A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton

Bon, Elisabeth; Recordon-Navarro, Patricia; Durrens, Pascal; Iwase, Masayuki; Toh‐e, Akio; Aigle, Michel

doi:10.1002/1097-0061(20000930)16:13<1229::aid-yea618>3.0.co;2-q

Cited by 42 publications

(49 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This indicates that the C-terminal sequences with the potential to form coiled-coils might fulfill a noncatalytic function in concert with sequences of the N-terminal half and affect proper localization and/or catalytic activity. Further functional analysis of the sequences outside the catalytic domain of Gyp5p would also be of interest, since previous studies suggested that the protein product of YPL249c (that we here identify to be the Ypt1-GAP Gyp5p) appears to interact with Rvs167p, a protein apparently involved in actin cytoskeleton function (39,40). In the case of Gyp1p of which a significant fraction is already bound to membranes, supposedly including the Golgi, the region N-terminal of the catalytic domain of Gyp1p is not re- quired to rescue, in synthetic media, a ⌬gyp1 deletion strain from growth inhibition at 37°C (34).…”

Section: Discussionmentioning

confidence: 95%

Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs

A¹,

Schmitzová

Trepte

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 ؋ 10 4 -fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 ؋ 10 4 -fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.

show abstract

Section: Discussionmentioning

confidence: 95%

Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs

A¹,

Schmitzová

Trepte

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Genes whose products are involved in cell wall formation, maintenance, and regulation, including members of the PKC͞ mitogen-activated protein (MAP) kinase cell integrity sensing pathway, constitute the largest of the Pho85 synthetic lethal gene classifications. Of the 24 direct substrates (Table 1), 7 have known roles in cell wall formation, maintenance, and regulation (36)(37)(38)(39)(40) and͞or interact with elements of the PKC͞MAP kinase pathway (41)(42)(43)(44)(45)(46) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Combining chemical genetics and proteomics to identify protein kinase substrates

Dephoure

Howson²,

Blethrow³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

View full text Add to dashboard Cite

Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.cell signaling ͉ phosphorylation ͉ cyclin-dependent kinase T he general criteria for establishing that a protein is a substrate of a given kinase are the ability of the kinase to phosphorylate the substrate in vitro and the dependence on the activity of the kinase for phosphorylation of the substrate in vivo (1). In vivo validation of kinase substrates continues to be laborious and time-consuming. Thus, it is crucial to be able to efficiently identify candidates before committing to this step. Indirect data, such as genetic and physical interactions, can provide insights into potential substrates, but many interacting proteins and genes are not substrates (1, 2). More direct approaches using in vitro kinase reactions have been attempted in many permutations (1, 2). Purified component reactions directly measure the ability of a kinase to phosphorylate a particular substrate and can be scaled for high-throughput formats, but such conditions often compromise reaction specificity and produce false-positive results (1, 2).We sought to improve on these techniques by carrying out a biochemical screen in an environment that more closely resembles the in vivo state. To do so, we carried out kinase reactions with near physiological levels of exogenous kinase in native whole-cell extracts. These extracts maintain protein-protein interactions that may affect substrate presentation and preserve a nearly full complement of cellular proteins, including potential adaptor proteins and cofactors that could affect the reaction. These reaction conditions also preserve the native relative protein abundances and a natural competition among substrates for limited kinase.A challenge of carrying out protein kinase reactions in wholecell lysates is that one cannot attribute phosphorylation of a protein to a particular kinase in the reaction, because all kinases in the extract are capable of cat...

show abstract

“…These data suggest that previous two-hybrid interactions between the BAR domain of Rvs167p and fulllength Rvs167p may have been indirect or an artifact of BAR domain overproduction. In support of this, Bon et al (2000) saw a two-hybrid interaction between Rvs167p and Rvs167p when both genes were on high-copy plasmids but not when they were on low-copy plasmids.We also tested whether Rvs161p could homodimerize. Because Rvs161p and Rvs167p are the only BAR-domaincontaining proteins in yeast and because Rvs161p is required for fusion during mating and Rvs167p is not (Brizzio et al, 1998), we were especially interested in whether Rvs161p would form homodimers in cells during the mating response.…”

mentioning

confidence: 80%

Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

Friesen

Humphries

et al. 2006

MBoC

View full text Add to dashboard Cite

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response. INTRODUCTIONRVS161 and RVS167, which encode closely related proteins in Saccharomyces cerevisiae, were first identified in a screen for mutants that exhibited reduced viability upon starvation (Bauer et al., 1993). Mutation of RVS161 or RVS167 causes a phenotype consistent with a role for the Rvs proteins in cortical actin cytoskeleton organization and endocytosis: loss of viability and unusual cell morphology in poor growth medium or salt-containing medium, delocalized actin distribution under suboptimal growth conditions, abnormal (random) budding in diploids, and defects in endocytosis and sporulation (Bauer et al., 1993). Ultrastructural studies have revealed that rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction (Breton et al., 2001), suggesting an additional role for Rvs161p and Rvs167p in vesicle trafficking.Rvs161p and Rvs167p are members of the BAR-domain family of proteins, which includes Bin1, Amphiphysin, and Rvs proteins (Sivadon et al., 1997). Amphiphysins are enriched in the mammalian brain and seem to function in synaptic vesicle endocytosis (for review, see Zhang and Zelhof, 2002). Bin1 is a splice isoform of amphiphysin 2 that has features of a tumor suppressor (Sakamuro et al., 1996). Proteins in this family are characterized by the presence of a conserved BAR domain, and it is through their BAR domains that Rvs161p interacts with Rvs167p (Navarro et al., 1997;Sivadon et al., 1997;Colwill et al., 1999). The crystal structure of the BAR domain of Drosophila amphiphysin has recently been solved (Peter et al., 2004). It is a crescentshaped dimer, in which each monomer forms a coiled coil. The curved shape is only revealed upon dimerization of the two slightly kinked ...

show abstract

A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton

Cited by 42 publications

References 75 publications

Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs

Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs

Combining chemical genetics and proteomics to identify protein kinase substrates

Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

Contact Info

Product

Resources

About