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.

Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein

1994 ◽  
Vol 6 (4) ◽  
pp. 555-565 ◽  
Author(s):  
Thomas Merkle ◽  
Thomas Haizel ◽  
Tomohiro Matsumoto ◽  
Klaus Harter ◽  
Geza Dallmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Yeast Cell ◽  
Fission Yeast ◽  
Binding Protein ◽  
Yeast Cell Cycle ◽  
Gtp Binding Protein ◽  
Regulatory Mutant ◽  
Fission Yeast Cell ◽  
Gtp Binding

scholarly journals The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach

1992 ◽  
Vol 103 (3) ◽  
pp. 785-796 ◽  
Author(s):  
C. Antony ◽  
C. Cibert ◽  
G. Geraud ◽  
A. Santa Maria ◽  
B. Maro ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
G Protein ◽  
Cell Lines ◽  
Binding Protein ◽  
Rab Proteins ◽  
Gtp Binding Protein ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Golgi Network ◽  
Exocytic Pathway

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.

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 Gene for 16S rRNA Methyltransferase (ksgA) Functions as a Multicopy Suppressor for a Cold-Sensitive Mutant of Era, an Essential RAS-Like GTP-Binding Protein in Escherichia coli

1998 ◽  
Vol 180 (19) ◽  
pp. 5243-5246 ◽  
Author(s):  
Qing Lu ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Binding Protein ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Gtp Binding Protein ◽  
Sensitive Mutant ◽  
E Coli ◽  
Gtp Binding ◽  
Cold Sensitive

ABSTRACT Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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