: 567-584Crossref PubMed Scopus (77) Google Scholar)) and Rvs167p (small cortical patches that polarize upon bud emergence (12Balguerie A. Sivadon P. Bonneu M. Aigle M. J. Cell Sci. 1999; 112: 2529-2537Crossref PubMed Google Scholar)). The two proteins apparently do not colocalize in the cell; however, in this study we have provided evidence that these two proteins do function together in vivo. This discrepancy might be explained if one considers that Rvs161p localization is mainly cytosolic. Therefore, it might also be localized to the Rvs167p-containing patches but is not concentrated there. In the case of Rvs167p, the bright fluorescence caused by its high concentration in cortical patches might make it difficult to detect an additional weak diffuse cytosolic staining. Therefore, it is possible that a certain amount of Rvs167p is localized to the cytosol and interacts with Rvs161p. Interestingly, overexpression of either Rvs161p or Rvs167p alone in a WT strain has no major effect on the internalization step of endocytosis. However, co-overexpression of both proteins reduced the α-factor internalization rate to two-thirds of WT levels. Also, overexpression of Rvs161p in an rvs167Δ strain, as well as overexpression of Rvs167p in an rvs161Δ strain, exacerbated the endocytic defect found in the mutated strains. Since the amount of protein upon overexpression is similar in all the strains (WT, rvs161Δ, and rvs167Δ, data not shown), we conclude that both the amount and the ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. We have tested the strains used for the endocytosis assays for an actin and a growth phenotype. None of the strains overexpressing the Rvs proteins exhibited an obvious change in their actin cytoskeleton when compared with the strains with the empty vectors. Also the growth rates were almost identical with the exception of a mild defect detected upon overexpression of Rvs167p in the rvs161Δ strain. The endocytic defect we detect in some of the strains overexpressing the Rvs proteins might be explained in two ways. One possibility would be that overexpression of the Rvs proteins causes a very subtle actin defect not detectable by immunofluorescence but affecting endocytosis. Another possibility would be that by overexpressing the Rvs proteins another protein required for endocytosis is titrated out and therefore endocytosis is affected. By using the two-hybrid system, we detected an interaction of Rvs167p with itself mediated via the BAR domain as previously described (13Colwill K. Field D. Moore L. Friesen J. Andrews B. Genetics. 1999; 152: 881-893Crossref PubMed Google Scholar). Since the same domain also mediates the interaction with Rvs161p, we wanted to determine whether the Rvs167p-Rvs167p interaction is direct or mediated via Rvs161p. Two-hybrid analysis in an rvs161Δstrain demonstrated that Rvs161p is not required for this interaction and, therefore, Rvs167p either interacts directly with itself or the interaction is mediated via a protein other than Rvs161p. Interestingly, amphiphysin I has also been shown to interact with itself in mammalian cells (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar). Mutations in rvs161 and rvs167 have been shown to affect the actin cytoskeleton (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). A previous study has shown that Rvs167p interacts with actin via its SH3 domain (14Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar), and it was suggested that the actin-binding protein Abp1p could mediate this Rvs167p-actin interaction (15Lila T. Drubin D.G. Mol. Biol. Cell. 1997; 8: 367-385Crossref PubMed Scopus (145) Google Scholar). Here we show that in the two-hybrid system Rvs167p interacts with actin in an abp1Δ strain showing that Abp1p is not required for this interaction. There are several possible explanations for this finding. First, it could be that in the absence of Abp1p another protein takes over its function. Second, there could be more than one protein involved in mediating this interaction. Third, Rvs167p could interact directly with actin. Another potential candidate to mediate the Rvs167p-actin interaction could be Las17p, since it was shown to interact with the GPA/SH3-domains of Rvs167p in the two-hybrid system (32Madania A. Dumoulin P. Grava S. Kitamoto H. Schärer-Brodbec C. Soulard A. Moreau V. Winsor B. Mol. Biol. Cell. 1999; 10: 3521-3538Crossref PubMed Scopus (138) Google Scholar). rvs mutant cells show pleiotropic phenotypes including sensitivity to nonoptimal growth conditions, defects in the organization of the actin cytoskeleton, defects in bud-site selection, and endocytosis (7Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar, 8Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar, 10Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Interestingly, Rvs167p was identified as a target of the Pho85 cyclin-dependent kinase and thus might be involved in the regulation of the actin cytoskeleton early in cell cycle (28Lee J. Colwill K. Aneliunas V. Tennyson C. Moore L. Ho Y. Andrews B. Curr. Biol. 1998; 8: 1310-1321Abstract Full Text Full Text PDF PubMed Google Scholar). Also its mammalian homolog, amphiphysin, has been shown to be regulated by phosphorylation (27Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar, 29Bauerfeind R. Takei K. De Camilli P. J. Biol. Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In addition, amphiphysin I has been implicated in the autoimmune Stiff-Man syndrome disorder associated with breast cancer, and amphiphysin II has been shown to interact with the MYC oncoprotein implicating it in cell cycle control (2Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (308) Google Scholar, 30David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar). Taken together these data suggest that some general functions and regulation of these proteins have been conserved through evolution. In addition, these findings implicate the members of this protein family not only as important players in intracellular membrane trafficking but also in mediating signals during the cell cycle. In summary, we demonstrate for the first time that the yeast amphiphysin homologs Rvs161p and Rvs167p function together in vivo. The interaction between the proteins seems to be crucial for their stability. Furthermore, we show that the amount and ratio of Rvs161p and Rvs167p are critical parameters for the internalization step of endocytosis. We thank the members of the Riezman laboratory for discussions and Kathleen D'Hondt for critical reading of the manuscript.