The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae (original) (raw)
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Characterization of an ADP-ribosylation Factor-like 1 Protein in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997
ADP-ribosylation factors (ARFs) are highly conserved ϳ20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Several ARF-like proteins (ARLs) have been cloned from Drosophila, rat, and human; however, the biological functions of ARLs are unknown. We have identified a yeast gene (ARL1) encoding a protein that is structurally related (>60% identical) to human, rat, and Drosophila ARL1. Biochemical analyses of purified recombinant yeast ARL1 (yARL1) protein revealed properties similar to those ARF and ARL1 proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL1 protein did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. yARL1 was not recognized by antibodies against mammalian ARLs or yeast ARFs. Anti-yARL1 antibodies did not cross-react with yeast ARFs, but did react with human ARLs. On subcellular fractionation, yARL1, similar to yARF1, was localized to the soluble fraction. The amino terminus of yARL1, like that of ARF, was myristoylated. Unlike Drosophila Arl1, yeast ARL1 was not essential for cell viability. Like rat ARL1, yARL1 might be associated in part with the Golgi complex. However, yARL1 was not required for endoplasmic reticulum-to-Golgi protein transport, and it may offer an opportunity to define an ARL function in another kind of vesicular trafficking, such as the regulated secretory pathway. ADP-ribosylation factors (ARFs) 1 are members of the Ras superfamily of ϳ20-kDa guanine nucleotide-binding proteins,
Eukaryotic Cell, 2012
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 sp...
Saccharomyces cerevisiae Gcs1 is an ADP-Ribosylation Factor GTPase-Activating Protein
Proceedings of The National Academy of Sciences, 1996
Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011
High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferated structures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expression enhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ER and Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealed that it depended on an intact unfolded protein response, because their assembly was reported to be lethal to yeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affected global ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybrid composition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normally cycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling of novel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, which regulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteins biosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response overexpression of a GPI-anchor remodelase in S. cerevisiae.
Characterization of a Novel ADP-ribosylation Factor-like Protein (yARL3) in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1999
ADP-ribosylation factors (ARFs) are highly conserved, ϳ20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several cDNAs for ARF-like proteins (ARLs) have been cloned from human, Drosophila, rat, and yeast, although the biological function(s) of ARLs is unknown. We have identified a yeast gene (yARL3) encoding a protein that is structurally related (>43% identical) to the mammalian ARF-like protein ARP. Biochemical studies of purified recombinant yARL3 protein revealed properties similar to those of ARF and ARL proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL3 did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. Anti-yARL3 antibodies did not cross-react with yARFs or yARL1. yARL3 was not essential for cell viability, but disruption of yARL3 resulted in cold-sensitive cell growth. At the nonpermissive temperature, processing of alkaline phosphatase and carboxypeptidase Y in arl3 mutant was slowed. yARL3 might be required for protein transport from endoplasmic reticulum to Golgi or from Golgi to vacuole at nonpermissive temperatures. On subcellular fractionation, unlike its mammalian homologue ARP, yARL3 was detected in the soluble fraction but not in the plasma membrane. Indirect immunofluorescence analysis revealed that yARL3 when overexpressed was associated in part with the endoplasmic reticulum-nuclear envelope. Thus, the structural and functional characteristics of yARL3 indicate that it may have a unique role(s) in vesicular trafficking. ADP-ribosylation factors (ARFs) 1 are a family of ϳ20-kDa GTP-binding proteins (or GTPases) that includes both ARFs and ARF-like proteins (ARLs) (for recent reviews, see Refs. 1 and 2). ARFs were originally identified and purified on the
The Journal of Cell Biology, 1991
Saccharomyces cerevisiae sec7 mutants exhibit pleiotropic deficiencies in the transit of proteins through the Golgi apparatus, and elaborate an array of Golgi apparatus-like cisternae at a restrictive growth temperature (37 degrees C). The SEC7 gene encodes an essential high-molecular weight protein (227 kD) that is phosphorylated in vivo. In cell lysates, Sec7 protein (Sec7p) is recovered in both sedimentable and soluble fractions. A punctate immunofluorescent pattern of Sec7p-associated structures seen in SEC cells coalesces in sec14 mutant yeast that accumulate exaggerated Golgi cisternae at 37 degrees C. Sec7p may function as a peripheral membrane protein that cycles between a soluble, cytosolic pool and a sedimentable, membrane-associated complex for its essential role in vesicular traffic through the Golgi apparatus. The transmembrane Kex2 protease, which processes precursors of secreted peptides within the yeast secretory pathway, is also localized by indirect immunofluoresce...
The EMBO Journal
Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.