scholarly journals A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments.

1994 ◽  
Vol 125 (2) ◽  
pp. 225-237 ◽  
Author(s):  
C Nuoffer ◽  
H W Davidson ◽  
J Matteson ◽  
J Meinkoth ◽  
W E Balch
Keyword(s):  
Secretory Pathway ◽  
Bound State ◽  
Competitive Inhibitor ◽  
Small Gtpase ◽  
Protein Export ◽  
Nucleotide Exchange ◽  
Vesicle Budding ◽  
Vesicular Traffic

Rab1 is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rab1 we have analyzed the function of a mutant (rab1a[S25N]) containing a substitution which perturbs Mg2+ coordination and reduces the affinity for GTP, resulting in a form which is likely to be restricted to the GDP-bound state. The rab1a(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rab1 during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rab1 function. Both rab1a and rab1b (92% identity) were able to antagonize the inhibitory activity of the rab1a(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rab1 mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rab1 function in vesicular traffic.

scholarly journals Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

2006 ◽  
Vol 81 (2) ◽  
pp. 558-567 ◽  
Author(s):  
George A. Belov ◽  
Nihal Altan-Bonnet ◽  
Gennadiy Kovtunovych ◽  
Catherine L. Jackson ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Brefeldin A ◽  
Rna Replication ◽  
Bound State ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Virus Growth ◽  
Guanine Nucleotide Exchange ◽  
Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

scholarly journals Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

2003 ◽  
Vol 161 (5) ◽  
pp. 889-897 ◽  
Author(s):  
Satoshi Yoshida ◽  
Ryuji Ichihashi ◽  
Akio Toh-e
Keyword(s):  
Small Gtpase ◽  
Mitotic Exit ◽  
Effector Protein ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Downstream Effector ◽  
Nucleotide Exchange Factor ◽  
Family Protein

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.

scholarly journals Arf3p GTPase is a key regulator of Bud2p activation for invasive growth in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (15) ◽  
pp. 2328-2339 ◽  
Author(s):  
Jia-Wei Hsu ◽  
Fang-Jen S. Lee
Keyword(s):  
Bound State ◽  
Invasive Growth ◽  
Nucleotide Exchange ◽  
General Applicability ◽  
Glucose Depletion ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Adp Ribosylation Factor

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. The regulatory mechanisms leading to Bud2p activation, however, are poorly understood. In this study, we report that ADP-ribosylation factor 3p (Arf3p) acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3Δ or bud2Δ cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p–Bud2p interaction is important for invasive growth and facilitates the Bud2p–Bud1p association in vivo. Finally, we show that under glucose depletion–induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state, and the activation is independent of Arf3p guanine nucleotide-exchange factor Yel1p. Thus we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion–induced invasive growth.

scholarly journals Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 91-91
Author(s):  
Wolfgang Bergmeier ◽  
David S Paul ◽  
Lucia Stefanini ◽  
Raymond F. Robledo ◽  
E. Ricky Chan ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
P2y12 Inhibitors ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments.

1994 ◽  
Vol 125 (1) ◽  
pp. 51-65 ◽  
Author(s):  
O Kuge ◽  
C Dascher ◽  
L Orci ◽  
T Rowe ◽  
M Amherdt ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Transitional Region ◽  
Nucleotide Exchange ◽  
New Members ◽  
Vesicle Budding ◽  
Guanine Nucleotide Exchange ◽  
Vesicular Carriers

Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transport. The requirement for novel components at different stages of the secretory pathway may reflect the recently recognized differences in protein transport between the Golgi stacks as opposed to the selective sorting and concentration of protein during export from the ER.

scholarly journals Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack.

1994 ◽  
Vol 126 (6) ◽  
pp. 1393-1406 ◽  
Author(s):  
F Peter ◽  
C Nuoffer ◽  
S N Pind ◽  
W E Balch
Keyword(s):  
Secretory Pathway ◽  
Gel Filtration ◽  
Small Gtpase ◽  
Rab Proteins ◽  
Guanine Nucleotide ◽  
Direct Role ◽  
Golgi Stack ◽  
Vesicular Traffic ◽  
Guanine Nucleotide Dissociation Inhibitor

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.

scholarly journals Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

2020 ◽  
Author(s):  
Lukas Weiß ◽  
Tina Reiner ◽  
Julia Mergner ◽  
Bernhard Kuster ◽  
Attila Fehér ◽  
...  
Keyword(s):  
Amino Acid ◽  
Posttranslational Modification ◽  
Individual Case ◽  
Bound State ◽  
Amino Acid Residues ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Developmental Defects

AbstractSmall RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major pathways regulate G-protein activity: canonical switching of the nucleotide bound state and posttranslational modification (PTM). PTMs can support or suppress RHO signaling, depending on each individual case. In plants, regulation of Rho of plants (ROPs) has been shown to act through nucleotide exchange and hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP Binding Kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is present in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa. Since this highly conserved amino acid residue is likewise ubiquitinated in mammalian Rac1 and Rac3, we suggest that RHO family proteins from different kingdoms could be generally regulated by ubiquitination of this site.

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).

Overexpression of the ARF1 exchange factor ARNO inhibits the early secretory pathway and causes the disassembly of the Golgi complex

1998 ◽  
Vol 111 (22) ◽  
pp. 3427-3436 ◽  
Author(s):  
S. Monier ◽  
P. Chardin ◽  
S. Robineau ◽  
B. Goud
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Cell Fractionation ◽  
Exchange Factor ◽  
Early Secretory Pathway ◽  
Membrane Budding

The small GTPase ARF1 is a key regulator of intracellular membrane traffic. In its active, GTP-bound form, ARF1 is associated with Golgi membranes and promotes the recruitment of the cytosolic coat protein complex, which will result in membrane budding and vesicle formation. ARNO (ARF nucleotide site opener) has been shown to act in vitro as a GTP exchange factor for ARF1. Here, we have investigated the function of ARNO in vivo. By immunofluorescence and cell fractionation, ARNO was found to be mostly cytosolic in HeLa cells. Its overexpression led to a strong inhibition of the secretion of SEAP (secreted form of alkaline phosphatase). Newly synthesized SEAP failed to acquire endoglycosidase H resistance, indicating a block in the early secretory pathway. This effect on secretion was accompanied by a disassembly of the Golgi complex and a redistribution of Golgi resident proteins into the endoplasmic reticulum (ER). On the other hand, ARNO overexpression did not affect the early endocytic pathway. These results show that ARNO functions in vivo in Golgi to ER transport. Its behavior is then consistent with ARNO being an exchange factor for ARF1.

scholarly journals Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

2018 ◽  
Vol 115 (49) ◽  
pp. E11551-E11560 ◽  
Author(s):  
Qiong Wang ◽  
Yuying Li ◽  
Kazuya Ishikawa ◽  
Ken-ichi Kosami ◽  
Kazumi Uno ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Domain Architecture ◽  
Nucleotide Binding ◽  
Small Gtpase ◽  
Leucine Rich Repeat ◽  
Nucleotide Exchange ◽  
Downstream Signaling

Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.

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