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Todd R. Graham - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular Transport
Journal of Cell Biology, 2013Co-Authors: Ryan D Baldridge, Richard J Chi, Christopher G Burd, Todd R. GrahamAbstract:Vesicle-mediated Protein Transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, Protein Transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for Protein Transport in this pathway are unknown. Here, we show that the Arf GTPase-activating Protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related Protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular Transport between the EE and the TGN.
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linking phospholipid flippases to vesicle mediated Protein Transport
Biochimica et Biophysica Acta, 2009Co-Authors: Babyperiyanayaki Muthusamy, Paramasivam Natarajan, Xiaoming Zhou, Todd R. GrahamAbstract:Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases (flippases) implicated in the generation of phospholipid asymmetry in biological membranes. P4-ATPases are typically the largest P-type ATPase subgroup found in eukaryotic cells, with five members in Saccharomyces cerevisiae, six members in Caenorhabditis elegans, 12 members in Arabidopsis thaliana and 14 members in humans. In addition, many of the P4-ATPases require interaction with a noncatalytic subunit from the CDC50 gene family for their Transport out of the endoplasmic reticulum (ER). Deficiency of a P4-ATPase (Atp8b1) causes liver disease in humans, and studies in a variety of model systems indicate that P4-ATPases play diverse and essential roles in membrane biogenesis. In addition to their proposed role in establishing and maintaining plasma membrane asymmetry, P4-ATPases are linked to vesicle-mediated Protein Transport in the exocytic and endocytic pathways. Recent studies have also suggested a role for P4-ATPases in the nonvesicular intracellular trafficking of sterols. Here, we discuss the physiological requirements for yeast P4-ATPases in phospholipid translocase activity, Transport vesicle budding and ergosterol metabolism, with an emphasis on Drs2p and its noncatalytic subunit, Cdc50p.
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flippases and vesicle mediated Protein Transport
Trends in Cell Biology, 2004Co-Authors: Todd R. GrahamAbstract:The best-understood mechanisms for generating Transport vesicles in the secretory and endocytic pathways involve the localized assembly of cytosolic coat Proteins such as clathrin, coat Protein complex (COP)I and COPII onto membranes. These coat Proteins can deform membranes by themselves, but accessory Proteins might help to generate the tight curvature needed to form a vesicle. Enzymes that pump phospholipid from one leaflet of the bilayer to the other (flippases) can deform membranes by creating an imbalance in the phospholipid number between the two leaflets. Recent studies describe a requirement for the yeast Drs2p family of P-type ATPases in both phospholipid translocation and Protein Transport in the secretory and endocytic pathways. This indicates that flippases work with coat Proteins to form vesicles.
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an arf1δ synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar Protein Transport in saccharomyces cerevisiae
Genetics, 1998Co-Authors: Chihying Chen, Todd R. GrahamAbstract:ADP-ribosylation factor (ARF) is a small GTP-binding Protein that is thought to regulate the assembly of coat Proteins on Transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial Protein Transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in Transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or Transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole Transport pathway for carboxypeptidase Y.
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brefeldin a reversibly blocks early but not late Protein Transport steps in the yeast secretory pathway
The EMBO Journal, 1993Co-Authors: Todd R. Graham, P A ScottAbstract:We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in Protein Transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde Transport of the alpha 1,6 mannosyl-transferase into the ER. We found that Transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.
Gustavo Egea - One of the best experts on this subject based on the ideXlab platform.
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βiii spectrin regulates the structural integrity and the secretory Protein Transport of the golgi complex
Journal of Biological Chemistry, 2013Co-Authors: Laia Salcedosicilia, Eugenia Mato, Susana Granell, Marko Jovic, Adria Sicart, Ludger Johannes, Tamas Balla, Gustavo EgeaAbstract:A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi Proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of Protein Transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi Protein Transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.
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regulation of Protein Transport from the golgi complex to the endoplasmic reticulum by cdc42 and n wasp
Molecular Biology of the Cell, 2002Co-Authors: Ana Luna, Olga B Matas, Jose A Martinezmenarguez, Eugenia Mato, Juan M Duran, J Ballesta, Michael Way, Gustavo EgeaAbstract:Actin is involved in the organization of the Golgi complex and Golgi-to-ER Protein Transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER Protein Transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated Transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde Transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident Proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER Transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(ΔWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER Protein Transport in an N-WASP–dependent manner.
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regulation of Protein Transport from the golgi complex to the endoplasmic reticulum by cdc42 and n wasp
Molecular Biology of the Cell, 2002Co-Authors: Ana Luna, Olga B Matas, Jose A Martinezmenarguez, Eugenia Mato, Juan M Duran, J Ballesta, Michael Way, Gustavo EgeaAbstract:Actin is involved in the organization of the Golgi complex and Golgi-to-ER Protein Transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoske...
Randy Schekman - One of the best experts on this subject based on the ideXlab platform.
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bi directional Protein Transport between the er and golgi
Annual Review of Cell and Developmental Biology, 2004Co-Authors: Elizabeth A. Miller, Jonathan Goldberg, L Orci, Randy SchekmanAbstract:▪ Abstract The endoplasmic reticulum (ER) and the Golgi comprise the first two steps in Protein secretion. Vesicular carriers mediate a continuous flux of Proteins and lipids between these compartments, reflecting the Transport of newly synthesized Proteins out of the ER and the retrieval of escaped ER residents and vesicle machinery. Anterograde and retrograde Transport is mediated by distinct sets of cytosolic coat Proteins, the COPII and COPI coats, respectively, which act on the membrane to capture cargo Proteins into nascent vesicles. We review the mechanisms that govern coat recruitment to the membrane, cargo capture into a Transport vesicle, and accurate delivery to the target organelle.
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SEC21 is a gene required for ER to Golgi Protein Transport that encodes a subunit of a yeast coatomer
Nature, 1992Co-Authors: Midori Hosobuchi, Thomas E Kreis, Randy SchekmanAbstract:NON-CLATHRIN coated vesicles have been implicated in early steps of intercompartmental Transport1–4. A distinct set of coat Proteins are peripherally associated with the exterior of purified mammalian intra-Golgi Transport vesicles5. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (β-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat6. Here we describe the characterization of SEC21, an essential gene required for Protein Transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat Protein plays an essential role in intercompartmental Transport.
Toshiya Endo - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of the cdna for pea chloroplast seca evolutionary conservation of the bacterial type seca dependent Protein Transport within chloroplasts
FEBS Letters, 1995Co-Authors: Tetsuya Nohara, Masato Nakai, Akira Goto, Toshiya EndoAbstract:We report here the isolation of the cDNA for pea chloroplast SecA. Pea SecA encodes a polypeptide of 1,011 amino acids and shows high sequence similarity with cyanobacterial SecA. Pea SecA was synthesized as a larger precursor and was imported into isolated chloroplasts in vitro. The purified pea SecA, which was expressed in Escherichia coli cells, stimulated the in vitro import of the 33 kDa Protein of the oxygen-evolving complex into thylakoids. These results indicate that higher plant chloroplasts contain a bacterial-type SecA Protein-dependent system for the intraorganellar Protein Transport into thylakoids.
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identification of the seca Protein homolog in pea chloroplasts and its possible involvement in thylakoidal Protein Transport
Journal of Biological Chemistry, 1994Co-Authors: Masato Nakai, Akira Goto, Tetsuya Nohara, D Sugita, Toshiya EndoAbstract:Abstract Recently, we identified the SecA and SecY Proteins in the cyanobacterium Synechococcus PCC7942. Antibodies raised against cyanobacterial SecA specifically reacted with a 110-kDa Protein of pea chloroplasts, suggesting the presence of SecA in higher plant chloroplasts. A part of the pea secA cDNA was polymerase chain reaction-amplified with degenerated oligonucleotide primers and with pea cDNA as a template. The deduced amino acid sequence shows 62% identity with cyanobacterial SecA and 52% identity with Escherichia coli SecA. Antibodies raised against the pea SecA fragment, which was expressed in E. coli cells from the obtained polymerase chain reaction-amplified cDNA, reacted with the 110-kDa chloroplast Protein; the 110-kDa Protein was mainly found in the stroma but partly in the thylakoid membrane. The anti-pea SecA IgG inhibited the in vitro import of the 33-kDa Protein of the oxygen-evolving complex, but not of the 23-kDa Protein of the oxygen-evolving complex, into thylakoids. These results suggest that SecA facilitates Transport of a subset of thylakoid lumenal Proteins including the 33-kDa Protein into thylakoids. We propose that a bacterial-type Sec Protein-dependent Transport system operates for Protein Transport into thylakoids in higher plant chloroplasts.
Franck Perez - One of the best experts on this subject based on the ideXlab platform.
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bml 265 and tyrphostin ag1478 disperse the golgi apparatus and abolish Protein Transport in human cells
Frontiers in Cell and Developmental Biology, 2019Co-Authors: Gaelle Boncompain, Nelly Gareil, Sarah Tessier, Aurianne Lescure, Thouis R Jones, Oliver Kepp, Guido Kroemer, Elaine Del Nery, Franck PerezAbstract:: The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde Transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory Protein Transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and Transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and Protein Transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.
