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Institute for Molecular Bioscience,1 ARC Special Research Centre for Functional and Applied Genomics,2 School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia,3 UMR7156, Centre National Recherche Scientifique, Université Louis Pasteur, Strasbourg 67084, France4
SUMMARY INTRODUCTION BUDDING YEAST Rvs PROTEINS Nutrient Availability and the Control of Cell Proliferation Rvs161p and Rvs167p Proteins and Their Common BAR Domain BAR Domains of Rvs161p and Rvs167p Assemble into Heterodimers Loss of Rvs161p and Loss of Rvs167p Cause Similar and Diverse Spectra of Phenotypes Reduced viability upon starvation (Rvs–). Growth sensitivity to salt (especially Na+). Growth sensitivity to cytotoxic compounds. Growth sensitivity to elevated temperature. Growth on nonfermentable carbon sources. Meiosis and sporulation. Heterogeneous cell size and morphology upon starvation or exposure to Na+. Loss of actin cytoskeleton polarization to sites of polarized growth. Delocalized cell wall chitin deposition. Loss of bipolar bud site selection. Defective fluid-phase and receptor-mediated endocytosis. Inefficient cell-cell fusion during mating. Rvs161p and Rvs167p Structure-Function Relationships Rvs Proteins Localize to the Cortical Actin Cytoskeleton Subcellular localization of Rvs161p and Rvs167p. Interactions between Rvs167p and actin patch proteins. Roles of Rvs161p and Rvs167p in Endocytosis Rvs161p and Rvs167p in receptor-mediated internalization of alpha-factor. Role for Rvs161p and Rvs167p in postinternalization trafficking through endosomes? Roles of Rvs161p and Rvs167p in the Secretory Pathway Rvs161p and Rvs167p are not essential for all secretory membrane traffic. Rvs proteins may be required for polarized secretion during cell division. Rvs proteins and post-Golgi apparatus traffic. Rvs proteins and ER-to-Golgi apparatus traffic. Rvs proteins and mating. rvs Mutations Interact with Actin and Myosin Mutations Rvs– phenotypes are associated with mutations affecting other actin cytoskeleton proteins. Genetic interactions between Rvs proteins and actin. Genetic interactions between Rvs proteins and yeast myosins. Rvs Proteins and Membranes Association of cortical actin cytoskeleton with membranes. Fractionation of Rvs proteins with membranes. Rvs proteins and lipid rafts. Suppression of Rvs– phenotypes by mutations affecting glycosphingolipid biosynthesis. Regulation of Rvs167p by Phosphorylation Regulation of Rvs167p by Ubiquitination Genomic and Proteomic Approaches and the Diverse Network of Rvs Interactions Large-scale two-hybrid screens for Rvs161p- and Rvs167p-interacting proteins. Identification of Rvs167p SH3 domain-interacting proteins by phage display. Identification of Rvs161p- and Rvs167p-interacting proteins by high-throughput proteomics. Mapping the genetic interactions of Rvs161p and Rvs167p by synthetic genetic array. Rvs161p and Rvs167p physical and genetic interactions suggest multiple functions in vivo. FISSION YEAST BAR DOMAIN PROTEINS Hob1p AND Hob3p Hob3p, Fission Yeast Ortholog of Rvs161p Hob3p has a domain structure similar to that of Rvs161p. Hob3p functions in polarization of the cortical actin cytoskeleton but not endocytosis. Functional conservation between Hob3p, Rvs161p, and human Bin3. Hob1p, Fission Yeast Ortholog of Rvs167p Hob1p has a domain structure similar to that of Rvs167p. Hob1p is not required for polarization of the cortical actin cytoskeleton or endocytosis. Hob1p interacts with actin assembly proteins and protein kinases that regulate cell polarity. Hob1p functions in regulating cell cycle progression in response to nutrient availability. Functional Conservation of Hob1p and Bin1 and Divergence of Rvs167p Hob1p functions in regulation of cell cycle progression in response to DNA damage. Hob1p functions in a cell stress signal transduction pathway upstream of Hob3p. VERTEBRATE BAR DOMAIN PROTEIN AMPHIPHYSIN 1 Endocytic Recycling of Synaptic Vesicles Role of dynamin 1 in synaptic vesicle recycling. Discovery of Amphiphysin 1 in Chicken Brain Chicken amphiphysin 1 has a domain structure similar to that of budding yeast Rvs167p. Chicken amphiphysin 1 is expressed in neurons. Minor pool of chicken amphiphysin 1 is tightly associated with synaptic vesicles. Discovery of Human Amphiphysin 1 Human amphiphysin 1 is the autoantigen in stiff-man syndrome with breast cancer. Human amphiphysin 1 has a domain structure similar to that of yeast Rvs167p. Mammalian amphiphysin 1 is highly expressed in the brain but is also expressed in other tissues. Amphiphysin 1 interacts with the endocytic proteins dynamin 1 and synaptojanin 1. Amphiphysin 1 Interacts with Clathrin and the AP-2 Adaptor Regulation of Amphiphysin 1 Interactions by Phosphorylation Amphiphysin 1 Subcellular Localization in Presynaptic Terminals Amphiphysin 1 localizes to endocytic intermediates. Amphiphysin 1 also localizes to the actin-rich cytomatrix. Amphiphysin 1 functions in polarized growth. Amphiphysin 1 Functions in Endocytosis Amphiphysin 1 interaction with dynamin 1 is essential for endocytosis in neurons. Amphiphysin interaction with dynamin is also essential for endocytosis in nonneuronal cells. Amphiphysin 1 interacts with clathrin and AP-2. Appendage domain of alpha-adaptin mediates interaction with amphiphysin 1. Amphiphysin 1 interaction with clathrin and AP-2 is essential for endocytosis. Amphiphysin 1 interacts with endophilin A1/SH3p4/SH3GL2. Amphiphysin 1 forms homodimers. Regulation of amphiphysin 1 complex formation by phosphorylation and dephosphorylation. Protein phosphatase 2B/calcineurin is responsible for dephosphorylation of amphiphysin 1, dynamin 1, and synaptojanin 1 upon nerve stimulation. Proline-rich central insert domain of amphiphysin 1 is phosphorylated by Cdk5/p35 in vivo. Amphiphysin 1 Knockout Mice Amphiphysin 1 is important but not essential for synaptic vesicle recycling in vivo. Essential role for amphiphysin 1 in learning. Amphiphysin 1 and Molding of Membranes Dynamin 1 binds membranes and evaginates tubules in vitro. Amphiphysin 1 binds membranes and evaginates tubules in vitro. Amphiphysin 1 binding to membranes is dependent on lipid composition. Amphiphysin 1 and dynamin 1 coassemble into rings on membrane tubules. Amphiphysin 1 stimulates assembly of dynamin 1 into thick rings in solution. Amphiphysin 1 coordinates clathrin bud and dynamin 1 tubule formation. Amphiphysin 1 stimulates the activity of dynamin 1 in membrane tubule fission. In vivo role for amphiphysin 1 in membrane tubule formation. Membrane curvature and amphiphysin 1 binding. Amphiphysin 1 tubulation of membranes is dependent on lipid composition. Domains of amphiphysin 1 required for membrane tubulation. Domains of amphiphysin 1 required for coassembly with dynamin 1 into rings in solution. SECOND ISOFORM OF AMPHIPHYSIN, Bin1 Discovery of Bin1 Bin1+6a+12+13/Amphiphysin 2/Amphiphysin II/BRAMP2 in the Brain Distribution of Bin1+12+13 in the brain. Localization of Bin1+6a+12 on purified plasma membranes. Association of Bin1 with membranes. Recruitment of Bin1+6a+12 to the plasma membrane. Interaction of Bin1+12 with Other Brain Proteins Bin1+12 (with or lacking exons 6a and 13) interacts with dynamin 1 and synaptojanin 1 in the brain. Bin1+12 (with or lacking exons 6a and 13) interacts with the alpha-adaptin subunit of AP-2 in the brain. Bin1+12 (with or lacking exons 6a and 13) interacts with clathrin heavy chain in the brain. Interaction of Bin1+12 with endophilin A1/SH3p4/SH3GL2 in the brain. Bin1+6a+12 forms homodimers and heterodimers in the brain. Interaction of amphiphysin 1/Bin1+6a+12 heterodimers with dynamin 1 in the brain. Bin1+6a+12 is regulated by phosphorylation and dephosphorylation. Bin1+6a+12 functions in synaptic vesicle recycling/endocytosis. Bin1+12 may function in postinternalization transport through endosomes. Bin1+10+13/Bin1 Identification of Bin1+10+13/Bin1. Domain structure of Bin1+10+13. Interaction of Bin1+10+13 with c-Myc. Bin1+10+13 is a tumor suppressor. Bin1+10+13 induces apoptosis specifically in tumor cells. Subcellular localization of Bin1+10+13. Stability of Bin1+10+13 in vivo. Phosphorylation of Bin1+10+13. Role for Bin1+10+13 in muscle differentiation. Bin1+10+13 localization in adult skeletal muscle. Bin1+10+13 and T-tubule formation in muscle. Bin1+10+13 and lipid rafts. Bin1+10+13 binds and tubulates liposomes in vitro. Bin1–10–12+13/SH3p9 Bin1–10–12–13/ALP1/Amphiphysin IIm Bin1–10–12–13 in regulation of the actin cytoskeleton. Bin1–10–12–13 functions in phagocytosis in macrophages. AMPHIPHYSIN-RELATED PROTEIN Bin2 AMPHIPHYSIN-RELATED PROTEIN Bin3 ENDOPHILIN FAMILY OF PROTEINS Identification of Endophilin A1/SH3p4/SH3GL2 Endophilin A and B Family Proteins Bind and Tubulate Membranes Endophilin A Family Proteins Function in Receptor-Mediated Endocytosis OTHER BAR DOMAIN PROTEINS Rich-1/Nadrin 1 SNX1 Other BAR Domain Proteins CRYSTAL STRUCTURE OF THE AMPHIPHYSIN BAR DOMAIN Structure of the BAR Domain Coupling of the BAR Domain with Additional Lipid-Binding Sequences Membrane Binding and GTPase Binding of BAR Domains: Independent or Associated? BAR Domain and IMD Structure MEMBRANE TUBULATION AND BAR DOMAIN PROTEIN FUNCTION IN VIVO CONCLUSIONS AND FUTURE PERSPECTIVES ACKNOWLEDGMENTS REFERENCES
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-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. | INTRODUCTION |
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-helical domain of 250 to 280 amino acids named after the founding members of this family: Bin1, Amphiphysin, and Rvs167 (BAR) (278, 298). BAR domain proteins have been implicated in an extraordinary diversity of cellular processes, including fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning/memory, tissue differentiation, ion flux across membranes, and tumor suppression. The conserved features of the BAR domain suggest there may exist an underlying common molecular mechanism that is provided by the BAR domain and that has been adapted for use in these different physiological processes. Insight into what this common molecular mechanism may be has come from recent key discoveries. The first discovery was that BAR domains bind liposomes in vitro and convert low-curvature spheres to high-curvature tubules (316). The second discovery was that the BAR domain is itself curved (banana-shaped) and is therefore exquisitely designed both to sense membrane curvature and to actively influence membrane curvature (237). Furthermore, subsequent structure comparisons revealed that many proteins that bind GTPases do so through a similar protein fold (109). These discoveries have created great excitement, since it may now be possible to explain the diverse cellular roles of BAR domain proteins in terms of sensing membrane curvature, binding GTPases, and actively molding cellular membranes.
BAR domain proteins are encoded by most, if not all, eukaryotic genomes. They are found in organisms from lower unicellular eukaryotes such as budding yeast and fission yeast to insects, plants, and vertebrates. The phylogeny of BAR domain proteins has been the subject of an excellent recent review (109). Database homology searches with known BAR domains have not yet identified an obvious BAR domain encoded by the genome of any prokaryote. Sequence homology between known BAR domains is, however, relatively modest. The sequence features that confer folding of a polypeptide into a structure that can bind and bend membranes are still not fully understood. It is possible that proteins with similar properties exist also in prokaryotes but that low sequence homology has made them difficult to recognize. The apparent origin of BAR domain proteins in eukaryotes suggests a function(s) unique to eukaryotes. Some of the roles of BAR domain proteins (e.g., in membrane traffic) would be relevant only in eukaryotic cells. Hence, the evolution of BAR domain proteins may parallel the evolution of cellular compartmentation and complexity.
This article will first review the physiological roles and interactions of the BAR domain proteins, focusing primarily on the amphiphysins (including the Bin1-3 proteins) and the endophilins. Other BAR domain proteins, such as the sorting nexins (SNXs), will be discussed only briefly. In particular, this article focuses on the roles of BAR domain proteins in yeasts (budding yeast and fission yeast) and in mammals (with occasional reference to their roles in flies). This article then reviews the three-dimensional structure of the BAR domain. The article concludes with a somewhat speculative attempt to explain the known physiological roles of BAR domain proteins in yeasts and in mammals in terms of binding membranes and generating membrane tubules in vivo.
| BUDDING YEAST Rvs PROTEINS |
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To identify genes important for cell cycle arrest in response to starvation, Michel Aigle and colleagues screened a random collection of UV-induced mutants for those specifically defective in their ability to adapt to starvation conditions. Mutants that were fully viable in the presence of nutrients but that lost viability more rapidly than wild-type cells when starved of glucose (carbon), ammonium (nitrogen), or sulfate (sulfur) were retained. Two mutants found to exhibit a Reduced Viability upon Starvation (Rvs–) phenotype and to carry mutations in distinct genes were named rvs161 and rvs167 the corresponding wild-type genes are RVS161 and RVS167, respectively). In cell cycle terminology, the proteins Rvs161p and Rvs167p are negative cell cycle regulators that link nutrient availability to cell cycle progression (12, 42).
-helical structure (12, 47, 220, 298). The N-terminal homologous domain was initially named the Rvs domain (299). Because vertebrate amphiphysin 1 and Bin1 (see below) also feature a homologous domain, Sakamuro et al. renamed the Rvs domain the Bin1/Amphiphysin/Rvs167 (BAR) domain (Fig. 1) (278). The designation BAR domain is now commonly used to describe this homologous domain in both yeast and nonyeast proteins. More recently, this domain has been subdivided into a short N-terminal amphipathic -helix (
40 to 45 residues) and the BAR domain itself, as some BAR domain proteins lack the N-terminal amphipathic -helix (Fig. 1).
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-helical potential in both the Rvs161p BAR domain (residues 22 to 65 and 127 to 183) and the Rvs167p BAR domain (residues 30 to 57 and 144 to 191) (47, 220). Moreover, the COILS algorithm revealed that the BAR domain -helices are amphipathic and have a propensity to form coiled-coil structures that may mediate interaction of the BAR domain with other coiled-coil proteins. Two-hybrid analysis revealed that Rvs167p and Rvs161p interact and that the interaction is mediated by their BAR domains. In this study, neither Rvs161p nor Rvs167p interacted with itself, suggesting that the Rvs proteins form obligate heterodimers. The in vivo association of Rvs161p with Rvs167p was confirmed by coimmunoprecipitation of Rvs161p with Rvs167p from yeast lysates in vitro (220). Subsequently, no fewer than eight other two-hybrid studies have analyzed interactions between Rvs161p and Rvs167p and each protein with itself (18, 38, 59, 84a, 97, 136, 180, 339). All these studies confirmed interaction between Rvs161p and Rvs167p via the respective BAR domains. In contrast to the initial study (220), two of these later studies found that Rvs167p also forms homodimers via the BAR domain (38, 180). Furthermore, two-hybrid interaction of Rvs167p with itself persists when RVS161 is deleted, indicating that Rvs167p-Rvs167p interaction is either direct or mediated by a protein other than Rvs161p (180). Two later studies, however, concluded that Rvs167p does not form homodimers in vegetatively growing cells (i.e., it only forms heterodimers) (84a, 97). Interestingly, in cells lacking Rvs161p the steady-state level of Rvs167p is considerably reduced due to accelerated proteolysis and vice versa (180). These results represent strong evidence that in vivo Rvs161p and Rvs167p form heterodimers and function in concert.
) or the RVS167 gene (rvs167) in almost every case reveals a phenotype identical to that with the original mutant allele (12, 42, 53, 84a).
Reduced viability upon starvation (Rvs–).
As introduced above, rvs161 and rvs167 mutants were originally isolated based on their inability to maintain viability when starved of a source of nitrogen, carbon, or sulfur. For example, rvs161 cells grown to stationary phase in glucose-limited or nitrogen-limited medium and then left in the depleted medium for 60 h exhibit only 20 and 65% viability, respectively. In contrast, wild-type cells subjected in the same way to either glucose or nitrogen starvation maintain close to 100% viability. A similar loss of viability under starvation conditions has been shown for rvs167 cells. In nitrogen- or glucose-limited minimal medium, 90 to 100% of wild-type cells arrest without buds after 50 h. In contrast, under each condition 20% of rvs161 cells still exhibit a bud. Again, a similar defect in starvation-induced cell cycle arrest was found for rvs167 cells. Hence, under starvation conditions rvs mutants still attempt to transit the cell cycle and likely die as a consequence of their failure to arrest (12, 42).
Small GTPases of the Ras family regulate response to starvation in yeast. They do this by regulating adenylyl cyclase activity and production of the second messenger cyclic AMP (282). Yeast mutants with constitutively activated Ras exhibit reduced viability upon starvation and an inability to mount a normal physiological response to nutrient deprivation similar to rvs mutants. Wild-type yeast cells accumulate the storage carbohydrate glycogen when starved (177). One characteristic of the constitutively activated ras phenotype is the failure to accumulate glycogen upon glucose starvation (185, 325, 329). Can rvs mutant cells accumulate glycogen when nutrients become limiting, e.g., when cultures reach stationary phase and the cells cease growth? When they reach stationary phase both rvs161 and rvs167 mutant cells do indeed accumulate glycogen normally (12, 42, 53). This suggests the defect in rvs mutant cells is distinct from that in hyperactivated ras mutants or mutants in which adenylyl cyclase activity is constitutive. The molecular basis of the defect in response to starvation in rvs mutants is not yet known.
Growth sensitivity to salt (especially Na+). rvs mutants are not only sensitive to starvation. They are also more sensitive to a range of other stresses that include the presence of high concentrations of various salts (e.g., NaCl, KCl, MgCl2, and Na2SO4) in the medium. This sensitivity is to salt rather than to osmotic strength, because rvs mutants are not sensitive to sorbitol at high osmotic strength. Moreover, there is selectivity for certain cations. For example, rvs161 mutants exhibit morphological defects at fivefold lower concentrations of NaCl (0.25 M) than of KCl (1.2 M) while wild-type cells are unaffected by either salt. Furthermore, rvs161 mutants are no more sensitive to LiCl than wild-type cells (although both are sensitive) (42). In this review the rvs salt sensitivity will be referred to as Na+ sensitivity to reflect this. The molecular basis of the enhanced sensitivity of rvs mutants to Na+ is not known.
Growth sensitivity to cytotoxic compounds. The growth of both rvs161 and rvs167 mutants is hypersensitive to the presence of various cytotoxic compounds, including 3-amino-1,2,4-triazole (3-AT) and canavanine (12, 42). 3-AT is a histidine biosynthesis inhibitor that induces an artificial starvation response when added to cells. Canavanine is an arginine analog that can be incorporated into proteins in place of arginine and produce nonfunctional proteins. Yeast mutants defective in ubiquitin-mediated proteolysis are especially sensitive to canavanine and overexpression of ubiquitin confers canavanine resistance (32, 115). In addition, rvs161 cells have been reported to be hypersensitive to the effects of sinefungin, which completely blocks growth of rvs161 cells at 0.1 µM (rvs167 cells were not tested) (42). Sinefungin is a toxic analog of S-adenosylmethionine that inhibits methylation reactions. In yeast, sinefungin has recently been shown to inhibit methylation of guanosine to form m7GpppN, which is used to cap the 5' end of mRNAs. Capping of mRNAs is in turn important for mRNA stability and efficient translation (36). Whether hypersensitivity to these compounds is specific or reflects a general hypersensitivity of rvs161 and rvs167 cells to all cytotoxic compounds is not known.
Growth sensitivity to elevated temperature. The standard growth temperature for budding yeast is 30°C. Elevated temperature presents a stress to yeast cells and while wild-type cells continue to grow well at 37°C they stop dividing at 42°C. Yeast mutants with constitutively activated ras mutations exhibit not only reduced viability upon starvation, but also an inability to grow at normal temperature after a short heat shock at 55°C. In contrast, rvs161 and rvs167 mutant cells both survive heat shock at 55°C. In two reports, rvs161 cells did not exhibit defects in growth at low (e.g., 15°C) or normal (28°C) temperature and neither rvs161 nor rvs167 cells exhibit defects in growth at elevated (e.g., 36°C) temperature (12, 42).
The ability of rvs161 and rvs167 cells to grow at elevated temperature may be dependent on genetic background or the accumulation of second-site suppressor mutations. In a somewhat different genetic background rvs161 and rvs167 cells did not grow at 37°C (215). Exposure to elevated temperature causes induction of heat shock proteins (HSP) which aid in the refolding of damaged proteins. However, HSP induction upon exposure to elevated temperature was found to be normal in rvs167 mutant cells (12). rvs mutants are not sensitive to all environmental stresses, e.g., rvs161 and wild-type yeast cells are equally resistant to extremes of pH (12, 42).
Growth on nonfermentable carbon sources. Both rvs161 and rvs167 mutants grow well on a range of fermentable carbon sources (e.g., glucose, galactose, mannose, and sucrose), but are unable to grow on nonfermentable carbon sources (e.g., glycerol, lactate, or acetate) (12, 42). An inability to utilize nonfermentable carbon sources suggests a possible mitochondrial defect. These carbon sources are metabolized in mitochondria (370). rvs161 mutant cells possess a normal spectrum of mitochondrial cytochromes, but respire threefold more slowly than wild-type cells as measured by oxygen consumption. It is not clear, however, whether this level of respiratory defect is sufficient to fully account for the observed failure to utilize nonfermentable carbon sources. Defects in glycerol utilization are a common phenotype of mutations that affect the actin cytoskeleton, but the molecular basis for the defect is still unclear. Mitochondrial defects alone cannot account for the Rvs– phenotype since deletions in the mitochondrial genome ([rho–]) that abolish growth on nonfermentable carbon sources do not give rise to the other Rvs– phenotypes such as reduced viability upon starvation (12, 42).
Meiosis and sporulation. In the laboratory, budding yeast cells can be maintained as either stable diploids or stable haploids. Diploid yeast cells can be induced to undergo sporulation (meiosis) by nitrogen starvation on nonfermentable carbon sources. Sporulation has been well studied in yeast because it represents a cellular differentiation process that can be studied in budding yeast and that may have molecular mechanisms in common with more complex cellular differentiation pathways used in vertebrate development. Sporulation involves switching off the expression of blocks of genes required for vegetative growth and initiating a developmental program in which spore-specific genes are expressed in a highly regulated pattern. In addition to meiosis, sporulation also involves de novo bilayer membrane biogenesis to form the plasma membrane of the resultant haploid cell and also cell wall biogenesis to form the spore wall.
Desfarge et al. reported that homozygous rvs161/rvs161 diploid cells do not sporulate upon nitrogen starvation (53). Subsequently, Colwill et al. showed that rvs167/rvs167 homozygous diploid cells, although not entirely compromised for sporulation, form spores with only 10% the frequency of wild-type diploids (38). rvs/rvs homozygous diploids may not sporulate because they are unable to utilize the nonfermentable carbon source provided or because they lose viability upon nitrogen starvation. The sporulation defect in these mutants could be indirect. However, genetic interaction studies have suggested that the Rvs proteins play a direct role in sporulation (53) (discussed below). The molecular mechanisms that underlie this defect have yet to be elucidated.
Heterogeneous cell size and morphology upon starvation or exposure to Na+. Although rvs161 and rvs167 mutant cells exhibit the relatively uniform size and ellipsoid shape of wild-type cells under optimal growth conditions, when starved or exposed to Na+ (or the cytotoxic compounds referred to above) cultures of both rvs161 and rvs167 cells accumulate a high proportion of cells that are either grossly enlarged with swollen vacuoles or abnormally tiny. Some mother cells also have multiple buds. Each bud has a nucleus but is apparently unable to complete cytokinesis and separate from the mother cell (12, 42). In wild-type yeast, there is a minimum cell size that is required at Start for commitment to a new cell division cycle (137). The rvs mutations appear to compromise this cell cycle regulation with under-sized mother cells continuing to divide.
Loss of actin cytoskeleton polarization to sites of polarized growth. Yeast cells possess an actin cytoskeleton comprising filamentous actin (F-actin) and a diverse set of actin-associated proteins (71, 213, 371). Yeasts express orthologs of many, although not all, vertebrate actin-associated proteins, including the Arp2/3 complex and its activators, both conventional (filament forming) and unconventional myosins, profilin, tropomyosin, fimbrin, capping protein, and cofilin. When yeast cells are stained with fluorophore-conjugated phalloidin (an F-actin-specific reagent) several distinct structures are visible. Actin patches are small highly motile spots located at or near the cortex. Actin cables are long thick fibers comprising bundled actin filaments that extend through the cortical cytoplasm. Although individual actin patches and actin cables turn over, patches and cables are visible throughout the cell cycle.
Dividing yeast cells display a third F-actin structure known as the contractile actomyosin ring (16, 178). This ring structure localizes at the neck between the mother cell and bud. Contraction of the actomyosin ring occurs during cytokinesis and is accompanied by deposition of new cell wall material (septum). Septum deposition eventually separates the cytoplasm of the mother cell and bud and cleavage of the septum allows cell separation.
During the cell division cycle, the distribution of actin patches and actin cables changes. Immediately prior to bud emergence, cables align with their tips focused at the nascent bud site and patches concentrate at this site. When the bud starts to emerge, the cables align with their tips inside the growing bud and the patches localize at the bud tip. In G2 the actin patches remain polarized to the bud but switch from a polarized to an isotropic distribution within the bud. After the switch the bud expands laterally as well as at the tip. During mitosis actin is recruited to the actomyosin ring, cables become randomly oriented, and patches distribute randomly throughout the mother cell and bud. Finally, upon exit from mitosis the patches in the mother cell and bud repolarize to either side of the bud neck and cables in the mother cell and bud realign with their tips focused to the bud neck. At this stage of the cell cycle the actomyosin ring contracts to a dot, septum is deposited and cleaved, and the cells divide. The newly divided mother and daughter cells transiently retain polarized actin patches and cables, and then polarity is lost until a new bud site is selected (16, 178, 371).
When rvs161 and rvs167 mutant cells were first stained to visualize F-actin it was apparent that the actin cytoskeleton in these cells was abnormal. Actin patches were not as polarized at nascent bud sites and growing buds. Actin cables were more difficult to visualize and appeared less well aligned than those in wild-type cells. When rvs161 or rvs167 mutant cells were exposed to high levels of salt (e.g., NaCl) or starved, the loss of actin cytoskeleton polarity became complete. Under both stress conditions actin cables disappeared completely and actin patches depolarized fully (12, 298).
Delocalized cell wall chitin deposition. In wild-type yeast cells the cell wall polysaccharide chitin is specifically deposited at the site of bud formation during both bud emergence and bud growth, and to seal the scar after separation of the mother cell and the bud. In rvs167 mutant cells, however, staining of chitin with Calcofluor and microscopic examination reveal that chitin is not restricted to sites of active bud formation and at scars from previous budding events. There appears to be an accumulation of chitin distributed evenly throughout the cell wall and this defect is exacerbated by the presence of a sublethal concentration of Na+ in the growth medium (12, 84a).
Loss of bipolar bud site selection. Yeast cells do not bud randomly. After a bud has emerged from a mother cell, a scar made of chitin is left on the surface of both mother (bud scar) and daughter (birth scar). These scars are permanent and can be visualized by use of the fluoresent stain Calcofluor. Bud scars on mother cells accurately record all sites where previous buds have emerged. In wild-type haploid cells, the bud sites are visualized as a cluster at one pole of the cell in an "axial" pattern, which is generated when budding occurs in new mothers at a site adjacent to the birth scar and in old mothers at a site adjacent to the previous bud scar. In diploid cells the bud sites form clusters at both poles of the cell in a "bipolar" pattern, which is generated when budding occurs in new mothers at a site on the opposite pole to the birth scar and in old mothers at a site that alternates between opposite cell poles.
Mutant studies have shown that the requirements for axial and bipolar bud site selection are distinct. Mutations in the genes BUD1, BUD2, and BUD5 cause haploid cells to bud at random sites, while mutations in BUD3 and BUD4 cause haploid cells to bud in a bipolar pattern (29, 30). So some genes are required for axial budding and some for all patterns of budding. Interestingly, RVS161 and RVS167 were among the first genes discovered that are specifically required for bipolar bud site selection. Haploid rvs161 and rvs167 mutant cells bud in an axial pattern like wild-type cells. However, rvs161/rvs161 and rvs167/rvs167 homozygous mutant diploids bud at random sites (12, 53, 65, 298). The defect is not specific to diploid cells, but to the process of bipolar bud site selection. When haploid cells are induced to undergo bipolar budding (e.g., by mutation of BUD3 or BUD4) additional mutation of RVS161 or RVS167 results in random budding (65).
It is possible that spatial landmarks at the cell poles are not formed properly in rvs161 or rvs167 mutant cells. This would mean that when the next division ensues the cell can no longer "remember" where the previous bud formed to place the next bud site appropriately. Alternatively, spatial landmarks may be formed in rvs mutant cells, but not recognized or interpreted correctly. Subsequently, a close correlation has been established between those mutations that affect actin patch polarization and those that abolish bipolar bud site selection (375).
Defective fluid-phase and receptor-mediated endocytosis.
Endocytosis is the process by which cells internalize plasma membrane material as well as ligands, particles, and fluid from the extracellular environment via invagination of the plasma membrane and formation of endocytic vesicles. To identify genes important for endocytosis, Riezman and colleagues conducted a screen for yeast mutants unable to internalize plasma membrane receptor-ligand complexes. A bank of random yeast mutants was screened using an assay for receptor-mediated internalization of -factor, the peptide ligand secreted by haploid yeasts of the mating type. One of several mutants recovered from this screen carried a mutation in ENDocytosis-defective 6 (END6). Isolation and characterization of the END6 gene showed that END6 is identical to RVS161 and that the end6 mutation affects polarization of the actin cytoskeleton as observed for rvs161. A previously isolated rvs167 mutant was also blocked in internalization of -factor. Hence, efficient receptor-mediated endocytosis in budding yeast requires both Rvs161p and Rvs167p. Uptake and accumulation in the vacuole of the membrane-impermeant fluid-phase endocytic dye lucifer yellow are also blocked in both rvs161 and rvs167 mutant cells (215).
Inefficient cell-cell fusion during mating.
Haploid budding yeast cells exist in two mating types, a (MATa) and (MAT), that have an identical physical appearance. MATa cells secrete a peptide pheromone known as a-factor that binds to the a-factor receptor (a G-protein-coupled receptor called Ste3p) expressed only by MAT cells. Conversely, MAT cells secrete a peptide pheromone known as -factor that binds to the -factor receptor (a G-protein-coupled receptor called Ste2p) expressed only by MATa cells. Binding of each pheromone to its receptor activates a mitogen-activated protein kinase signal transduction cascade that has two main readouts. First, cell cycle progression is arrested in G1 to ensure that each mating cell has a normal 1C DNA content in preparation for nuclear fusion. Second, expression of various proteins specifically required for mating is induced, e.g., proteins that promote cell-cell adhesion and fusion (41, 365).
During mating, each haploid cell chooses a mate from the surrounding cells based on the level of pheromone each cell secretes. Yeast cells are able to detect gradients of pheromone with extraordinary sensitivity and form a tube-like projection at their surface known as a mating projection or shmoo. This projection grows up the gradient to the source of pheromone, i.e., towards the cell that produces the highest level. When mating yeast cells are treated with synthetic mating pheromone they mate at random with cells of the opposite mating type even if those cells produce no pheromone. This behavior, known as "default" mating, is approximately 10-fold less efficient than normal pheromone gradient mating. Supersensitive 2 (sst2) mutants are hypersensitive to endogenous pheromone and are consequently unable to accurately detect subtle pheromone gradients. They mate by default with random partners even in the presence of endogenous pheromone gradients (56).
In a search for mutants defective in default mating, candidate mutants were tested for their ability to mate with a "pheromoneless" partner in the presence of synthetic pheromone. One of the mutants tested was rvs161. Earlier work had shown rvs161 sst2 double mutants (which mate by default due to sst2) mate extremely inefficiently. Indeed, the rvs161 mutant was specifically defective in default mating since rvs161 and wild-type cells mated with efficiency similar to that of wild-type cells of the opposite mating type (56).
In an independent study, a screen was performed for mutants defective in mating (using endogenous pheromone) but only when both parents carry the mutation (i.e., bilateral mating defect) (158). One such mutation blocked mating at the stage of cell-cell fusion and was named fusion 7 (fus7) (91). Further work showed fus7 is a mutation in RVS161 (21). The mating defect of rvs161 cells was not apparent in the study of Dorer et al. (56) because only mating of rvs161 cells to wild-type cells was tested (which only detects unilateral mating defects). Interestingly, rvs167 cells do not exhibit a mating defect in either test, one case where rvs161 and rvs167 cells differ in phenotype.
The ability of the various truncated Rvs167p fragments to rescue the loss of actin cables and depolarization of actin patches in rvs167 mutant cells exposed to a sublethal concentration of Na+ was also examined. In general, the same Rvs167p fragments that were functional in growth assays were also able to rescue the actin cytoskeleton defects, i.e., the BAR domain alone was able to correct the actin cytoskeleton defects, but rescue by the BAR domain was not as complete as rescue by full-lengthRvs167p. The fragment comprising the GPA-rich and SH3 domains was not able to correct the actin cytoskeleton defects. Expression of the BAR domain alone restored bipolar bud site selection in 75% of rvs167/rvs167 homozygous diploid cells but full rescue of bipolar budding required full-lengthRvs167p. The GPA-rich and SH3 domain fragment did not rescue bipolar bud site selection (299).
In a later study, various Rvs167p constructs were tested for their ability to restore growth in the presence of Na+, bipolar bud site selection, fluid-phase endocytosis, and sporulation to cells lacking Rvs167p (rvs167). In addition to a truncated Rvs167p construct lacking an SH3 domain (BAR-GPA), this study also employed a full-length Rvs167p construct featuring a P473L substitution in the SH3 domain that abolishes binding to proline-rich motifs. Unlike the BAR-GPA fragment, the full-length P473L mutant construct was expressed at the same steady-state level as full-length Rvs167p and fully rescued all rvs167 defects. This study concluded that Rvs167p is fully functional without a functional SH3 domain if expressed at normal levels.
Do the GPA-rich and SH3 domains contribute to Rvs167p function? The GPA-rich and SH3 fragment (GPA-SH3) did not rescue growth in the presence of Na+, bipolar bud site selection, or endocytosis, but unexpectedly was able to fully rescue the sporulation defect of rvs167. The SH3 domain alone was also able to rescue the sporulation defect, showing that the SH3 domain was sufficient for this function (38).
The BAR domains of Rvs161p and Rvs167p are highly homologous. Sivadon et al. tested the effect of swapping the BAR domains. The Rvs167p GPA-rich and SH3 domains were fused to Rvs161p to create an artificial Rvs167p-like protein (Rvs161p-GPA-SH3). The fragment of Rvs167p comprising the BAR domain only was used as the corresponding Rvs161p-like protein (Rvs167p-BAR). Rvs161p-GPA-SH3 retained full ability to rescue the growth, actin cytoskeleton, and bud site selection defects of rvs161, but did not rescue these defects in rvs167 cells. Rvs167p-BAR was partially functional in rescuing the phenotypes of rvs167 cells, but it lacked the ability to rescue the defects of rvs161 cells. Nor could Rvs167p-BAR and Rvs161p-GPA-SH3 when coexpressed rescue the growth, actin cytoskeleton, or bud site selection defects in rvs161 rvs167 double mutant cells (299). Clearly, the two BAR domains have important differences.
Enforced overexpression of Rvs167p from a strong promoter induces lethality at normal growth temperature. In contrast, overexpression of the GPA-SH3 construct does not affect growth. However, the SH3 domain is important for the overexpression phenotype of full-length Rvs167p. Overexpression of the full-length P473L mutant has milder deleterious effects than wild-type Rvs167p and these only become apparent at elevated temperature (38). The mechanism by which Rvs167p overexpression inhibits growth is not known. Consistent with an important function for the Rvs167p SH3 domain, a recent study identified conditions under which the Rvs167p SH3 domain becomes important for growth (84a).
In growing cells a fusion protein comprising Rvs161p and GFP (Rvs161p-GFP) was reported to exhibit a predominantly diffuse cytoplasmic distribution in cells without buds (Fig. 2A). In cells with small buds Rvs161p-GFP localized to patches at the bud neck, but these patches were no longer apparent in large buds. Expression of Rvs161p-GFP fully rescued the cell fusion defect of an rvs161 mutant (see below), suggesting this fusion protein is functional (at least in cell fusion). This study focused on the role of Rvs161p in cell fusion during mating, so the ability of this fusion protein to rescue other rvs161 defects was not tested. Both N- and C-terminal Rvs161p fusions to GFP exhibited the same subcellular distribution (21). A more recent report demonstrated localization of a similar Rvs161p-GFP fusion protein to numerous small cortical patches (Fig. 2B). These patches exhibited polarization to nascent bud sites and small buds during bud emergence and to the bud neck in dividing cells. However, this particular Rvs161p-GFP fusion protein was not able to rescue the defects of an rvs161 mutant, so it is nonfunctional (10).
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An Rvs167p-GFP fusion protein localizes in vegetatively growing cells to cortical actin patches (Fig. 3). The Rvs167p-GFP fusion protein was able to fully rescue the various defects of rvs167 cells and hence appears to be functional (11). This gives one confidence that this fusion protein displays an authentic Rvs167p subcellular localization. Double labeling of F-actin and Rvs167p-GFP shows that most patches that contain F-actin also contain Rvs167p-GFP and vice versa, although the relative signal intensity of the two proteins varies from one patch to another (11). This difference may reflect the age of the patch, since a number of studies have shown that the protein composition of an individual patch can vary during its lifetime (132, 139, 143, 144).
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Interactions between Rvs167p and actin patch proteins. How does Rvs167p localize to actin patches? As loss of Rvs161p does not perturb actin patch localization of Rvs167p, it seems unlikely that interaction with Rvs161p mediates actin patch localization of Rvs167p (11).
Does Rvs167p associate with actin? In one study a two-hybrid screen was performed to identify actin-interacting proteins and a fragment encoding the Rvs167p SH3 domain was recovered. Hence, there is some evidence Rvs167p associates with actin. The Rvs167p-actin interaction requires the Rvs167p SH3 domain since deletion of the SH3 domain-encoding sequence abolishes the two-hybrid interaction (4). SH3 domains interact with short proline-rich motifs (e.g., PXXP) (331). The actin sequence contains only a single motif (PMNP) that might mediate SH3 domain interaction. Subsequently, it was proposed that the Rvs167p-actin interaction is indirect (38, 176, 180). A third protein might bind actin directly and contain proline-rich motifs that then bind the Rvs167p SH3 domain.
Systematic "charged-to-alanine" scanning mutagenesis of the yeast ACTin gene (ACT1) has been performed (363). Charged residues predicted to be surface exposed and potentially able to engage in interactions with other proteins were replaced singly and in clusters with uncharged alanine residues and a collection of 35 act1 mutations was generated. These act1 mutants vary in phenotype (e.g., some are inviable and others are without obvious phenotype). Two-hybrid analysis using these 35 mutant act1 genes and a panel of actin-interacting proteins revealed that different interactions are affected by different act1 mutations. Unexpectedly, all those act1 mutations that abolish interaction with Rvs167p also abolish interaction with another actin-binding protein, profilin. Conversely, the act1 mutations that do not affect interaction with profilin do not affect interaction with Rvs167p (4). So either Rvs167p binds to actin in the same way as profilin (i.e., makes the same contacts) or Rvs167p binds to profilin and the Rvs167p-actin interaction is indirect and mediated by profilin. To date, interaction between Rvs167p and profilin has not been demonstrated.
One actin patch component that has been proposed to mediate actin-Rvs167p interaction is Abp1p, encoded by the Actin Binding Protein 1 (ABP1) gene. Abp1p was first identified biochemically as a protein that binds with high affinity to F-actin in vitro (60). The Rvs167p SH3 domain recognizes Abp1p in a Far-Western blot and in two-hybrid screens. Consistent with a model that Abp1p contributes to Rvs167p function in vivo, loss of Abp1p results in defects in sporulation, growth in the presence of Na+, and growth on nonfermentable carbon sources, similar to but weaker than defects associated with loss of Rvs167p. Abp1p and Rvs167p also exhibit functional redundancy with the same set of proteins (i.e., with Sla1p, Sla2p, and Sac6p). Moreover, loss of Rvs167p reduces the deleterious effect of Abp1p overexpression on growth and cell morphology, which suggests that the deleterious effect of Abp1p overexpression requires an intact Rvs167p-Abp1p complex (38, 176). A recent study identified conditions under which Rvs167p-Abp1p interaction becomes important for vegetative growth (84a).
Despite the appeal of models in which Abp1p recruits Rvs167p into actin patches via interaction with the Rvs167p SH3 domain, experimental evidence suggests other mechanisms must also exist: deletion of ABP1 (abp1) does not affect Rvs167p localization to actin patches (11), two-hybrid interaction between the Rvs167p SH3 domain and actin does not require Abp1p (180), the act1 two-hybrid data suggest that Rvs167p interaction with actin is mediated by a protein that binds monomeric actin (e.g., profilin) while Abp1p is an F-actin binding protein, localization of Rvs167p to cortical patches does not require F-actin, the SH3 domain of Rvs167p alone does not associate with actin patches in vivo, and the SH3 domain of Rvs167p is not essential for localization to actin patches (11, 38). Abp1p may function in Rvs167p localization to actin patches, but its role may be redundant with that of other proteins (see Table 1 for a list of other cortical actin patch proteins known to interact with Rvs167p).
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Sla1p associates w
