scholarly journals Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

2013 ◽  
Vol 24 (17) ◽  
pp. 2703-2713 ◽  
Author(s):  
Philip D. Fox ◽  
Christopher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Jenny L. Higgins ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Surface Proteins ◽  
Hek 293 ◽  
Cortical Endoplasmic Reticulum

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

scholarly journals Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

2002 ◽  
Vol 70 (8) ◽  
pp. 4353-4361 ◽  
Author(s):  
Michael J. Wichroski ◽  
Jody A. Melton ◽  
Carolyn G. Donahue ◽  
Rodney K. Tweten ◽  
Gary E. Ward
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Surface Proteins ◽  
Alpha Toxin ◽  
Clostridium Septicum ◽  
Ultrastructural Studies

ABSTRACT As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondii tachyzoites. The mechanisms by which T. gondii GPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown. As a first step in developing tools to study these processes, we show here that Clostridium septicum alpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active against T. gondii tachyzoites (50% effective concentration, 0.2 nM). Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite. Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin. Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1. Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed. Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking in T. gondii and other susceptible protozoa.

scholarly journals Golgi complex–plasma membrane trafficking directed by an autonomous, tribasic Golgi export signal

2014 ◽  
Vol 25 (6) ◽  
pp. 866-878 ◽  
Author(s):  
Hirendrasinh B. Parmar ◽  
Christopher Barry ◽  
FuiBoon Kai ◽  
Roy Duncan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Transmembrane Proteins ◽  
Complex Plasma ◽  
Sorting Signals ◽  
Linear Motifs ◽  
Export Signal

Although numerous linear motifs that direct protein trafficking within cells have been identified, there are few examples of linear sorting signals mediating directed export of membrane proteins from the Golgi complex to the plasma membrane. The reovirus fusion-associated small transmembrane proteins are simple, single-pass transmembrane proteins that traffic through the endoplasmic reticulum–Golgi pathway to the plasma membrane, where they induce cell–cell membrane fusion. Here we show that a membrane-proximal, polybasic motif (PBM) in the cytosolic tail of p14 is essential for efficient export of p14 from the Golgi complex to the plasma membrane. Extensive mutagenic analysis reveals that the number, but not the identity or position, of basic residues present in the PBM dictates p14 export from the Golgi complex, with a minimum of three basic residues required for efficient Golgi export. Results further indicate that the tribasic motif does not affect plasma membrane retention of p14. Furthermore, introduction of the tribasic motif into a Golgi-localized, chimeric ERGIC-53 protein directs export from the Golgi complex to the plasma membrane. The p14 PBM is the first example of an autonomous, tribasic signal required for Golgi export to the plasma membrane.

scholarly journals Endoplasmic Reticulum/Plasma Membrane Junctions Function as Membrane Protein Trafficking Hubs

2013 ◽  
Vol 104 (2) ◽  
pp. 619a
Author(s):  
Philip D. Fox ◽  
Christpher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Elizabeth J. Akin ◽  
Matthew J. Kennedy ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Protein Trafficking ◽  
Membrane Protein Trafficking

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals A Region Within the Third Extracellular Loop of Rat AQP6 Precludes Its Trafficking to Plasma Membrane in a Heterologous Cell Line.

2021 ◽  
Author(s):  
David Soler ◽  
Thomas Kowatz ◽  
Andrew Sloan ◽  
Thomas McCormick ◽  
Kevin Cooper ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Line ◽  
Membrane Trafficking ◽  
Extracellular Loop ◽  
Reporter System ◽  
The Third ◽  
Heterologous Cell ◽  
Retention Signal

Abstract The inability to over-express AQP6 in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail. Using the AGR reporter system we have identified a region within loop C of AQP6 that is responsible for severely hampering its plasma membrane localization. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous cells.

Patching plasma membrane disruptions with cytoplasmic membrane

2000 ◽  
Vol 113 (11) ◽  
pp. 1891-1902 ◽  
Author(s):  
P.L. McNeil ◽  
S.S. Vogel ◽  
K. Miyake ◽  
M. Terasaki
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Sea Urchin ◽  
Membrane Trafficking ◽  
Cytoplasmic Membrane ◽  
Yolk Granule ◽  
High Threshold ◽  
Surface Markers ◽  
Plasma Membrane Protein ◽  
Cell Cytoplasm

Vesicle-vesicle fusion initiated in cell cytoplasm by high Ca(2+) can rapidly erect large membrane boundaries. These might be used as a ‘patch’ for resealing plasma membrane disruptions. Three central predictions of this ‘patch’ hypothesis are here established in sea urchin eggs. First, we show that surface markers for plasma membrane protein and lipid are initially absent over disruption sites after resealing is complete. Second, we demonstrate that resealing capacity is strongly dependent upon local availability of fusion competent cytoplasmic organelles, specifically the reserve or yolk granule. Lastly, we demonstrate that the reserve granule is capable of rapid (t(1/2) <1 second), Ca(2+)-regulated (high threshold) fusion capable of erecting large (>1000 μm(2)), continuous membrane boundaries. Production of patch vesicles for resealing may proceed by an ‘emergency’ fusion mechanism distinct from that utilized for the much slower, highly regulated, cytosol-requiring organelle-organelle fusion events typical of constitutive membrane trafficking pathways.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

Identification of a Cellubrevin/Vesicle Associated Membrane Protein 3 Homologue in Human Platelets

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Audrey M. Bernstein ◽  
Sidney W. Whiteheart
Keyword(s):  
Membrane Proteins ◽  
Complex Formation ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Human Platelets ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Transport Vesicle ◽  
Exocytic Pathway ◽  
Conserved Core

Abstract Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N–terminal domain. Northern blot analysis showed that the 2.5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in -SNAP–dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most -granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.

scholarly journals Autophagosome formation in relation to the endoplasmic reticulum

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Yo-hei Yamamoto ◽  
Takeshi Noda
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
De Novo ◽  
Transmembrane Protein ◽  
Lipid Transfer ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Selective Autophagy ◽  
The Relationship ◽  
Er Membrane

Abstract Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

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