Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.
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PMID:ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70. 969 77

Cancer chemotherapy often results in the development of multidrug resistance (MDR), which is commonly associated with overexpression of P-glycoprotein (P-gp), a plasma membrane drug efflux ATPase. It was shown recently that glycosphingolipids are elevated in MDR cells. Sphingolipids are major constituents of caveolae and of detergent-insoluble, glycosphingolipid-rich membrane domains. Here we report that multidrug-resistant HT-29 human colon adenocarcinoma cells exhibit a 12-fold overexpression of caveolin-1, a 21-kDa coat/adaptor protein of caveolae. Similar observations were made in adriamycin-resistant MCF-7 human breast adenocarcinoma cells. Caveolin-2 expression is also up-regulated in MCF-7-AdrR cells, but neither caveolin-1 nor caveolin-2 were detected in MCF-7 cells stably transfected with P-gp. The up-regulation of caveolins is associated with a 5-fold increase in the number of caveolae-like structures observed in plasma membrane profiles of HT-29-MDR cells and with the appearance of a comparable number of caveolae in MCF-7-AdrR cells. A significant fraction (approximately 40%) of cellular P-gp is localized in low density detergent-insoluble membrane fractions derived from either HT-29-MDR or MCF-7-AdrR cells. The distribution of recombinant P-gp in stably transfected MCF-7 cells was similar, even though these cells do not express caveolins and are devoid of caveolae. The possibility that caveolae contribute to the multidrug resistance and thus are co-selected with P-gp during the acquisition of this phenotype is discussed.
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PMID:Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells. 982 65

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.
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PMID:Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein. 1055 5

Inhibition of Na(+),K(+)-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic alpha-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the alpha-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing alpha-subunit phosphorylation, but by impaired endocytosis of alpha-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit. Phosphorylation of the alpha-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of alpha-subunit phosphorylation; increased InsP(6) in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.
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PMID:G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin. 1071 25

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.
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PMID:Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking. 1082 93

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.
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PMID:Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity. 1117 28

A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.
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PMID:Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli. 1167 35

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.
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PMID:Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis. 1185 87

Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.
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PMID:Subunit H of the V-ATPase binds to the medium chain of adaptor protein complex 2 and connects Nef to the endocytic machinery. 1203 42

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the beta-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the beta-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For beta-adaptins, this fragment binds to cytoplasmic di-leucine-based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine-based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold.
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PMID:Subunit H of the V-ATPase involved in endocytosis shows homology to beta-adaptins. 1205 68


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