scholarly journals Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon-COP.

1994 ◽  
Vol 125 (6) ◽  
pp. 1213-1224 ◽  
Author(s):  
Q Guo ◽  
E Vasile ◽  
M Krieger
Keyword(s):  
Brefeldin A ◽  
Membrane Traffic ◽  
Temperature Sensitive ◽  
Intracellular Membrane ◽  
Cho Cell ◽  
Animal Cells ◽  
Cell Temperature ◽  
Ldl Receptors ◽  
Golgi Transport ◽  
Golgi Structure

The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.

The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast

2001 ◽  
Vol 114 (12) ◽  
pp. 2241-2253 ◽  
Author(s):  
Anne Peyroche ◽  
Régis Courbeyrette ◽  
Alain Rambourg ◽  
Catherine L. Jackson
Keyword(s):  
Membrane Dynamics ◽  
Guanine Nucleotide Exchange Factors ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Golgi Transport ◽  
Golgi Structure ◽  
And Function

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics. Their precise molecular roles in different trafficking steps within the cell have not been elucidated. We present a functional analysis of two members of this family, Gea1p and Gea2p, in the yeast Saccharomyces cerevisiae. Gea1p and Gea2p can functionally replace each other, but at least one is necessary for viability. Temperature sensitive gea mutants were generated and found to have defects in ER-Golgi and intra-Golgi transport. Similar to mutants in COPI subunits in yeast, gea mutants had a cargo-selective secretion defect, in that some proteins continued to be secreted whereas others were blocked in the ER or early Golgi. Like yeast arf mutants, the rate of transport of those proteins that continued to be secreted was slowed. In addition, the structure of Golgi elements was severly perturbed in gea mutants. We conclude that Gea1p and Gea2p play an important role in the structure and functioning of the Golgi apparatus in yeast.

Cell surface membrane homeostasis and intracellular membrane traffic balance in mouse L929 cells

1999 ◽  
Vol 112 (14) ◽  
pp. 2431-2440
Author(s):  
G.T. Coupin ◽  
C.D. Muller ◽  
A. Remy-Kristensen ◽  
J.G. Kuhry
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Membrane Trafficking ◽  
Surface Membrane ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Intracellular Membrane ◽  
Simple Method ◽  
Membrane Area ◽  
Membrane Probe

We have developed a simple method for synchronizing L929 mouse fibroblasts. Cultured as monolayers, these cells stop growing at confluency and arrest at the end of the G1 phase. Upon seeding at low density, they enter the S phase simultaneously. Using these cells we then looked at the evolution of the surface membrane area during the cell cycle using the fluorescence membrane probe TMA-DPH. In contact with cells, this probe partitions between the membrane (probe fluorescent) and the external medium (non-fluorescent), delivering a signal proportional to the membrane area. This area was constant until just before mitosis, when it increased at once. With the same probe as an endocytic marker, we examined how this membrane homeostasis could be consistent with intracellular membrane trafficking. The study was limited to one selected period of the cell cycle (6-9 hours). We observed that 14% of the membrane endocytosed was not recycled, but was replaced at the cell surface by newly formed membrane from biosynthetic pathways. Brefeldin A modified the membrane traffic, but not the overall membrane homeostasis. The results are discussed in the framework of a maturation model.

Distribution of the intermediate elements operating in ER to Golgi transport

1991 ◽  
Vol 100 (3) ◽  
pp. 415-430 ◽  
Author(s):  
J. Saraste ◽  
K. Svensson
Keyword(s):  
Golgi Apparatus ◽  
Close Association ◽  
Brefeldin A ◽  
Temperature Sensitive ◽  
Cellular Distribution ◽  
Transport Pathway ◽  
Golgi Transport ◽  
Golgi Region ◽  
Viral Glycoproteins ◽  
Rough Er

We have used a 58 kDa membrane protein (p58) as a marker to study the transport pathway between the rough endoplasmic reticulum (ER) and the Golgi apparatus. Immunolocalization of p58 in fibroblasts showed its presence in a single cisterna and in small tubular and vesicular elements at the cis side of the Golgi apparatus. In addition, the protein was detected in large (200–500 nm in diameter) tubulovesicular structures, clustered in the Golgi region but also found in peripheral locations. These represent intermediates in ER to Golgi transport since they contained newly synthesized viral glycoproteins, arrested in cells at 15 degrees C. The peripheral structures accumulated at low temperature but reclustered rapidly to the Golgi region upon shift of cells back to 37 degrees C. This movement involved long intracellular distances and was efficiently inhibited by nocodazole, indicating that it requires the integrity of microtubules. In contrast, reclustering was unaffected by brefeldin A (BFA), suggesting that this compound affects ER to Golgi transport prior to the temperature-sensitive step. In BFA-treated cells p58 was localized to scattered, tubular, smooth ER clusters, found in close association with rough ER cisternae. The cellular distribution of the intermediate elements indicates that the sites of protein exit are widely distributed within the rough ER network. We suggest that the smooth ER locations where p58 accumulates in BFA-treated cells could represent such peripheral exit sites.

scholarly journals Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

2002 ◽  
Vol 157 (3) ◽  
pp. 405-415 ◽  
Author(s):  
Daniel Ungar ◽  
Toshihiko Oka ◽  
Elizabeth E. Brittle ◽  
Eliza Vasile ◽  
Vladimir V. Lupashin ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Cho Cell ◽  
Golgi Transport ◽  
Cog Complex ◽  
Transport Assay ◽  
Golgi Structure ◽  
And Function ◽  
Transport Complex

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. “Deep etch” EM of purified COG revealed an ∼37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.

scholarly journals A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 833-841
Author(s):  
Yu Jiang ◽  
Al Scarpa ◽  
Li Zhang ◽  
Shelly Stone ◽  
Ed Feliciano ◽  
...  
Keyword(s):  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Suppression ◽  
Golgi Transport ◽  
New Gene ◽  
Insight Into ◽  
Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

scholarly journals Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures

1999 ◽  
Vol 112 (5) ◽  
pp. 589-600 ◽  
Author(s):  
T. Farmaki ◽  
S. Ponnambalam ◽  
A.R. Prescott ◽  
H. Clausen ◽  
B.L. Tang ◽  
...  
Keyword(s):  
Protein Transport ◽  
Protein Export ◽  
Sensitive Assay ◽  
Plasma Membrane Protein ◽  
Animal Cells ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Intermediate Compartment ◽  
First Time ◽  
Mitotic Cells

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

scholarly journals Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport

2020 ◽  
Vol 133 (14) ◽  
pp. jcs239814 ◽  
Author(s):  
Laura M. Westrate ◽  
Melissa J. Hoyer ◽  
Michael J. Nash ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Coated Vesicle ◽  
First Person ◽  
Coat Proteins ◽  
Animal Cells ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Copii Vesicles ◽  
Export System

ABSTRACTSecretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.

Membrane traffic and polarization of lipid domains during cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 395-399 ◽  
Author(s):  
Arnaud Echard
Keyword(s):  
Intracellular Transport ◽  
Membrane Traffic ◽  
Membrane Composition ◽  
Intercellular Bridge ◽  
Animal Cells ◽  
Lipid Domain ◽  
Complex Events ◽  
Cytoskeletal Elements ◽  
Membrane Sealing ◽  
Endocytic Pathways

Growing evidence indicates that membrane traffic plays a crucial role during the late post-furrowing steps of cytokinesis in animal cells. Indeed, both endocytosis and exocytosis contribute to stabilizing the intercellular bridge that connects the daughter cells and to the final abscission in diverse organisms. The need for several intracellular transport routes probably reflects the complex events that occur during the late cytokinesis steps such as local remodelling of the plasma membrane composition, removal of components required for earlier steps of cytokinesis and membrane sealing that leads to daughter cell separation. In this mini-review, I will focus on recent evidence showing that endocytic pathways, such as the Rab35-regulated recycling pathway, contribute to the establishment of a PtdIns(4,5)P2 lipid domain at the intercellular bridge which is involved in the localization of cytoskeletal elements essential for the late steps of cytokinesis. Possible cross-talk between Rab35 and other endocytic pathways involved in cytokinesis are also discussed.

Sign in / Sign up

Export Citation Format

Share Document