Yeast Amphiphysin

Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. rvs167 shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism (Bauer, 1993).

The actin cytoskeleton cells are altered in rvs161 mutant yeast, with the defect becoming more pronounced under unfavorable growth conditions, as described for the rvs167 mutant. The cytoskeletal alteration has no apparent effect on invertase secretion and polarized growth. Mutations in RVS161, just as in RVS167, lead to a random budding pattern in a/alpha diploid cells. This behavior is not observed in a/a diploid cells homozygous for the rvs161-1 or rvs167-1 mutations. In addition, sequence comparisons reveals that amphiphysin, a protein first found in synaptic vesicles of chicken and shown to be the autoantigen of Stiff Man syndrome, presents similarity with both Rvs proteins. Furthermore, limited similarities with myosin heavy chain and tropomyosin alpha chain from higher eukaryotic cells allow for the definition of a possible consensus sequence. The finding of related sequences suggests the existence of a function for these proteins that is conserved among eukaryotic organisms (Sivadon, 1995).

Mutations in RVS161 and RVS167 yeast genes induce identical phenotypes associated to actin cytoskeleton disorders. The whole Rvs161 protein is similar to the amino-terminal part of Rvs167p, thus defining a RVS domain. In addition to this domain, Rvs167p contains a central glycine-proline-alanine rich domain and a SH3 domain. To assess the function of these different domains recombinant Rvs proteins were expressed in rvs mutant strains. Phenotype analysis has shown that the RVS and SH3 domains are necessary for phenotypical complementation, whereas the GPA domain is not. Moreover, the RVS domains from Rvs161p and Rvs167p have distinct roles, and the SH3 domain needs the specific RVS domain of Rvs167p to function. These results suggest that Rvs161p and Rvs167p play distinct roles, while acting together in a common function (Sivadon, 1997).

In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. Evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that binds in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. A site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic 'synthetic lethal' interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations that specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. It is concluded that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. This work illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions (Lila, 1997).

The yeast rvs mutants display phenotypes close to those described for the actin mutants: disorganization of the actin cytoskeleton, random budding of the diploids, loss of polarity and sensitivity to salt. Mutations in the RVS genes lead to synthetic lethality with a set of mutations in the actin gene, ACT1. This synthetic lethality is allele-specific regarding the act1 mutations, pointing to a region on the actin molecule where contacts with the myosin head have been described. The possible involvement of a myosin in a vital function fulfilled both by the Rvsp proteins and actin is strengthened further by the fact that the double mutants rvs167;myo1 and rvs167;myo2 are, respectively, lethal and severely affected in growth. These data support the idea that actin, myosin and Rvsp proteins are linked in a common functional pathway in yeast (Breton, 1998).

The yeast amphiphysin-like protein Rvs167p is localized mainly in small cortical patches throughout the cell in unbudding cells. During budding, the patches are polarized at the bud emergence site. During mating, Rvs167p is concentrated at the tip of the shmoo. Rvs167p colocalizes with actin patches during yeast vegetative growth and mating. Complete disruption of the actin cytoskeleton using Latrunculin-A does not affect Rvs167p localization in patches throughout the cell. In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected. These observations suggest that Rvs167p may localize the actin cortical complex properly. Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, is required but not sufficient for the correct localization of the protein (Balguerie, 1999).

The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologs of higher eukaryotes' amphiphysins, associate with actin and appear to be involved in several functions related to the actin cytoskeleton. In order to identify partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors were screened using either Rvs167p or Rvs161p as a bait. The selected candidates, representing 34 ORFs, were then tested against both Rvsp proteins, as well as domains of Rvs167p or Rvs161p. Among the most significant candidates, 24 ORFs were specific prey of Rvs167p only and two gave interactions with Rvs161p only. Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167, LAS17, YNL094w, YMR192w and YPL249c). Analysis of putative functions of the candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but also opens possible new fields, such as nuclear functions (Bon, 2000).

The Rvs161 and Rvs167 proteins are known to play a role in actin cytokeleton organization and endocytosis. Moreover, Rvs167p functionally interacts with the myosin Myo2p. The involvement of the Rvs proteins in vesicle traffic and in cell integrity was explored. The rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction. They are synthetic-lethal with the slt2/mpk1 mutation, which affects the MAP kinase cascade controlled by Pkc1p and is required for cell integrity. The phenotype of the double mutants is close to that described for the pkc1 mutant. Synthetic defects for growth are also observed with mutation in KRE6, a gene coding for a glucan synthase, required for cell wall construction. These data support the idea that the Rvs proteins are involved in the late targeting of vesicles whose cargoes are required for cell wall construction (Breton, 2001).

Identification of mammalian Amphiphysins

To obtain access to novel proteins of the neuronal synapse, antisera have been raised against proteins of synaptic plasma membranes and used for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but is not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which the name amphiphysin is proposed (Lichte, 1992).

Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. This study reports characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brain-specific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis (Ramjaun, 1997).

Mammalian Amphiphysin I functions in synaptic vesicle endocytosis

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. A specific, SH3 domain-mediated, interaction between amphiphysin and dynamin has been demonstrated by gel overlay and affinity chromatography. In addition, the two proteins are colocalized in nerve terminals and coprecipitate from brain extracts consistent with their interactions in situ. A region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis (David, 1996).

Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. The SH3 domain of amphiphysin, but not a mutant SH3 domain, binds with high affinity to a single site in the long proline-rich region of human dynamin I -- this site is distinct from the binding sites for other SH3 domains, and the mutation of two adjacent amino acids in dynamin I is sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). These data demonstrate that the long proline-rich stretch present in dynamin I contains multiple SH3 domain binding sites that recognize interacting proteins with high specificity (Grabs, 1997).

The role of amphiphysin in receptor-mediated endocytosis in vivo has been examined. To address the importance of the amphiphysin SH3 domain in dynamin recruitment, a transferrin and epidermal growth factor (EGF) uptake assay was carried out in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence revealed that cells transfected with the amphiphysin SH3 domain show a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all fail to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin can associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis. These results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. It is proposed that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis (Wigge, 1997).

Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Using a cell-free system, direct morphological evidence has been provided in support of this hypothesis. Unexpectedly, it was also found that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis (Takei, 1999).

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. This study defines amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin results in a segregation of clathrin, which acquires a diffuse distribution, from AP-2, which accumulates at patches also positive for Eps15. These effects correlate with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produces a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 does not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin (Slepnev, 2000).

Amphiphysin 1 is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. Mammalian amphiphysin 1 interacts with the cdk5-activating subunit p35 and this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells (Floyd, 2001).

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin has been supported by dominant-negative interference studies. amphiphysin 1 knockout mice have been generated; lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation. These defects suggest impairments at multiple stages of synaptic vesicle recycling. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions (Di Paolo, 2002).

At the molecular level, a striking consequence of the lack of amphiphysin 1 expression in brain is the nearly complete disappearance of amphiphysin 2. Since levels of amphiphysin 2 mRNA are unchanged in the brains of mutant mice, this phenomenon is likely to result from decreased stability of amphiphysin 2 and is consistent with the reported occurrence of amphiphysin 1 and 2 primarily as stable heterodimers in brain. No downregulation of amphiphysin 2 occurs in skeletal muscle where this protein is normally expressed at very high levels without amphiphysin 1. It remains to be determined whether the stability of this skeletal muscle isoform, which differs from the major brain amphiphysin 2 isoform, is accounted for by its greater intrinsic resistance to proteolytic degradation or different proteolytic machinery in muscle, by heterodimerization with another BAR domain-containing protein, or by its localization in a subcellular compartment which protects it from high turnover (Di Paolo, 2002).

These results strongly support the hypothesis that one of the functions of brain amphiphysin is to act as a multifunctional adaptor connecting various cytosolic components of the endocytic machinery to each other and to the lipid bilayer. They suggest that amphiphysin may contribute to the recruitment of clathrin, AP-2, and synaptojanin 1 to the membrane. Moreover, although binding of dynamin to lipid bilayers is not dependent upon amphiphysin under the in vitro conditions of this study, affinity purification experiments on the proline-rich tail of dynamin demonstrate that amphiphysin can indeed act as a bridge between clathrin coat components and dynamin. Thus, amphiphysin may help to coordinate the function of all these proteins at the membrane. Furthermore, the direct binding of dynamin to membrane in situ may undergo regulation, and a contribution of amphiphysin to dynamin recruitment in vivo cannot be ruled out (Di Paolo, 2002).

Mammalian Amphiphysin II is implicated in the function of the actin cytoskeleton

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homolog of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. This study describes the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasma membrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function (Butler, 1997).

The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, the crystal structure of the amphiphysin-2 (Amph2) SH3 domain has been resolved to 2.2 Å. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. The interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. These data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis (Owen, 1998).

p73 is a nuclear protein that is similar in structure and function to p53. Notably, the C-terminal region of p73 has a regulatory function, through interactions with a positive or negative regulator. The yeast two-hybrid technique has been used to identify a novel p73beta binding protein, designated amphiphysin IIb-1. Amphiphysin IIb-1 is one of the splicing variants of amphiphysin II, and has a shorter protein product than amphiphysin IIb, which has been previously reported. Amphiphysin IIb-1 binds full-length p73beta, both in vitro and in vivo. This association is mediated via the SH3 domain of amphiphysin IIb-1 and C-terminal amino acids 321-376 of p73beta. Double immunofluorescence patterns reveal that p73beta is relocalized to the cytoplasm in the presence of amphiphysin IIb-1. Overexpression of amphiphysin IIb-1 significantly inhibits the transcriptional activity of p73beta in a dose-dependent manner. In addition, the cell death function of p73beta is inhibited by amphiphysin IIb-1. These findings offer a new insight into the regulation mechanism of p73beta, and suggest that amphiphysin IIb-1 modulates p73beta function by direct binding (Kim, 2001).

Amphiphysin : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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