scholarly journals α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

2010 ◽  
Vol 21 (11) ◽  
pp. 1850-1863 ◽  
Author(s):  
Nandhakumar Thayanidhi ◽  
Jared R. Helm ◽  
Deborah C. Nycz ◽  
Marvin Bentley ◽  
Yingjian Liang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Direct Action ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Protein Receptors

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

2005 ◽  
Vol 72 ◽  
pp. 1-13 ◽  
Author(s):  
Krysten J. Palmer ◽  
Peter Watson ◽  
David J. Stephens
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Motor Proteins ◽  
Microtubule Cytoskeleton ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Intracellular Compartments ◽  
Retrograde Traffic

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.

scholarly journals Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

1998 ◽  
Vol 9 (10) ◽  
pp. 2819-2837 ◽  
Author(s):  
Sara Jones ◽  
Celeste J. Richardson ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Assay

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7and GYP1, nor is it influenced by mutations inSEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

scholarly journals HtrA2 Regulates β-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

2007 ◽  
Vol 282 (38) ◽  
pp. 28285-28295 ◽  
Author(s):  
Henri J. Huttunen ◽  
Suzanne Y. Guénette ◽  
Camilla Peach ◽  
Christopher Greco ◽  
Weiming Xia ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Proteasome Inhibition ◽  
Precursor Protein ◽  
Amyloid Peptide ◽  
Early Secretory Pathway ◽  
App Metabolism ◽  
Microsomal Membranes ◽  
Β Amyloid

Alzheimer disease-associated β-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Aβ secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

scholarly journals rsly1 Binding to Syntaxin 5 Is Required for Endoplasmic Reticulum-to-Golgi Transport but Does Not Promote SNARE Motif Accessibility

2004 ◽  
Vol 15 (1) ◽  
pp. 162-175 ◽  
Author(s):  
Antionette L. Williams ◽  
Sebastian Ehm ◽  
Noëlle C. Jacobson ◽  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Protein Function ◽  
Snare Complex ◽  
Golgi Transport ◽  
Snare Motif ◽  
Sm Protein ◽  
Snare Complex Formation

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE–complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1–syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.

scholarly journals Integrity of the Early Secretory Pathway Promotes, but Is Not Required for, Severe Acute Respiratory Syndrome Coronavirus RNA Synthesis and Virus-Induced Remodeling of Endoplasmic Reticulum Membranes

2009 ◽  
Vol 84 (2) ◽  
pp. 833-846 ◽  
Author(s):  
Kèvin Knoops ◽  
Cindy Swett-Tapia ◽  
Sjoerd H. E. van den Worm ◽  
Aartjan J. W. te Velthuis ◽  
Abraham J. Koster ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Severe Acute Respiratory Syndrome ◽  
Rna Synthesis ◽  
Secretory Pathway ◽  
Electron Tomography ◽  
Viral Rna ◽  
Direct Role ◽  
Ultrastructural Studies ◽  
Early Secretory Pathway

ABSTRACT To accommodate its RNA synthesis in the infected cell, severe acute respiratory syndrome coronavirus (SARS-CoV) induces a cytoplasmic reticulovesicular network (RVN) that is derived from endoplasmic reticulum (ER) membranes. We set out to investigate how the early secretory pathway interacts with the RVN and the viral replication/transcription complex (RTC) that is anchored to it. When the secretory pathway was disrupted by brefeldin A (BFA) treatment at the start of infection, RVN formation and viral RTC activity were not blocked and continued up to 11 h postinfection, although RNA synthesis was reduced by ca. 80%. In vitro RTC assays, using membrane fractions from infected cells, demonstrated that BFA does not directly interfere with the activity of the viral RNA-synthesizing enzymes. Confocal microscopy studies showed that early secretory pathway components are not associated with SARS-CoV-induced replication sites, although our studies revealed that infection induces a remarkable redistribution of the translocon subunit Sec61α. Ultrastructural studies, including electron tomography, revealed that the formation of the RVN and all its previously documented features can occur in the presence of BFA, despite differences in the volume and morphology of the network. We therefore conclude that early secretory pathway proteins do not play a direct role in RVN morphogenesis or the functionality of the SARS-CoV RTC. The BFA-induced disruption of ER integrity and functionality probably affects the overall quality of the membrane scaffold that is needed to support the viral RTC and/or the availability of specific host factors, which in turn compromises viral RNA synthesis.

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).

TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells

1999 ◽  
Vol 112 (2) ◽  
pp. 147-156 ◽  
Author(s):  
H. Field ◽  
B.R. Ali ◽  
T. Sherwin ◽  
K. Gull ◽  
S.L. Croft ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Phylogenetic Analyses ◽  
Small Gtpases ◽  
Snare Complex ◽  
Rab Proteins ◽  
Over Expression ◽  
Golgi Transport

The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.

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.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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