scholarly journals Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2498-2512 ◽  
Author(s):  
Scott E. Phillips ◽  
Kristina E. Ile ◽  
Malika Boukhelifa ◽  
Richard P.H. Huijbregts ◽  
Vytas A. Bankaitis
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Membrane Binding ◽  
Bound State ◽  
Missense Mutations ◽  
Cis Acting ◽  
Domain Mapping ◽  
Phosphatidylinositol Transfer ◽  
Golgi Network

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203motif). Combination of the specificity elements with the W202W203motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262is required for PITPβ targeting to Golgi membranes.

scholarly journals Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

2012 ◽  
Vol 23 (12) ◽  
pp. 2327-2338 ◽  
Author(s):  
Amy J. Curwin ◽  
Julia von Blume ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Calcium Atpase ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Reduced Rate ◽  
Golgi Network

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.

scholarly journals Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

2002 ◽  
Vol 13 (3) ◽  
pp. 739-754 ◽  
Author(s):  
James G. Alb ◽  
Scott E. Phillips ◽  
Kathleen Rostand ◽  
Xiaoxia Cui ◽  
Jef Pinxteren ◽  
...  
Keyword(s):  
Mast Cell ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Embryonic Stem ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Homologous Proteins ◽  
Genetic Ablation ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITPα and PITPβ, two highly homologous proteins that are encoded by distinct genes. Insights into PITPα and PITPβ function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITPα and PITPβ in murine cells. Contrary to expectations, we find that ablation of PITPα function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITPα does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITPα-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITPβ alleles from murine cells and show that PITPβ deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITPβ is an essential housekeeping PITP in murine cells, whereas PITPα plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 968-969
Author(s):  
K. McCammon ◽  
M. Segal ◽  
J. Sambrook ◽  
M. J. Gething ◽  
A. McDowall
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Cysteine Residue ◽  
Homologous Sequence ◽  
Golgi Compartment ◽  
Golgi Network

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

GGA proteins: new players in the sorting game

2001 ◽  
Vol 114 (19) ◽  
pp. 3413-3418 ◽  
Author(s):  
Annette L. Boman
Keyword(s):  
Membrane Trafficking ◽  
Sequence Homology ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Protein Domains ◽  
Vesicle Formation ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A

1993 ◽  
Vol 106 (3) ◽  
pp. 789-802 ◽  
Author(s):  
M. Roa ◽  
V. Cornet ◽  
C.Z. Yang ◽  
B. Goud
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Atp Depletion ◽  
Cell Fractionation ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Organizing Center ◽  
Golgi Network

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10–15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

scholarly journals Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

2009 ◽  
Vol 185 (4) ◽  
pp. 601-612 ◽  
Author(s):  
Robin W. Klemm ◽  
Christer S. Ejsing ◽  
Michal A. Surma ◽  
Hermann-Josef Kaiser ◽  
Mathias J. Gerl ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Membrane Lipids ◽  
Secretory Vesicles ◽  
Shotgun Lipidomics ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Sorting Station ◽  
Golgi Network ◽  
First Time

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

Defects in early secretory pathway transport machinery components and neurodevelopmental disorders

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Bor Luen Tang
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Underlying Disease ◽  
Membrane Dynamics ◽  
Coat Proteins ◽  
Disease Manifestation ◽  
Vesicular Traffic ◽  
Early Secretory Pathway ◽  
Genes Encoding

Abstract The early secretory pathway, provisionally comprising of vesicular traffic between the endoplasmic reticulum (ER) and the Golgi apparatus, occurs constitutively in mammalian cells. Critical for a constant supply of secretory and plasma membrane (PM) materials, the pathway is presumably essential for general cellular function and survival. Neurons exhibit a high intensity in membrane dynamics and protein/lipid trafficking, with differential and polarized trafficking towards the somatodendritic and axonal PM domains. Mutations in genes encoding early secretory pathway membrane trafficking machinery components are known to result in neurodevelopmental or neurological disorders with disease manifestation in early life. Here, such rare disorders associated with autosomal recessive mutations in coat proteins, membrane tethering complexes and membrane fusion machineries responsible for trafficking in the early secretory pathway are summarily discussed. These mutations affected genes encoding subunits of coat protein complex I and II, subunits of transport protein particle (TRAPP) complexes, members of the YIP1 domain family (YIPF) and a SNAP receptor (SNARE) family member. Why the ubiquitously present and constitutively acting early secretory pathway machinery components could specifically affect neurodevelopment is addressed, with the plausible underlying disease etiologies and neuropathological mechanisms resulting from these mutations explored.

scholarly journals 1School of Life Science and Technology, ShanghaiTech University, Pudong, Shanghai 201210, China; [email protected] (X.Y.); [email protected] (Y.Q.)

2019 ◽  
Vol 20 (8) ◽  
pp. 2028 ◽  
Author(s):  
Yue ◽  
Qian ◽  
Gim ◽  
Lee
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Host Cells ◽  
Scaffolding Protein ◽  
Membrane Domain ◽  
Cellular Functions ◽  
Domain Organization ◽  
Bacterial Proliferation ◽  
Intracellular Organelles

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington’s disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

scholarly journals Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

2004 ◽  
Vol 15 (2) ◽  
pp. 637-648 ◽  
Author(s):  
Seema Dalal ◽  
Meredith F. N. Rosser ◽  
Douglas M. Cyr ◽  
Phyllis I. Hanson
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Atp Hydrolysis ◽  
Brefeldin A ◽  
Transmembrane Conductance Regulator ◽  
Organelle Biogenesis ◽  
Ubiquitinated Proteins ◽  
Organelle Assembly ◽  
Specific Assessment

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.

scholarly journals New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

2019 ◽  
Author(s):  
Laia Bassaganyas ◽  
Stephanie J. Popa ◽  
Max Horlbeck ◽  
Anupama Ashok ◽  
Sarah E. Stewart ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Basic Principles ◽  
Golgi Membranes ◽  
Genome Wide ◽  
A Genome ◽  
Powerful Strategy ◽  
Shed Light

AbstractProtein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathways function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to the Golgi complex. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion, and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

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