Jansen E J, Holthuis J C, McGrouther C, Burbach J P, Martens G J
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands.
J Cell Sci. 1998 Oct;111 ( Pt 20):2999-3006. doi: 10.1242/jcs.111.20.2999.
Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.
Ac45是一种与液泡H⁺-ATP酶相关的I型跨膜蛋白，液泡H⁺-ATP酶是一种质子泵，介导多种细胞内细胞器的酸化。在本研究中，我们检测了转染的CV-1成纤维细胞中Ac45的细胞内转运途径。稳态免疫标记显示Ac45位于质膜和近核区域的一个液泡区室中。抗体内化实验表明Ac45能迅速从细胞表面被回收，并靶向液泡结构。Ac45的26个氨基酸残基的胞质尾巴本身能够介导细胞表面蛋白Tac的内吞作用，这表明该尾巴包含一个自主内化信号。对表达羧基末端截短的Ac45突变体的细胞进行免疫定位研究表明，在胞质尾巴的膜远端区域存在重要的转运信息。对该缺乏公认的基于酪氨酸或双亮氨酸的分选基序的区域进行进一步的突变分析表明，多个位点而非短线性序列负责内化作用。总的来说，我们的结果表明Ac45的胞质尾巴包含与先前描述的转运决定因素不同的自主靶向信息。
