scholarly journals GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

1992 ◽  
Vol 119 (4) ◽  
pp. 749-761 ◽  
Author(s):  
E J Tisdale ◽  
J R Bourne ◽  
R Khosravi-Far ◽  
C J Der ◽  
W E Balch
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Accumulation ◽  
Rab Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Gtp Binding

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

scholarly journals Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport.

1997 ◽  
Vol 8 (7) ◽  
pp. 1305-1316 ◽  
Author(s):  
C Nuoffer ◽  
S K Wu ◽  
C Dascher ◽  
W E Balch
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Rab Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rab Protein ◽  
Guanine Nucleotide Exchange ◽  
Golgi Transport

Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab proteins in the secretory pathway. We have previously shown that Rab1A mutants defective in GTP-binding potently inhibit endoplasmic reticulum to Golgi transport, presumably by sequestering an unknown GEF regulating its function. We now demonstrate that these mutants stably associate with Mss4 both in vivo and in vitro and that Mss4 effectively neutralizes the inhibitory activity of the Rab1A mutants. An equivalent Rab3A mutant (Rab3A[N135I]), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A[N124I] mutant. Although Rab3A[N135I] prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transport, it has no effect on Rab1 function or endoplasmic reticulum to Golgi transport. Furthermore, quantitative immunodepletion of Mss4 fails to inhibit transport in vitro. We conclude that Mss4 and its yeast homologue, Dss4, are not GEFs mediating activation of Rab, but rather, they interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guanine nucleotide-free chaperones (GFCs).

scholarly journals Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

1991 ◽  
Vol 114 (4) ◽  
pp. 671-679 ◽  
Author(s):  
T Oka ◽  
S Nishikawa ◽  
A Nakano
Keyword(s):  
Temperature Sensitivity ◽  
Protein Function ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Transport Reaction ◽  
Golgi Transport ◽  
Gtp Binding

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

scholarly journals Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

2020 ◽  
Author(s):  
Levi J. McClelland ◽  
Kaiming Zhang ◽  
Tung-Chung Mou ◽  
Jake Johnston ◽  
Cindee Yates-Hansen ◽  
...  
Keyword(s):  
G Protein ◽  
Domain Architecture ◽  
Guanine Nucleotide Exchange Factor ◽  
Chaperone Activity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for Gα biogenesis and membrane localization2, 3. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)4. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation5. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to Gα, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gαi1 (isoform 1 of Gα family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of Gα, disrupting all three switch regions that convey Gα nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of Gα. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of Gα that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes Gα-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

scholarly journals Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes.

1992 ◽  
Vol 119 (5) ◽  
pp. 1077-1096 ◽  
Author(s):  
R Schwaninger ◽  
H Plutner ◽  
G M Bokoch ◽  
W E Balch
Keyword(s):  
G Protein ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Protein S ◽  
Rab Proteins ◽  
Heterotrimeric G Protein ◽  
Gtp Binding Proteins ◽  
Initial Event ◽  
Gtp Binding ◽  
Transport Vesicle

Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.

Genetic Interactions in Yeast Between Ypt GTPases and Arf Guanine Nucleotide Exchangers

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1543-1556 ◽  
Author(s):  
Sara Jones ◽  
Gregory Jedd ◽  
Richard A Kahn ◽  
Alex Franzusoff ◽  
Francesca Bartolini ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Genetic Interactions ◽  
Rab Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Domain Family ◽  
Vesicle Budding ◽  
Ability To Act ◽  
Arf Gtpases ◽  
Sec7 Domain

Abstract Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.

scholarly journals Trapp Stimulates Guanine Nucleotide Exchange on Ypt1p

2000 ◽  
Vol 151 (2) ◽  
pp. 289-296 ◽  
Author(s):  
Wei Wang ◽  
Michael Sacher ◽  
Susan Ferro-Novick
Keyword(s):  
Secretory Pathway ◽  
Free Form ◽  
Temperature Sensitive ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Transport Vesicles ◽  
Novel Complex

TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.

scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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