scholarly journals The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

1989 ◽  
Vol 109 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
R A Bacon ◽  
A Salminen ◽  
H Ruohola ◽  
P Novick ◽  
S Ferro-Novick
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Transport Defect

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

scholarly journals Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER.

1991 ◽  
Vol 114 (4) ◽  
pp. 663-670 ◽  
Author(s):  
C d'Enfert ◽  
L J Wuestehube ◽  
T Lila ◽  
R Schekman
Keyword(s):  
Membrane Protein ◽  
Protein Transport ◽  
Binding Protein ◽  
Integral Membrane Protein ◽  
Gtp Binding Protein ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Transport Vesicles

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.

scholarly journals Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

2015 ◽  
Vol 112 (28) ◽  
pp. E3679-E3688 ◽  
Author(s):  
Susanne de Boor ◽  
Philipp Knyphausen ◽  
Nora Kuhlmann ◽  
Sarah Wroblowski ◽  
Julian Brenig ◽  
...  
Keyword(s):  
Binding Protein ◽  
Rat Tissues ◽  
Lysine Acetylation ◽  
Nucleotide Exchange ◽  
Gtp Binding Protein ◽  
Cellular Effects ◽  
Gtp Binding ◽  
Cytoplasmic Transport

Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.

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 Reconstitution in vitro of a membrane-fusion event involved in constitutive exocytosis. A role for cytosolic proteins and a GTP-binding protein, but not for Ca2+

1992 ◽  
Vol 285 (2) ◽  
pp. 383-385 ◽  
Author(s):  
J M Edwardson ◽  
P U Daniels-Holgate
Keyword(s):  
Membrane Fusion ◽  
Binding Protein ◽  
Fusion Event ◽  
Gtp Binding Protein ◽  
In Vitro System ◽  
Cytosolic Proteins ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Transport Vesicles

The fusion of post-Golgi transport vesicles with the plasma membrane is perhaps the least well understood step in the network of intracellular membrane traffic. We have used an ‘in vitro’ system to study this membrane-fusion event. We show here that fusion requires the presence of cytosolic proteins, but not Ca2+, and is inhibited by the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate, which indicates the involvement of a GTP-binding protein.

Function of Tubulin Binding Proteins in Vivo

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 69-80 ◽  
Author(s):  
James A Fleming ◽  
Leticia R Vega ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gene Products ◽  
Salvage Pathway ◽  
Tubulin Binding ◽  
Mutant Cells ◽  
Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

scholarly journals Arf6 is necessary for high level Wingless signalling duringDrosophilawing development

2021 ◽  
Author(s):  
Julien Marcetteau ◽  
Tamàs Matusek ◽  
Frédéric Luton ◽  
Pascal P. Thérond
Keyword(s):  
Signal Transduction ◽  
Adherens Junctions ◽  
Developmental Model ◽  
Wing Margin ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Wide Range ◽  
High Level

AbstractWnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa.In vitrostudies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findingsin vivo, we analyse the consequence of the absence of Arf6 activity onDrosophilawing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

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 The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast.

1989 ◽  
Vol 109 (3) ◽  
pp. 1023-1036 ◽  
Author(s):  
A Salminen ◽  
P J Novick
Keyword(s):  
Secretory Pathway ◽  
Microsomal Fraction ◽  
Binding Protein ◽  
Protein Product ◽  
Loss Of Function ◽  
Partial Loss ◽  
Gtp Binding Protein ◽  
Gene Encoding ◽  
Vesicular Traffic ◽  
Gtp Binding

SEC15 function is required at a late stage of the yeast secretory pathway. Duplication of the gene encoding the ras-like, GTP-binding protein, Sec4, can suppress the partial loss of function resulting from the sec15-l mutation, but cannot suppress disruption of sec15. Analysis of the SEC15 gene predicts a hydrophilic protein product of 105 kD. Anti-Sec15 antibody recognizes a protein of 116-kD apparent molecular mass which is associated with a microsomal fraction of yeast in a strongly pH dependent fashion. Overproduction of Sec15 protein interferes with the secretory pathway, resulting in the formation of a cluster of secretory vesicles, and a patch of Sec15 protein revealed by immunofluorescence. The sec4-8 and sec2-4l mutations, but not mutations in other SEC genes, prevent formation of the Sec15 protein patch. We propose that Sec15 protein responds to the function of the Sec4 protein to control vesicular traffic.

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