Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.
We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.
Milla
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ME
Department of Biochemistry, University of Massachusetts Medical Center, Worcester 01655.
Hirschberg
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CB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003568:Cytidine Monophosphate / Q000494:pharmacology; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005615:Freezing; D006056:Golgi Apparatus / Q000648:ultrastructure*; D006358:Hot Temperature; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008099:Liver / Q000648:ultrastructure*; D009711:Nucleotides / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000378:metabolism*
D000227:Adenine Nucleotides; D008081:Liposomes; D009711:Nucleotides; D012794:Sialic Acids; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D010724:Phosphoadenosine Phosphosulfate; D003568:Cytidine Monophosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid