scholarly journals Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.

1994 ◽  
Vol 125 (5) ◽  
pp. 1077-1093 ◽  
Author(s):  
W Yamochi ◽  
K Tanaka ◽  
H Nonaka ◽  
A Maeda ◽  
T Musha ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Binding Protein ◽  
Yeast Cells ◽  
Restrictive Temperature ◽  
Cortical Actin ◽  
Gtp Binding Protein ◽  
Cell Functions ◽  
Gtp Binding

The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.

scholarly journals The Growth-Promoting and Stress Response Activities of the Bacillus subtilis GTP Binding Protein Obg Are Separable by Mutation

2008 ◽  
Vol 190 (20) ◽  
pp. 6625-6635 ◽  
Author(s):  
Shrin Kuo ◽  
Borries Demeler ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Binding Protein ◽  
Normal Growth ◽  
Gtp Binding Protein ◽  
Missense Change ◽  
Gtp Binding ◽  
Growth Promoting ◽  
Carboxy Terminal

ABSTRACT Bacillus subtilis Obg is a ribosome-associating GTP binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (GSR). It is unclear whether the roles of Obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. The present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for Obg in each process. The first allele [obg(G92D)] encodes a missense change in the protein's highly conserved “obg fold” region. This mutation impairs cell growth and the ability of Obg to associate with ribosomes but fails to block sporulation or the induction of the GSR. The second obg mutation [obg(Δ22)] replaces the 22-amino-acid carboxy-terminal sequence of Obg with an alternative 26-amino-acid sequence. This Obg variant cofractionates with ribosomes and allows normal growth but blocks sporulation and impairs the induction of the GSR. Additional experiments revealed that the block on sporulation occurs early, preventing the activation of the essential sporulation transcription factor Spo0A, while inhibition of the GSR appears to involve a failure of the protein cascade that normally activates the GSR to effectively catalyze the reactions needed to activate the GSR transcription factor (σB).

scholarly journals Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

1996 ◽  
Vol 15 (22) ◽  
pp. 6060-6068 ◽  
Author(s):  
H. Kohno ◽  
K. Tanaka ◽  
A. Mino ◽  
M. Umikawa ◽  
H. Imamura ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Putative Target

scholarly journals A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

1991 ◽  
Vol 11 (5) ◽  
pp. 2909-2912 ◽  
Author(s):  
T Sasaki ◽  
K Kaibuchi ◽  
A K Kabcenell ◽  
P J Novick ◽  
Y Takai
Keyword(s):  
Exchange Reaction ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Constitutive Secretion ◽  
Gdp Dissociation Inhibitor ◽  
Exchange Protein

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

scholarly journals ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4396-4403 ◽  
Author(s):  
H Hirano ◽  
K Tanaka ◽  
K Ozaki ◽  
H Imamura ◽  
H Kohno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Protein ◽  
Free Form ◽  
Nucleotide Sequencing ◽  
Temperature Sensitive ◽  
Gtp Binding Protein ◽  
Exchange Factor ◽  
Dominant Negative Mutation ◽  
Gtp Binding ◽  
Novel Protein

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein

1990 ◽  
Vol 10 (8) ◽  
pp. 4116-4122
Author(s):  
Y Matsui ◽  
A Kikuchi ◽  
S Araki ◽  
Y Hata ◽  
J Kondo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Bovine Brain ◽  
Subunit Structure ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Ras P21

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

scholarly journals Role of the Rab GTP-Binding Protein Ypt3 in the Fission Yeast Exocytic Pathway and Its Connection to Calcineurin Function

2002 ◽  
Vol 13 (8) ◽  
pp. 2963-2976 ◽  
Author(s):  
Hong Cheng ◽  
Reiko Sugiura ◽  
Wenlian Wu ◽  
Masaaki Fujita ◽  
Yabin Lu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Binding Protein ◽  
Leader Peptide ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Gtp Binding Protein ◽  
Cytoplasmic Staining ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Exocytic Pathway

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed thatypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1+ leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.

scholarly journals Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

1990 ◽  
Vol 10 (8) ◽  
pp. 4116-4122 ◽  
Author(s):  
Y Matsui ◽  
A Kikuchi ◽  
S Araki ◽  
Y Hata ◽  
J Kondo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Bovine Brain ◽  
Subunit Structure ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Ras P21

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

Purification and Partial Amino Acid Sequences of a Phospholipase CAssociated GTP-Binding Protein from Calf Thymocytes1

1989 ◽  
Vol 105 (3) ◽  
pp. 461-466 ◽  
Author(s):  
Peng Wang ◽  
Jun Nishihata ◽  
Eiji Takabori ◽  
Kazuo Yamamoto ◽  
Satoshi Toyoshima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Amino Acid Sequences ◽  
Gtp Binding Protein ◽  
Gtp Binding

A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein

1991 ◽  
Vol 11 (5) ◽  
pp. 2909-2912
Author(s):  
T Sasaki ◽  
K Kaibuchi ◽  
A K Kabcenell ◽  
P J Novick ◽  
Y Takai
Keyword(s):  
Exchange Reaction ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Constitutive Secretion ◽  
Gdp Dissociation Inhibitor ◽  
Exchange Protein

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

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