Rab10 function in tubular endosome formation requires the N-terminal K3 residue and is disrupted by N-terminal tagging

ABSTRACT 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.