We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.
Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes. We have developed pulse-chase and mating assays to follow the fate of existing peroxisomes, de novo–formed peroxisomes, and ER-derived preperoxisomal structures. We find that in wild-type (WT) cells, peroxisomes multiply by fission and do not form de novo. A marker for the maturation pathway, Pex3-GFP, is delivered from the ER to existing peroxisomes. Strikingly, cells lacking peroxisomes as a result of a segregation defect do form peroxisomes de novo. This process is slower than peroxisome multiplication in WT cells and is Drp independent. In contrast, peroxisome fission is Drp dependent. Our results show that peroxisomes multiply by growth and division under our assay conditions. We conclude that the ER to peroxisome pathway functions to supply existing peroxisomes with essential membrane constituents.
Pexophagy, peroxysome autophagy, is regulated in Saccharomyces cerevisiae by Atg36 by direct binding to peroxysome regulator Pex3, Atg8 and Atg11 of the core autophagy machinery.
The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.
We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the ␥-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH 2 -terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTP␥S, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.
Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disease characterized clinically by a disproportionately short stature primarily affecting the proximal parts of the extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Although some patients have single enzyme deficiencies, the majority of RCDP patients (86%) belong to a single complementation group (CG11, also known as complementation group I, Amsterdam nomenclature). Cells from CG11 show a tetrad of biochemical abnormalities: a deficiency of i) dihydroxyacetonephosphate acyltransferase, ii) alkyldihydroxyacetonephosphate synthase, iii) phytanic acid alpha-oxidation and iv) inability to import peroxisomal thiolase. These deficiencies indicate involvement of a component required for correct targeting of these peroxisomal proteins. Deficiencies in peroxisomal targeting are also found in Saccharomyces cerevisiae pex5 and pex7 mutants, which show differential protein import deficiencies corresponding to two peroxisomal targeting sequences (PTS1 and PTS2). These mutants lack their PTS1 and PTS2 receptors, respectively. Like S. cerevisiae pex cells, RCDP cells from CG11 cannot import a PTS2 reporter protein. Here we report the cloning of PEX7 encoding the human PTS2 receptor, based on its similarity to two yeast orthologues. All RCDP patients from CG11 with detectable PEX7 mRNA were found to contain mutations in PEX7. A mutation resulting in C-terminal truncation of PEX7 cosegregates with the disease and expression of PEX7 in RCDP fibroblasts from CG11 rescues the PTS2 protein import deficiency. These findings prove that mutations in PEX7 cause RCDP, CG11.
Summary Yeast peroxisomes multiply by fission. Fission requires two dynamin-related proteins, Dnm1p and Vps1p. We show that Dnm1p-dependent peroxisome fission requires Fis1p, Caf4p and Mdv1p using an in vivo fission assay. Fluorescence microscopy of cells expressing GFP-tagged Caf4p and Mdv1p revealed that their association with peroxisomes relies on Fis1p. Vps1p-dependent peroxisome fission occurs independently of these factors. Vps1p contributes most to fission of peroxisomes when cells are grown on glucose. Overexpression of Dnm1p suppresses the fission defect as long as Fis1p and either Mdv1p or Caf4p are present. Conversely, overexpression of Dnm1p does not restore the vacuolar fusion defect of vps1 cells, and Vps1p overexpression does not restore the mitochondrial fission defect of dnm1 cells. These data show that Vps1p and Dnm1p are part of independent fission machineries. Since the contribution of Dnm1p to peroxisome fission appears to be more pronounced in cells that proliferate peroxisomes in response to mitochondrial dysfunction, Dnm1p might be part of the mechanism that coordinates mitochondrial and peroxisomal biogenesis.
Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. INTRODUCTIONLike many viruses, human immunodeficiency virus (HIV)-1 has evolved strategies to avoid detection and destruction by the host. One such strategy is the down-regulation of major histocompatability complex (MHC) class I from the plasma membrane of HIV-1-infected cells, which prevents the cells from being attacked by cytotoxic T lymphocytes. MHC-I down-regulation has been shown to be a function of a virally encoded protein called Nef (Schwartz et al., 1996;Collins et al., 1998). Nef is one of the so-called "accessory proteins" of both human and simian immunodeficiency viruses: although not required for viral replication in vitro, Nef plays a key role in the development of acquired immunodeficiency syndrome. Nef is a small protein, only ϳ200 amino acids, but it has been reported to interact with a large number of host cell proteins, including several proteins that are involved in membrane traffic. These interactions are thought to be responsible for the ability of Nef to modulate the surface expression of MHC-I and other molecules (for review, see Collins and Baltimore, 1999;Piguet et al., 1999;Doms and Trono, 2000;Roeth and Collins, 2006).Among the binding partners that have been identified for Nef are the adaptor protein (AP) complexes. There are four AP complexes in mammalian cells, two of which, AP-1 and AP-2, are highly enriched in clathrin-coated vesicles (CCVs). AP-1 facilitates clathrin-mediated trafficking between the trans-Golgi network (TGN) and endosomes (although there is still some question about directionality), and AP-2 facilitates clathrin-mediated endocytosis (Robinson, 2004). AP-3, which seems to be able to act both in a clathrin-dependent and in a clathrin-independent manner (Dell'Angelica et al., 1998;Peden et al., 2...
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