ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whetherarf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined inarf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

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Requirement for Neo1p in Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0463 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4971-4983 ◽

Cited By ~ 65

Author(s):

Zhaolin Hua ◽

Todd R. Graham

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Protein Transport ◽

Retrograde Transport ◽

Transport Pathway ◽

Transport Defect ◽

Copii Vesicles ◽

P Type ◽

Adp Ribosylation Factor

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.

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Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e02-01-0027 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2518-2532 ◽

Cited By ~ 72

Author(s):

Ken Sato ◽

Akihiko Nakano

Keyword(s):

Endoplasmic Reticulum ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Retrograde Transport ◽

Subcellular Fractionation ◽

Terminal Region ◽

Growth Defect ◽

Critical Determinant ◽

Reading Frame ◽

Glycoprotein Secretion

The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca2+-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca2+ sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.

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The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.12.4.1035 ◽

2001 ◽

Vol 12 (4) ◽

pp. 1035-1045 ◽

Cited By ~ 41

Author(s):

Anne Spang ◽

Johannes M. Herrmann ◽

Susan Hamamoto ◽

Randy Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Protein Transport ◽

Retrograde Transport ◽

Guanine Nucleotide Exchange Factors ◽

Temperature Sensitive ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Membrane Bound

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identifiedGEA2 as a multicopy suppressor of asec21-3 temperature-sensitive mutant.SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat.GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 andGEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

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Atlastin-mediated membrane tethering is critical for cargo mobility and exit from the endoplasmic reticulum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908409116 ◽

2019 ◽

Vol 116 (28) ◽

pp. 14029-14038 ◽

Cited By ~ 17

Author(s):

Liling Niu ◽

Tianji Ma ◽

Feng Yang ◽

Bing Yan ◽

Xiao Tang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hereditary Spastic Paraplegia ◽

Critical Role ◽

Spastic Paraplegia ◽

Exit Site ◽

Cargo Proteins ◽

Membrane Tethering ◽

Copii Vesicles ◽

Er Export ◽

In Cells

Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.

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Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum

Cells ◽

10.3390/cells9051295 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1295

Author(s):

Sergio Lopez ◽

Ana Maria Perez-Linero ◽

Javier Manzano-Lopez ◽

Susana Sabido-Bozo ◽

Alejandro Cortes-Gomez ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Transport ◽

Retrograde Transport ◽

Secretory Proteins ◽

Dependent Manner ◽

Cellular Mechanisms ◽

Export Pathway ◽

Additional Layer ◽

Cargo Receptor ◽

Er Export

The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.

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Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

Molecular Biology of the Cell ◽

10.1091/mbc.e14-11-1568 ◽

2015 ◽

Vol 26 (17) ◽

pp. 3071-3084 ◽

Cited By ~ 45

Author(s):

Tetsuya Hirata ◽

Morihisa Fujita ◽

Shota Nakamura ◽

Kazuyoshi Gotoh ◽

Daisuke Motooka ◽

...

Keyword(s):

Protein Transport ◽

Transmembrane Proteins ◽

Retrograde Transport ◽

Hek293 Cells ◽

Protein Overexpression ◽

Surface Integral ◽

Anterograde Transport ◽

Genome Wide ◽

Golgi Network ◽

Garp Complex

The importance of endosome-to– trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

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PIG-W Is Critical for Inositol Acylation but Not for Flipping of Glycosylphosphatidylinositol-Anchor

Molecular Biology of the Cell ◽

10.1091/mbc.e03-03-0193 ◽

2003 ◽

Vol 14 (10) ◽

pp. 4285-4295 ◽

Cited By ~ 71

Author(s):

Yoshiko Murakami ◽

Uamporn Siripanyapinyo ◽

Yeongjin Hong ◽

Ji Young Kang ◽

Sonoko Ishihara ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Acyl Chain ◽

Critical Role ◽

Chinese Hamster ◽

Surface Proteins ◽

Acyltransferase Activity ◽

Acid Protein ◽

Luminal Side ◽

Mutant Cells

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.

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