Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase

Ding, Jiantao; Zhao, Weihong; Crider, Bill P.; Ma, Ying; Li, Xinji; Slaughter, Clive A.; Gong, Limin; Xie, Xiao‐Song

doi:10.1074/jbc.m910319199

Cited by 106 publications

(101 citation statements)

References 25 publications

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“…(iii) The erythrocyte Mg 2ϩ -ATPase does not seem to pump Mg 2ϩ , H ϩ , or other ions tested across the erythrocyte plasma membrane (30). (iv) PS stimulates ATP hydrolysis by ATPase II and the erythrocyte Mg 2ϩ -ATPase, suggesting that it is a substrate of these enzymes (25,29). (v) Phylogenetic analysis of the Drs2p family of P-type ATPases indicates a substantial divergence from ion and heavy-metal transporters (31).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Natarajan

Wang

Hua

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

184

222

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Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p͞ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.

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Section: Discussionmentioning

confidence: 99%

“…PE is able to stimulate ATP hydrolysis by purified ATPase II at Ϸ15% of the rate induced by PS (29). Perhaps the failure to detect translocation of NBD-PE by Drs2p was caused by the large excess of preferred substrate, because PS comprises Ϸ20% of the Golgi membranes used in the translocase assays described above.…”

Section: Figmentioning

confidence: 99%

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Natarajan

Wang

Hua

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

184

222

View full text Add to dashboard Cite

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“…Since phospholipids are pumped from the outer, lumenal leaflet to the inner leaflet, phospholipids should bind to the E2P form; that binding (and occlusion of the bound phospholipid) would then be the prerequisite for dephosphorylation of the enzyme. In fact, the addition of phospholipid has been shown to promote dephosphorylation of the E2P form of the P4 enzyme [6,7,10].…”

Section: Common Mechanism Of Phosphorylation and Dephosphorylationmentioning

confidence: 99%

Outside of the box: recent news about phospholipid translocation by P4 ATPases

Stone

Williamson

2012

J Chem Biol

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The P4 subfamily of P-type ATPases includes phospholipid transporters. Moving such bulky amphipathic substrate molecules across the membrane poses unique mechanistic problems. Recently, three papers from three different laboratories have offered insights into some of these problems. One effect of these experiments will be to ignite a healthy debate about the path through the enzyme taken by the substrate. A second effect is to suggest a counterintuitive model for the critical substrate-binding site. By putting concrete hypotheses into play, these papers finally provide a foundation for investigations of mechanism for these proteins.Keywords P4 ATPase . Phospholipid transport . ATPase reaction cycleFraming the problem P-type ATPases are ATP-dependent ion transporters that derive their name from an aspartate that becomes transiently phosphorylated during the reaction cycle [17]. The structures of several members of the family have been determined at atomic resolution in the last decade, and they all have a large tripartite cytoplasmic domain and a small extracellular domain connected by six to ten transmembrane helices. The P4 subfamily of P-type ATPases was first defined in 1996, when the gene encoding an aminophospholipid translocase, ATP8A1, from bovine chromaffin granules [26] was found to be a relative of the previously identified yeast P-type ATPase, Drs2p [23]. P4 type ATPases are found in all eukaryotic organisms but not in prokaryotes. In vertebrates, there are 14 genes that encode P4 ATPases [9] while in yeast there are five; Drs2, Dnf1, Dnf2, Dnf3, and Neo1 [19]. Unlike the other subfamilies of P-type ATPases, which all transport simple ions, the only known substrates of the P4 ATPases are phospholipids.The transport mechanism of the P-type ATPases depends on sequential partial reactions of ATP hydrolysis-phosphorylation and dephosphorylation of the critical aspartic acid residue in the cytoplasmic P domain-and linked sequential conformational changes; coordination between chemical and conformational steps drives substrate across the membrane against a concentration gradient. The conformational changes link a binding pocket for the transported ion to either the cytoplasm (the E1 conformation) or the extracytoplasmic, or lumenal space (the E2 conformation). In the classic Post-Albers model [1,22], binding of ions in the E1 conformation enables phosphorylation to the E1P form; this form is conformationally unstable, and converts to the E2P conformation. Loss of ions into the luminal space from that conformation is the key to dephosphorylation to the E2 form. The latter is also conformationally unstable, and the enzyme thus converts back into the E1 conformation [1,22].There is a subtlety about this sequence of events which should be mentioned here, particularly as it applies to the E2P conformation. This latter designation must define two conformations; indeed, both have been crystallized in the case of the Ca-ATPase [15,16,27]. One is the form that releases the cytoplasmically bound ions into th...

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“…However, this ATPase activity was insensitive to orthovanadate, a commonly used P-type ATPase inhibitor (16,17), and was completely inhibited by 1 mM azide (Fig. 1D).…”

Section: Drs2mentioning

confidence: 99%

“…A flippase activity toward spin-labeled PS and PE analogues has been reconstituted with a partially purified, but unidentified, Mg 2ϩ -ATPase from human red blood cells (14). ATPase II/Atp8a1, a Mg 2ϩ -ATPase that potentially catalyzes aminophospholipid translocase activity in bovine chromaffin granules (15), has been purified to homogeneity (16,17). Phylogenetic analysis of the ATPase II/Atp8a1 sequence indicates that it is a member of the P4-ATPase family, which includes Fic1/Atp8b1 and Drs2p from yeast (18,19).…”

mentioning

confidence: 99%

Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast

Zhou

Graham

2009

Proc. Natl. Acad. Sci. U.S.A.

145

163

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Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg 2؉ -ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.flippase ͉ membrane asymmetry ͉ P4-ATPase ͉ proteoliposome ͉ Cdc50p

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Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase

Cited by 106 publications

References 25 publications

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Outside of the box: recent news about phospholipid translocation by P4 ATPases

Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast

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