Hata Rinka, Sugawara Akira, Fukuda Mitsunori
Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
J Cell Sci. 2025 Feb 1;138(3). doi: 10.1242/jcs.263649. Epub 2025 Feb 7.
Various N-terminal tags have often been used to identify the functions and localization of Rab small GTPases, but their impact on Rab proteins themselves has been poorly investigated. Here, we used a knockout (KO)-rescue approach to systematically evaluate the effect of N-terminal tagging of two Rabs, Rab10 and Rab27A, on RAB10-KO HeLa cells and Rab27A-deficient melanocytes (melan-ash cells), respectively. The results showed that all of the N-terminal-tagged Rab27A proteins mediated actin-based melanosome transport in the melan-ash cells, but none of the N-terminal-tagged Rab10 proteins fully rescued the defect in tubular endosome formation in RAB10-KO cells. Although the N-terminal-tagged Rab10 proteins had the ability to localize tubular endosomes in wild-type HeLa cells, they sometimes exhibited a dominant-negative effect on tubular endosome formation. We also found that a conserved lysine residue at amino acid position 3 (K3) in the Rab10 proteins of different species is required for tubular endosome formation. Thus, it will be important to determine whether other Rab isoforms with N-terminal tags behave similarly to their corresponding untagged isoforms by performing appropriate KO-rescue experiments in future studies.
各种N端标签常被用于识别Rab小GTP酶的功能和定位，但其对Rab蛋白本身的影响却鲜有研究。在此，我们采用基因敲除（KO）-拯救方法，分别系统评估了两种Rab蛋白（Rab10和Rab27A）的N端标签对RAB10基因敲除的HeLa细胞和Rab27A缺陷的黑素细胞（黑素灰细胞）的影响。结果表明，所有N端带标签的Rab27A蛋白均介导黑素灰细胞中基于肌动蛋白的黑素小体转运，但N端带标签的Rab10蛋白均不能完全挽救RAB10基因敲除细胞中管状内体形成的缺陷。尽管N端带标签的Rab10蛋白能够在野生型HeLa细胞中定位于管状内体，但它们有时会对管状内体形成产生显性负效应。我们还发现，不同物种Rab10蛋白中第3位氨基酸处保守的赖氨酸残基（K3）是管状内体形成所必需的。因此，在未来的研究中通过进行适当的KO-拯救实验来确定其他带N端标签的Rab异构体是否与其相应的无标签异构体表现相似将很重要。
