TAILIEUCHUNG - Báo cáo khoa học: The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway

The vacuolar H + -ATPase (V-ATPase) is a multimeric enzyme complex that acidi®es organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interactwith themembrane sector of theV-ATPase inonlya subset of organelles. | Eur. J. Biochem. 269 1844-1853 2002 FEBS 2002 doi The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway Vincent Th. G. Schoonderwoert Eric J. R. Jansen and Gerard J. M. Martens Department of Animal Physiology Nijmegen Center for Molecular Life Sciences University of Nijmegen the Netherlands The vacuolar H -ATPase V-ATPase is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact w 46-kDa Ac45 was found to be N-linked glycosylated to w 62 kDa and a minor fraction to w 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of w 61 and w 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa the deglycosylated form is w 23 kDa and a previously not detected w 22-kDa N-terminal cleavage fragment the deglycosylated form is w 20 kDa . Degradation of the N-terminal fragment is rapid does not occur in lysosomes and is inhibited by brefeldin A. Both the N- and C-terminal fragment pass the medial Golgi as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process half-life of intact Ac45 is 4-6 h and takes place in the early secretory pathway as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process suggesting that Ac45 needs proper folding for the cleavage to occur. Together our results indicate that Ac45 folding and cleavage occur slowly and .

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