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 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 The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

scholarly journals mTrs130 Is a Component of a Mammalian TRAPPII Complex, a Rab1 GEF That Binds to COPI-coated Vesicles

2009 ◽  
Vol 20 (19) ◽  
pp. 4205-4215 ◽  
Author(s):  
Akinori Yamasaki ◽  
Shekar Menon ◽  
Sidney Yu ◽  
Jemima Barrowman ◽  
Timo Meerloo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Golgi Compartment ◽  
A Subunit ◽  
Protein Particle ◽  
First Time

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.

scholarly journals Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Divyanshu Mahajan ◽  
Boon Kim Boh ◽  
Yan Zhou ◽  
Li Chen ◽  
Tobias Carl Cornvik ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Active Form ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
And Function ◽  
Golgi Network

Abstract Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

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.

Identification and characterisation of novel Mss4-binding Rab GTPases

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Viktor Wixler ◽  
Ludmilla Wixler ◽  
Anika Altenfeld ◽  
Stephan Ludwig ◽  
Roger S. Goody ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Binding Studies ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

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 The Fission Yeast spo14 + Gene Encoding a Functional Homologue of Budding Yeast Sec12 Is Required for the Development of Forespore Membranes

2003 ◽  
Vol 14 (3) ◽  
pp. 1109-1124 ◽  
Author(s):  
Michiko Nakamura-Kubo ◽  
Taro Nakamura ◽  
Aiko Hirata ◽  
Chikashi Shimoda
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Vesicles ◽  
Spindle Pole ◽  
Nucleotide Exchange ◽  
Gene Encoding ◽  
Single Nucleotide Change ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cold Sensitive

The Schizosaccharomyces pombe spo14-B221 mutant was originally isolated as a sporulation-deficient mutant. However, thespo14 + gene is essential for cell viability and growth. spo14 + is identical to the previously characterizedstl1 + gene encoding a putative homologue of Saccharomyces cerevisiae Sec12, which is essential for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In the spo14 mutant cells, ER-like membranes were accumulated beneath the plasma membrane and the ER/Golgi shuttling protein Rer1 remained in the ER. Sec12 is a guanine nucleotide exchange factor for the Sar1 GTPase. Overproduction ofpsr1 + coding for an S. pombe Sar1 homologue suppressed both the sporulation defect ofspo14-B221 and cold-sensitive growth of newly isolatedspo14-6 and spo14-7 mutants. These results indicate that Spo14 is involved in early steps of the protein secretory pathway. The spo14-B221 allele carries a single nucleotide change in the branch point consensus of the fifth intron, which reduces the abundance of the spo14 mRNA. During meiosis II, the forespore membrane was initiated near spindle pole bodies; however, subsequent extension of the membrane was arrested before its closure into a sac. We conclude that Spo14 is responsible for the assembly of the forespore membrane by supplying membrane vesicles.

scholarly journals Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

2006 ◽  
Vol 26 (13) ◽  
pp. 4830-4842 ◽  
Author(s):  
Sonja G. Hunter ◽  
Guanglei Zhuang ◽  
Dana Brantley-Sieders ◽  
Wojciech Swat ◽  
Christopher W. Cowan ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rac1 Gtpase ◽  
Rho Family ◽  
Epha Receptor

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

scholarly journals Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

2009 ◽  
Vol 20 (5) ◽  
pp. 1388-1399 ◽  
Author(s):  
Mike Ngo ◽  
Neale D. Ridgway
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Binding Protein ◽  
Dominant Negative ◽  
Ph Domain ◽  
Oxysterol Binding Protein ◽  
Large Gene ◽  
Dominant Negative Variant

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

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