A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments:: class E vps phenotype in mammalian cells
A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments:: class E vps phenotype in mammalian cells
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DOI：
10.1242/jcs.00213
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发表时间：
2003-01-15
影响因子：
4
通讯作者：
Himeno, M
中科院分区：
文献类型：
--
作者：
Fujita, H;Yamanaka, M;Himeno, M
SKD 1是与细胞活动相关的ATP酶家族的成员，其酵母同源物Vps 4p已涉及内体/液泡膜转运。当缺乏ATP酶活性的SKD 1突变体[SKD 1（E235 Q）]在哺乳动物细胞中过表达时，其诱导以异常内体结构（表示为E235 Q隔室）为特征的显性阴性表型。SKD 1（E235 Q）的表达引起基底外侧再循环受体的积累，如去唾液酸糖蛋白受体和低密度脂蛋白在极化肝细胞和Madin-Darby犬肾细胞，分别在E235 Q隔室。此外，SKD 1（E235 Q）也废除，通过内涵体，运输到trans-Golgi网络，如TGN 38在E235 Q区室的积累所示。三条证据进一步证明SKD 1参与了从早期内体到晚期内体/溶酶体的膜转运：（1）晚期内体和溶酶体膜蛋白内毒素在E235 Q隔室中的重新分布;（2）由于受体在E235 Q隔室中的积累，抑制表皮生长因子受体的降解;以及（3）新合成的组织蛋白酶D的蛋白水解加工中的错误分选和缺陷。一个有趣的发现是，SKD 1（E235 Q）的表达导致溶酶体数量减少（至对照数量的六分之一），但其大小增加（直径比对照溶酶体大2.4倍）。实际上，超微结构分析显示SKD 1（E235 Q）的表达导致通过晚期内体和溶酶体之间的直接融合形成的杂合细胞器的积累。我们得出结论，SKD 1调节膜运输的早期内体和重组的溶酶体从一个混合细胞器的多个步骤。
SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.