Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.
(65/1930)
ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex. (+info)
Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.
(66/1930)
The surface and cytoplasmic expressions of the transducing chain (IFN-gamma R2) of the heterodimeric IFN-gamma receptor on human T lymphocytes have been investigated. We show that its surface expression is low, whereas high cytoplasmic levels are found in both resting and PHA-activated T lymphocytes. This low expression does not prevent activated T cells from responding to IFN-gamma, because it induces IFN-regulatory factor 1 expression. Low surface IFN-gamma R2 expression appears to be due to recycling between cytoplasmic stores and the cell surface, which does not depend on signals mediated by endogenous IFN-gamma, because IFN-gamma R2 surface expression is low, and its internalization is equally observed in patients with inherited IFN-gamma R1 gene deficiency and in healthy donors. Moreover, IFN-gamma R2 internalization in T lymphoblasts from healthy donors was not affected by the presence of anti-IFN-gamma-neutralizing or anti-IFN-gamma R1-blocking mAb. In conclusion, these data illustrate a new mechanism whereby human T cells limit the surface expression of IFN-gamma R2 in a ligand-independent manner. (+info)
The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway.
(67/1930)
Trafficking of the Na(+)/H(+) exchanger isoform 3 (NHE3) between sub-apical vesicles and apical membrane of epithelial cells is a suggested mechanism of regulation of NHE3 activity. When epitope-tagged NHE3 was stably expressed in NHE-deficient Chinese hamster ovary cells, a sizable fraction was found in recycling endosomes. This system was used to analyze the mechanism of endocytosis of NHE3. Immunofluorescence and radiolabeling experiments showed that inhibition of clathrin-mediated endocytosis using hypertonicity, acid treatment, or K(+) depletion inhibited internalization of NHE3. Moreover, transient transfection of an inhibitory mutant of dynamin (DynS45N) blocked the clathrin-mediated uptake of transferrin, as well as the endocytosis of NHE3. In ileal villus cells, endogenous NHE3 was also found to co-purify with isolated clathrin-coated vesicles, thereby confirming their association in native tissues. The role of COP-I subunits in the intracellular traffic of NHE3 was evaluated using ldlF cells, which bear a temperature-sensitive mutation in the epsilon-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of epsilon-COP, NHE3 was still internalized, but its subcellular distribution was altered. These results indicate that endocytosis of NHE3 occurs primarily via clathrin-coated pits and vesicles and that normal intracellular trafficking of NHE3 involves an epsilon-COP-dependent step. (+info)
Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells.
(68/1930)
Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells. (+info)
Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network.
(69/1930)
Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN. (+info)
Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II.
(70/1930)
Newly synthesized major histocompatibility complex class II molecules (MHC-II) are transported to MHC-II-containing endosomal and lysosomal compartments (MIICs) for the degradation of associated invariant chain and peptide loading. Subsequently MHC-II is transported to the plasma membrane, in part through direct fusion of MIICs with the plasma membrane. In search of potential alternative pathway(s) we studied the 3-dimensional structure of MIICs and the subcellular distribution of MHC-II by immuno electronmicroscopy on whole-mount preparations and cryosections of Mel JuSo cells. Intracellular MHC-II and invariant chain mainly localized to lamp-1 positive compartments suggesting that the majority of MHC-II exits the endocytic tract at lysosomes. Clathrin-coated lattices and buds were found to be associated with these organelles, but MHC-II was not found to be enriched in the clathrin-coated domains. Moreover, leupeptin, a drug that interferes with Ii-processing and delays delivery of newly synthesized MHC-II to the plasma membrane, was not found to decrease the relative amount of MHC-II in clathrin-coated areas. Together these data indicate clathrin-mediated exit site(s) from lysosomes but suggest that they do not selectively recruit mature MHC-II, consistent with the notion that transport to the plasma membrane occurs independently of the cytoplasmic domains of the MHC-II (&agr;) and (beta) chains. (+info)
Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.
(71/1930)
Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components. (+info)
Internalization of proximal tubular type II Na-P(i) cotransporter by PTH: immunogold electron microscopy.
(72/1930)
Physiological/pathophysiological alterations in proximal tubular P(i) reabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-P(i) cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in P(i) reabsorption by new synthesis and BBM insertion of type II Na-P(i) cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-P(i) cotransporters (NaPi-2). In kidney of rats on a chronic low-P(i) diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1-34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2. (+info)