Transport of adenosine triphosphate into endoplasmic reticulum proteoliposomes.
We have reconstituted a partially purified extract from rat liver endoplasmic reticulum membrane proteins into phosphatidylcholine liposomes. The resulting proteoliposomes, of an average diameter of 58 nm, transport intact ATP into their lumen in a temperature-dependent manner; transport was saturable (apparent Km = 0.72 microM) and highly specific: CMP-sialic acid and GTP were transported very slowly or not at all. Transport of ATP was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) but not by carboxyatractyloside. Previously, we showed that vesicles derived from rat liver and dog pancreas endoplasmic reticulum translocate ATP into their lumen in vitro but in these studies, following incubations with ATP, most of the phosphate was transferred to proteins because of the many kinases, endogenous acceptors for phosphorylation, and ATP binding proteins present in the vesicle membranes and lumen. This reconstituted system, which yielded a highly functional ATP transporter, can be used for further characterization and purification of this and probably other nucleotide transporters of the endoplasmic reticulum membrane. Previously used reconstitution protocols which were successful for Golgi membrane nucleotide transporters did not yield a functional endoplasmic reticulum ATP transporter.
Guillén
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Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655-0103, USA.
Hirschberg
C B
CB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D010179:Pancreas / Q000378:metabolism; D010766:Phosphorylation; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D008081:Liposomes; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D001278:Atractyloside; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C003853:carboxyatractyloside