scholarly journals Dominant-Interfering Hsc70 Mutants Disrupt Multiple Stages of the Clathrin-Coated Vesicle Cycle in Vivo

2001 ◽  
Vol 152 (3) ◽  
pp. 607-620 ◽  
Author(s):  
Sherri L. Newmyer ◽  
Sandra L. Schmid
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Initial Rate ◽  
Coated Vesicle ◽  
Coat Proteins ◽  
Heat Shock Cognate Protein ◽  
Vesicle Cycle ◽  
Cognate Protein ◽  
Multiple Stages

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation.

2005 ◽  
Vol 72 ◽  
pp. 65-70 ◽  
Author(s):  
Alexander Flett ◽  
Sophia Semerdjieva ◽  
Antony P. Jackson ◽  
Elizabeth Smythe
Keyword(s):  
Adaptor Protein ◽  
State Level ◽  
Coated Vesicle ◽  
Coat Proteins ◽  
Reversible Phosphorylation ◽  
Permeabilized Cells ◽  
Vesicle Cycle

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the $#x03BC;2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of $#x03BC;2 phosphorylation demonstrated that clathrin is a specific activator of the $#x03BC;2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of $#x03BC;2 phosphorylation. Furthermore, phosphorylated $#x03BC;2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-$#x03BC;2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.

Monoclonal Antibody Specifically Reacting against 73-Kilodalton Heat Shock Cognate Protein: Possible Expression on Mammalian Cell Surface

Hybridoma ◽  
1994 ◽  
Vol 13 (5) ◽  
pp. 373-381 ◽  
Author(s):  
NORIOKI TSUBOI ◽  
MASAKO ISHIKAWA ◽  
YASUAKI TAMURA ◽  
SHINICHI TAKAYAMA ◽  
HIROTOSHI TOBIOKA ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Heat Shock ◽  
Cell Surface ◽  
Mammalian Cell ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein

scholarly journals Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S

1998 ◽  
Vol 111 (13) ◽  
pp. 1877-1888 ◽  
Author(s):  
R. Pepperkok ◽  
M. Lowe ◽  
B. Burke ◽  
T.E. Kreis
Keyword(s):  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Coat Proteins ◽  
Gtp Hydrolysis ◽  
Dominant Mutant ◽  
Vesicular Stomatitis ◽  
Gamma S

Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

The role of nucleoplasmin in chromatin assembly and disassembly

1993 ◽  
Vol 339 (1289) ◽  
pp. 263-269 ◽  
Keyword(s):  
Nuclear Protein ◽  
Sperm Chromatin ◽  
Signal Presentation ◽  
Localization Signal ◽  
Heat Shock Cognate Protein ◽  
Chromatin Assembly And Disassembly ◽  
Cognate Protein

Nucleoplasmin is the most abundant nuclear protein in Xenopus oocytes and eggs. The term ‘molecular chaperone’ was coined to describe its role in the assembly of the nucleosome subunits of chromatin. Although histones and DNA can self-assemble into nucleosomes, nucleoplasmin can facilitate this process in vitro by competing against non-specific charge interactions. In vivo nucleoplasmin binds histones H2A and H2B and transfers them to DNA. Another acidic nuclear protein, N l, binds and transfers histones H3 and H4. Nucleoplasmin has at least one other role in modulating chromatin structure in Xenopus eggs. It is required for the first stage of sperm chromatin decondensation. It binds and removes sperm basic proteins and replaces them by histones H2A and H2B, again forming nucleosomes, and resulting in decondensation of the compacted sperm chromatin. In addition we propose that the properties of the nuclear localization signal of nucleoplasmin can be explained by a model in which heat shock cognate protein hsc70 has a chaperone role in signal presentation during nuclear transport.

Non-anticoagulant functions of heparin and heparan sulfate

1996 ◽  
Vol 16 (01) ◽  
pp. 6-14 ◽  
Author(s):  
E. Buddecke
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Structural Features ◽  
Coat Proteins ◽  
Antiproliferative Effects ◽  
Viral Coat ◽  
Adhesive Proteins

SummaryHeparin is known to bind a large number of proteins not involved in anticoagula-tion, such as growth factors, adhesive proteins of the extracellular matrix, viral coat proteins and other enzymes and proteins. In vivo predominantly heparan sulfate – the most ubiquitous cell surface glycosaminoglycan – takes the functional role of heparin. Structural features, sources and non-anticoagulant func-tions of heparin and heparan sulfate proteoglycan are described. The functional diversity of heparin and heparan sulfate is reviewed in the following sections: (I) heparin and heparan sulfate as partners in fibroblast growth factor action, (II) antiproliferative effects of heparan sulfate and heparin, (III) cell surface heparan sulfate as extracellular matrix receptor and coreceptor, (IV) proteoheparan sulfate in central and peripheral nervous system, (V) role of proteoheparan sulfate in binding and uptake of lipoproteins, (VI) virus and spirochete binding to heparin and heparan sulfate.

scholarly journals GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

1998 ◽  
Vol 140 (3) ◽  
pp. 565-575 ◽  
Author(s):  
Maria L. Wei ◽  
Frank Bonzelius ◽  
Rebecca M. Scully ◽  
Regis B. Kelly ◽  
Gary A. Herman
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Glucose Transporter ◽  
Immunofluorescence Microscopy ◽  
Neuroendocrine Cells ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Double Labeling

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

scholarly journals Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alina Khramova ◽  
Roberto Boi ◽  
Vincent Fridén ◽  
Anna Björnson Granqvist ◽  
Ulf Nilsson ◽  
...  
Keyword(s):  
Surface Layer ◽  
Kidney Disease ◽  
Cell Surface ◽  
Diabetic Kidney Disease ◽  
Exchange Chromatography ◽  
Diabetic Kidney ◽  
Endothelial Cell Surface ◽  
Filtration Barrier ◽  
Essential Components

AbstractAll capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.

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