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 Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

scholarly journals Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

1988 ◽  
Vol 8 (10) ◽  
pp. 4098-4109 ◽  
Author(s):  
K A Eakle ◽  
M Bernstein ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Cell Fractionation ◽  
Significant Similarity

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.

scholarly journals Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

1997 ◽  
Vol 8 (1) ◽  
pp. 13-31 ◽  
Author(s):  
L Hicke ◽  
B Zanolari ◽  
M Pypaert ◽  
J Rohrer ◽  
H Riezman
Keyword(s):  
Fusion Protein ◽  
Secretory Pathway ◽  
Protein A ◽  
Brefeldin A ◽  
Early Endosome ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Early Secretory Pathway ◽  
Late Endosomes

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

scholarly journals Changes in architecture of the Golgi complex and other subcellular organelles during myogenesis.

1993 ◽  
Vol 120 (2) ◽  
pp. 399-409 ◽  
Author(s):  
E Ralston
Keyword(s):  
Protein Synthesis ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Muscle Fibers ◽  
Spatial Relationship ◽  
Mouse Muscle ◽  
Subcellular Organelles ◽  
Flexor Digitorum Brevis

Myogenesis involves changes in both gene expression and cellular architecture. Little is known of the organization, in muscle in vivo, of the subcellular organelles involved in protein synthesis despite the potential importance of targeted protein synthesis for formation and maintenance of functional domains such as the neuromuscular junction. A panel of antibodies to markers of the ER, the Golgi complex, and the centrosome were used to localize these organelles by immunofluorescence in myoblasts and myotubes of the mouse muscle cell line C2 in vitro, and in intact single muscle fibers from the rat flexor digitorum brevis. Antibodies to the ER stained structures throughout the cytoplasm of both C2 myoblasts and myotubes. In contrast, the spatial relationship between nucleus, centrosome, and Golgi complex was dramatically altered. These changes could also be observed in a low-calcium medium that allowed differentiation while preventing myoblast fusion. Muscle fibers in vivo resembled myotubes except that the ER occupied a smaller volume of cytoplasm and no staining was found for one of the Golgi complex markers, the enzyme alpha-mannosidase II. Electron microscopy, however, clearly showed the presence of stacks of Golgi cisternae in both junctional and extrajunctional regions of muscle fibers. The perinuclear distribution of the Golgi complex was also observed in live muscle fibers stained with a fluorescent lipid. Thus, the distribution of subcellular organelles of the secretory pathway was found to be similar in myotubes and muscle fibers, and all organelles were found in both junctional and extrajunctional areas of muscle.

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 Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

1995 ◽  
Vol 128 (6) ◽  
pp. 1003-1017 ◽  
Author(s):  
P J Peters ◽  
V W Hsu ◽  
C E Ooi ◽  
D Finazzi ◽  
S B Teal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Membrane System ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Wild Type ◽  
Gtp Binding ◽  
Defective Mutant

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

scholarly journals α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding

2011 ◽  
Vol 194 (1) ◽  
pp. 89-103 ◽  
Author(s):  
Iwona M. Pranke ◽  
Vincent Morello ◽  
Joëlle Bigay ◽  
Kimberley Gibson ◽  
Jean-Marc Verbavatz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Interaction ◽  
Membrane Curvature ◽  
Yeast Cells ◽  
Amphipathic Helices ◽  
Early Secretory Pathway ◽  
Intrinsic Capacity ◽  
In Cells

Membrane curvature sensors have diverse structures and chemistries, suggesting that they might have the intrinsic capacity to discriminate between different types of vesicles in cells. In this paper, we compare the in vitro and in vivo membrane-binding properties of two curvature sensors that form very different amphipathic helices: the amphipathic lipid-packing sensor (ALPS) motif of a Golgi vesicle tether and the synaptic vesicle protein α-synuclein, a causative agent of Parkinson’s disease. We demonstrate the mechanism by which α-synuclein senses membrane curvature. Unlike ALPS motifs, α-synuclein has a poorly developed hydrophobic face, and this feature explains its dual sensitivity to negatively charged lipids and to membrane curvature. When expressed in yeast cells, these two curvature sensors were targeted to different classes of vesicles, those of the early secretory pathway for ALPS motifs and to negatively charged endocytic/post-Golgi vesicles in the case of α-synuclein. Through structures with complementary chemistries, α-synuclein and ALPS motifs target distinct vesicles in cells by direct interaction with different lipid environments.

scholarly journals Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product

1988 ◽  
Vol 8 (10) ◽  
pp. 4098-4109
Author(s):  
K A Eakle ◽  
M Bernstein ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Cell Fractionation ◽  
Significant Similarity

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

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