A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis
A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis
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DOI：
10.1074/jbc.ra119.012233
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发表时间：
2020-09-04
影响因子：
4.8
通讯作者：
Fukuda, Mitsunori
中科院分区：
文献类型：
--
作者：
Oguchi, Mai E.;Okuyama, Koki;Fukuda, Mitsunori
初级纤毛是细胞外环境中化学和机械信号的传感器。初级纤毛的形成（即纤毛发生）需要动态的膜运输事件，和几个Rab小GTP酶，膜运输的关键调节剂，最近已被报道参与纤毛发生。然而，在纤毛发生过程中Rab介导的膜运输的确切机制在很大程度上仍然未知。在本研究中，我们使用了一组针对62个人Rabs的siRNA，对调节血清饥饿诱导的人端粒酶逆转录酶视网膜色素上皮1（hTERT-RPE 1）细胞中纤毛发生的Rabs进行了全面的敲除筛选，并成功地将Rab 34鉴定为必需的Rab。在hTERT-RPE 1细胞中，Rab 34基因的敲除（KO），而Rabs基因（如Rab 8和Rab 10）的敲除（KO），可显著削弱血清饥饿诱导的纤毛发生。Rab 34也需要在NIH/3 T3细胞和MCF 10A细胞中血清饥饿诱导的纤毛发生，但不需要在Madin-Darby犬肾（MDCK）-II囊肿中的纤毛发生。然后，我们试图通过对Rab 34进行缺失和突变分析来鉴定Rab 34的特定区域，该区域对于纤毛发生是必需的。出乎意料的是，取代开关II区域中的特定序列，其通常对于识别效应蛋白（例如Rab相互作用溶酶体蛋白[RILP]）是重要的，在保守的GTdR结构域之前Rab 34的独特的长N-末端区域被发现是必需的。这些发现表明Rab 34是一种非典型的Rab，通过其独特的N-末端区域调节血清饥饿诱导的纤毛发生。
Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e.ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g.Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g.Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.