The N-terminal Leu-Pro-Gln sequence of Rab34 is required for ciliogenesis in hTERT-RPE1 cells-猫眼课题宝

DOI：

10.1080/21541248.2021.1894910

发表时间：

2021-04

期刊：

Small GTPases

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Mai E Oguchi;Y. Homma;M. Fukuda

我们以前已经证明Rab 34是纤毛发生的重要调节因子，其独特的长N-末端区域（氨基酸1-49）是某些培养的哺乳动物细胞中纤毛发生所必需的。在本研究中，我们进行了深入的缺失分析的N-末端区域的Rab 34连同基于Ala的定点突变，以确定所需的血清饥饿诱导的hTERT-RPE 1细胞中的纤毛发生的必需氨基酸。结果显示，缺少N-末端18个氨基酸的Rab 34突变体和携带LPQ至AAA突变（氨基酸16-18）的Rab 34突变体不能拯救Rab 34-KO表型（即，纤毛发生缺陷）。我们的研究结果表明Rab 34的LPQ序列对hTERT-RPE 1细胞中的纤毛发生至关重要。缩略语：AA，氨基酸; ac-Tub，乙酰化微管蛋白; bsr，杀稻瘟菌素S耐药基因; HRP，辣根过氧化物酶; hTERT-RPE 1，人端粒酶逆转录酶视网膜色素上皮1; KO，敲除; NS，不显着; PBS，磷酸盐缓冲液; puro，嘌呤霉素耐药基因

ABSTRACT 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. Abbreviations: 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

The N-terminal Leu-Pro-Gln sequence of Rab34 is required for ciliogenesis in hTERT-RPE1 cells

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