Translocation of ATP into the lumen of rough endoplasmic reticulum-derived vesicles and its binding to luminal proteins including BiP (GRP 78) and GRP 94.
Rat liver and canine pancreas rough endoplasmic reticulum-derived vesicles, which were sealed and of the same topographical orientation as in vivo, were used in a system in vitro to demonstrate translocation of ATP into their lumen. Translocation of ATP is saturable (apparent Km: 3-4 microM and Vmax: 3-7 pmol/min/mg of protein) and protein mediated because treatment of intact vesicles with Pronase, N-ethylmaleimide, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibit transport. The entire ATP molecule is being translocated; this was shown by high performance liquid chromatography analysis and the use of a nonhydrolyzable analog. Control experiments rule out that translocation of ATP attributed to rough endoplasmic reticulum-derived vesicles is due to contamination by mitochondria and Golgi vesicles. Following translocation of ATP into the lumen of the vesicles, binding to luminal proteins including BiP (immunoglobulin heavy chain-binding protein-glucose-regulated protein 78) and glucose-regulated protein 94 was observed. This binding appeared to be specific because similar experiments with GTP were negative. These studies strongly suggest that translocation of ATP into the rough endoplasmic reticulum lumen may serve as a mechanism for making ATP available in proposed energy requiring reactions within the lumen.
Clairmont
C A
CA
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655.
De Maio
A
A
Hirschberg
C B
CB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002458:Cell Fractionation; D003432:Cross-Linking Reagents; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains
D003432:Cross-Linking Reagents; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid