Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte.
Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte.
复制标题
DOI：
10.1083/jcb.136.6.1227
复制
发表时间：
1997-03-24
期刊：
影响因子：
--
通讯作者：
Stahl PD
中科院分区：
文献类型：
--
作者：
Mukhopadhyay A;Barbieri AM;Funato K;Roberts R;Stahl PD
为了探索GTP酶在内吞作用中的作用，我们开发了一种使用注射重组蛋白的非洲爪蟾卵母细胞来跟踪液相标记物HRP的摄取的测定。在注射GTPγS或与氟化铝孵育的细胞中，HRP摄取被抑制，表明GTP酶在内吞作用中的一般作用。将Rab 5注射到卵母细胞中，以及Rab 5：Q79 L，一种具有降低的GT酶活性的突变体，增加HRP摄取。注射Rab 5：S34 N（显性负突变体）抑制HRP摄取。注射N-乙基马来酰亚胺敏感因子（NSF）刺激HRP的摄取，ATP酶缺陷的NSF突变体抑制HRP的摄取时，共注射Rab 5：Q79 L，证实了NSF的内吞作用的要求。令人惊讶的是，注射Rab 7：WT刺激HRP的摄取和降解/活化。后者似乎是由于增强运输到晚期内体/前溶酶体降解室，这是莫能菌素敏感。由于Rab 7的刺激作用被Rab 5：S34 N共注射阻断，Rab 7的摄取增强似乎通过Rab 5敏感途径在卵母细胞中起作用。Rab 5：S34 N可阻断Rab 5对细胞摄取的刺激作用，但Rab 7：T22 N则不能阻断Rab 5对细胞摄取的刺激作用。我们的研究结果表明，Rab 7，而Rab 5的下游功能，可能是限制率的卵母细胞的内吞作用。
To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.