Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition, and Behaviour and Nijmegen Centre for Molecular Life Sciences (NCMLS), Faculty of Science, Radboud University Nijmegen, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands.
J Biol Chem. 2012 Aug 10;287(33):27537-46. doi: 10.1074/jbc.M112.356105. Epub 2012 Jun 26.
The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.
液泡（H(+))-ATP 酶（V-ATPase）对于维持细胞内细胞器的酸性微环境至关重要，而其膜结合的 V(0)-部分则参与 Ca(2+)-依赖性膜融合。在分泌途径中，V-ATPase受其 I 型跨膜和 V(0)-相关辅助亚基 Ac45 调节。为了执行其功能，完整的 Ac45 蛋白被蛋白水解加工成裂解的 Ac45，从而释放其 N 端结构域。在这里，我们通过分析一组接近体内情况的缺失突变体（即在转基因非洲爪蟾中间垂体黑素细胞中）来寻找 Ac45 中的功能域。完整的 Ac45 加工不良并在转基因黑素细胞的内质网中积累。相比之下，裂解的 Ac45 有效地通过分泌途径运输，导致 V-ATPase在质膜处积累，并减少多巴胺能抑制 Ca(2+)-依赖性肽分泌。令人惊讶的是，从完整的 Ac45 中去除 C 尾会导致细胞表型也类似于裂解的 Ac45，而从裂解的 Ac45 中去除 C 尾仍允许其运输到质膜，但会阻止 V-ATPase 招募到分泌途径，并使多巴胺能抑制细胞不受影响。我们得出结论，位于 Ac45 蛋白的 N-和 C-末端的结构域指导其运输、V-ATPase 招募和 Ca(2+)-依赖性调节的胞吐作用。
