scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals RAB6 and microtubules restrict secretion to focal adhesions

2018 ◽  
Author(s):  
L. Fourrière ◽  
A. Kasri ◽  
N. Gareil ◽  
S. Bardin ◽  
J. Boulanger ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

ABSTRACTTo ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

1985 ◽  
Vol 232 (1) ◽  
pp. 71-78 ◽  
Author(s):  
J A Hedo ◽  
I A Simpson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Receptor ◽  
Golgi Complex ◽  
Microsomal Fraction ◽  
Primary Cultures ◽  
Sodium Dodecyl ◽  
High Density ◽  
Low Density ◽  
Adipose Cells

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

scholarly journals C. elegansparaoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

2016 ◽  
Author(s):  
Yushu Chen ◽  
Shashank Bharill ◽  
Zeynep Altun ◽  
Robert O'Hagan ◽  
Brian Coblitz ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Functional Expression ◽  
Similar Protein ◽  
Touch Sensitivity ◽  
Additional Protein

Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by themec-4(d)mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on theXenopusoocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components.

scholarly journals Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

2020 ◽  
Vol 31 (9) ◽  
pp. 2044-2064 ◽  
Author(s):  
Suzie J. Scales ◽  
Nidhi Gupta ◽  
Ann M. De Mazière ◽  
George Posthuma ◽  
Cecilia P. Chiu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
High Frequency ◽  
Lipid Droplets ◽  
Human Kidney ◽  
Cytoplasmic Face ◽  
Large Panel ◽  
Surface Localization ◽  
Apolipoprotein L1

BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

scholarly journals Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

1997 ◽  
Vol 17 (9) ◽  
pp. 5210-5226 ◽  
Author(s):  
V I Titorenko ◽  
D M Ogrydziak ◽  
R A Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Protein Secretion ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mycelial Form ◽  
Secretory Pathways ◽  
Associated Proteins ◽  
Yeast Yarrowia Lipolytica

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

A small GTPase involved in mitochondrial morphology and function

2015 ◽  
Vol 43 (1) ◽  
pp. 108-110 ◽  
Author(s):  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Essential Role ◽  
Small Gtpase ◽  
Mitochondrial Morphology ◽  
Interconnected Network ◽  
Contact Sites ◽  
Close Proximity ◽  
And Function ◽  
Adp Ribosylation Factor

Mitochondria are the powerhouse of cells as they produce the bulk of ATP which is consumed by the cell. They form a highly interconnected network that is governed by fission and fusion processes. In addition, mitochondria and the endoplasmic reticulum (ER) are found in close proximity to each other and it is thought that they maintain contact sites to exchange molecules. The regulation and the function of these contact sites need to be further explored. The small GTPase Arf1 (ADP-ribosylation factor 1), which is best known for its essential role in the generation of coatomer protein I (COPI)-coated vesicles at the Golgi complex appears to be also essential for the dynamics and maintenance of mitochondrial function, presumably at ER–mitochondrial contact sites.

scholarly journals Evidence for Coupled Biogenesis of Yeast Gap1 Permease and Sphingolipids: Essential Role in Transport Activity and Normal Control by Ubiquitination

2007 ◽  
Vol 18 (8) ◽  
pp. 3068-3080 ◽  
Author(s):  
Elsa Lauwers ◽  
Guido Grossmann ◽  
Bruno André
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Surface ◽  
Essential Role ◽  
Surface Proteins ◽  
Transport Activity ◽  
Amino Acid Permease ◽  
Down Regulation ◽  
General Amino Acid Permease ◽  
And Control

Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease.

scholarly journals Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
Author(s):  
C S Copeland ◽  
R W Doms ◽  
E M Bolzau ◽  
R G Webster ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Subunit Interactions ◽  
Oligomer Formation ◽  
Influenza Hemagglutinin ◽  
Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

scholarly journals TspanC8 tetraspanins differentially regulate ADAM10 endocytosis and half-life

2019 ◽  
Vol 3 (1) ◽  
pp. e201900444 ◽  
Author(s):  
Etienne Eschenbrenner ◽  
Stéphanie Jouannet ◽  
Denis Clay ◽  
Joëlle Chaker ◽  
Claude Boucheix ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Notch Signaling ◽  
Essential Role ◽  
Tissue Homeostasis ◽  
Substrate Selectivity ◽  
C Terminus ◽  
Opposite Action ◽  
Chimeric Molecules ◽  
Palmitoylation Site

ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other’s expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins.

Rab proteins of the endoplasmic reticulum: functions and interactors

2012 ◽  
Vol 40 (6) ◽  
pp. 1426-1432 ◽  
Author(s):  
Carolina Ortiz Sandoval ◽  
Thomas Simmen
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Retrograde Trafficking ◽  
Rab Family ◽  
Intermediate Compartment

Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER–Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.

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