Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Small GTPases. 2022 Jan;13(1):77-83. doi: 10.1080/21541248.2021.1894910. Epub 2021 Apr 16.
We have previously shown that Rab34 is an important regulator of ciliogenesis and that its unique long N-terminal region (amino acids 1-49) is essential for ciliogenesis in certain cultured mammalian cells. In the present study, we performed an in-depth deletion analysis of the N-terminal region of Rab34 together with Ala-based site-directed mutagenesis to identify the essential amino acids that are required for serum-starvation-induced ciliogenesis in hTERT-RPE1 cells. The results showed that a Rab34 mutant lacking an N-terminal 18 amino acids and a Rab34 mutant carrying an LPQ-to-AAA mutation (amino acids 16-18) failed to rescue a Rab34-KO phenotype (i.e., defect in ciliogenesis). Our findings suggest that the LPQ sequence of Rab34 is crucial for ciliogenesis in hTERT-RPE1 cells. AA, amino acid(s); ac-Tub, acetylated tubulin; bsr, blasticidin S-resistant gene; HRP, horseradish peroxidase; hTERT-RPE1, human telomerase reverse transcriptase retinal pigment epithelium 1; KO, knockout; NS, not significant; PBS, phosphate-buffered saline; puro, puromycin-resistant gene.
我们之前已经证明 Rab34 是纤毛发生的重要调节因子，其独特的长 N 端区域（氨基酸 1-49）对于某些培养的哺乳动物细胞中的纤毛发生是必需的。在本研究中，我们对 Rab34 的 N 端区域进行了深入的缺失分析，同时进行了基于丙氨酸的定点突变，以确定在 hTERT-RPE1 细胞中血清饥饿诱导的纤毛发生所必需的关键氨基酸。结果表明，缺失 N 端 18 个氨基酸的 Rab34 突变体和携带 LPQ-to-AAA 突变（氨基酸 16-18）的 Rab34 突变体均不能挽救 Rab34-KO 表型（即纤毛发生缺陷）。我们的研究结果表明，Rab34 的 LPQ 序列对于 hTERT-RPE1 细胞中的纤毛发生至关重要。AA，氨基酸；ac-Tub，乙酰化微管；bsr，博来霉素抗性基因；HRP，辣根过氧化物酶；hTERT-RPE1，人端粒酶逆转录酶视网膜色素上皮 1；KO，敲除；NS，不显著；PBS，磷酸盐缓冲盐水；puro，嘌呤霉素抗性基因。
