Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum
Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum
复制标题
DOI：
10.1091/mbc.8.7.1343
复制
发表时间：
1997-07-01
影响因子：
3.3
通讯作者：
Cardelli, J
中科院分区：
文献类型：
--
作者：
Buczynski, G;Bush, J;Cardelli, J
哺乳动物小分子量GTTRab 7（酵母中的Ypt 7）参与调节内体途径沿着内化后膜运输。从盘基网柄藻（Dictyosteelium discoideum）中克隆了一个编码与哺乳动物Rab 7在氨基酸水平上85%相同的蛋白质的cDNA。亚细胞分级和免疫荧光显微镜表明，Rab 7富集在溶酶体，postylsosomes，和成熟的吞噬体。产生过表达Rab 7野生型（WT）、Rab 7 Q67 L（组成型活性形式）和Rab 7 T22 N（显性阴性形式）蛋白的细胞系。Rab 7 T22 N细胞系以对照细胞的三分之一的速率内化流体相标记物和乳胶珠（吞噬作用），而Rab 7 WT和Rab 7 Q67 L细胞系在摄取速率方面是正常的，但比对照细胞更快地胞吐流体相。与此相反，流体相标记物驻留在酸性隔室中的时间更长，并且与对照细胞相比，从Rab 7 T22 N细胞中胞吐的速度更慢。光学显微镜检查表明Rab 7表达细胞系含有形态学改变的内体区室。与对照细胞相比，表达Rab 7 WT和Rab 7 Q67 L的细胞中溶酶体后大小的囊泡（>2.5 μ m）数量减少，而较小的囊泡数量增加，其中许多是非酸性的;在对照细胞中，>90%的较小囊泡是酸性的。与此相反，Rab 7 T22 N细胞含有相对于非酸性囊泡的大酸性囊泡的比例增加。放射性标记脉冲追踪实验表明，所有细胞系均正常加工并靶向溶酶体α-甘露糖苷酶，这表明Rab 7在靶向途径中缺乏重要作用;然而，Rab 7 WT和Rab 7 T22 N细胞系中成熟溶酶体水解酶的保留受到影响。与液相流出实验观察到的结果相反，Rab 7 T22 N细胞过度分泌α-甘露糖苷酶，而Rab 7 WT细胞与对照细胞相比保留了这种水解酶。这些数据支持Rab 7可能调节溶酶体酶和V型H+-ATP酶从后溶酶体到溶酶体的逆行转运以及从细胞中有效释放流体相的模型。
The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postylsosomes, and maturing phagosomes. Cell lines were generated that overexpressed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles the size of postlysosomes (>2.5 mu m) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, >90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H+-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.