Multicopy suppression screen in a Saccharomyces cerevisiae strain lacking the Rab GTPase-activating protein Msb3p

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

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Delineating the folding perturbations and molecular mechanisms of Thr-Ala 642 mutation in Rab-GTPase activating protein Akt substrate of 160kDa and its impact on the aetiology of diabetes

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2020.1726814 ◽

2020 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Utkarsh Nagpal ◽

D. Kamalesh ◽

P. Raghuraman ◽

Sriroopreddy Ramireddy ◽

C. Sudandiradoss

Keyword(s):

Molecular Mechanisms ◽

Rab Gtpase ◽

Gtpase Activating Protein

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AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain

Biochemical Journal ◽

10.1042/bj20050887 ◽

2005 ◽

Vol 391 (1) ◽

pp. 87-93 ◽

Cited By ~ 273

Author(s):

Cristinel P. Mîinea ◽

Hiroyuki Sano ◽

Susan Kane ◽

Eiko Sano ◽

Mitsunori Fukuda ◽

...

Keyword(s):

Glucose Transporter ◽

Protein Domain ◽

Rab Gtpase ◽

Significant Activity ◽

Glut4 Translocation ◽

Gtpase Activating Protein ◽

Akt Phosphorylation ◽

Intracellular Vesicles ◽

Glucose Transporter Glut4 ◽

Protein Kinase Akt

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

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Cell cycle–dependent phosphorylation of Sec4p controls membrane deposition during cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201602038 ◽

2016 ◽

Vol 214 (6) ◽

pp. 691-703 ◽

Cited By ~ 9

Author(s):

Dante Lepore ◽

Olya Spassibojko ◽

Gabrielle Pinto ◽

Ruth N. Collins

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Membrane Trafficking ◽

Intracellular Trafficking ◽

Rab Gtpase ◽

Rab Gtpases ◽

Positive Regulator ◽

Physiological Relevance ◽

A Cell ◽

Cycle Dependent

Intracellular trafficking is an essential and conserved eukaryotic process. Rab GTPases are a family of proteins that regulate and provide specificity for discrete membrane trafficking steps by harnessing a nucleotide-bound cycle. Global proteomic screens have revealed many Rab GTPases as phosphoproteins, but the effects of this modification are not well understood. Using the Saccharomyces cerevisiae Rab GTPase Sec4p as a model, we have found that phosphorylation negatively regulates Sec4p function by disrupting the interaction with the exocyst complex via Sec15p. We demonstrate that phosphorylation of Sec4p is a cell cycle–dependent process associated with cytokinesis. Through a genomic kinase screen, we have also identified the polo-like kinase Cdc5p as a positive regulator of Sec4p phosphorylation. Sec4p spatially and temporally localizes with Cdc5p exclusively when Sec4p phosphorylation levels peak during the cell cycle, indicating Sec4p is a direct Cdc5p substrate. Our data suggest the physiological relevance of Sec4p phosphorylation is to facilitate the coordination of membrane-trafficking events during cytokinesis.

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IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4303 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4303-4313 ◽

Cited By ~ 211

Author(s):

K Tanaka ◽

M Nakafuku ◽

F Tamanoi ◽

Y Kaziro ◽

K Matsumoto ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Cyclic Amp ◽

Negative Regulator ◽

Gtpase Activating Protein ◽

Ras Gtpase ◽

Shock Sensitivity ◽

Dna Libraries ◽

Increased Sensitivity ◽

Multicopy Vector

The IRA1 gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the ira1 mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA1 protein. The region homologous between the IRA1 protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the arg1 locus on the left arm of chromosome XV and was found to be allelic to glc4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (iii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRA1 and IRA2 indicated that IRA1 and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.

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Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0062 ◽

2015 ◽

Vol 26 (13) ◽

pp. 2535-2549 ◽

Cited By ~ 21

Author(s):

Meenakshi Rana ◽

Jens Lachmann ◽

Christian Ungermann

Keyword(s):

Endoplasmic Reticulum ◽

Endocytic Pathway ◽

Rab Gtpase ◽

Guanine Nucleotide ◽

Membrane Recycling ◽

Nucleotide Exchange ◽

Gtpase Activating Protein ◽

Guanine Nucleotide Exchange ◽

Vacuole Fusion ◽

Nucleotide Exchange Factor

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.

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