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Rab5和Rab7的顺序作用调节非洲爪蟾卵母细胞的内吞作用。

Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte.

作者信息

Mukhopadhyay A, Barbieri A M, Funato K, Roberts R, Stahl P D

机构信息

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

出版信息

J Cell Biol. 1997 Mar 24;136(6):1227-37. doi: 10.1083/jcb.136.6.1227.

DOI:10.1083/jcb.136.6.1227
PMID:9087439
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2132516/
Abstract

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 GTPgammaS 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.

摘要

为了探究GTP酶在胞吞作用中的作用，我们开发了一种检测方法，利用注射了重组蛋白的非洲爪蟾卵母细胞来追踪液相标记物辣根过氧化物酶（HRP）的摄取情况。在注射了GTPγS或与氟化铝孵育的细胞中，HRP摄取受到抑制，这表明GTP酶在胞吞作用中具有普遍作用。将Rab5以及GTP酶活性降低的突变体Rab5:Q79L注射到卵母细胞中，可增加HRP摄取。注射显性负性突变体Rab5:S34N可抑制HRP摄取。注射N - 乙基马来酰亚胺敏感因子（NSF）可刺激HRP摄取，而ATP酶缺陷型NSF突变体与Rab5:Q79L共同注射时可抑制HRP摄取，这证实了NSF在胞吞作用中的必要性。令人惊讶的是，注射野生型Rab7可刺激HRP的摄取和降解/激活。后者似乎是由于转运增强至对莫能菌素敏感的晚期内体/溶酶体前降解区室。Rab7对摄取的增强作用似乎通过卵母细胞中Rab5敏感的途径发挥作用，因为Rab7的刺激作用可被Rab5:S34N共同注射所阻断。Rab5对摄取的刺激作用可被Rab5:S34N阻断，但不能被Rab7:T22N阻断。我们的结果表明，Rab7虽然在Rab5下游发挥作用，但可能是卵母细胞胞吞作用的限速因素。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e18c/2132516/1027f2167d6e/JCB.mukhopadhyay9.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e18c/2132516/06724c748f5a/JCB.mukhopadhyay1.jpg
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