Membrane Targeting and GTPase Activity of Rab7 Are Required for Its Ubiquitination by RNF167

• Published in: International journal of molecular sciences Vol. 23; no. 14; p. 7847
• Main Authors: Ghilarducci, Kim, Cabana, Valérie C., Harake, Ali, Cappadocia, Laurent, Lussier, Marc P.
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 16-07-2022; MDPI
• Subjects: Charcot-Marie-Tooth disease; Charcot–Marie–Tooth Type 2B Rab7 variant; Depletion; DNA repair; endosome; Endosomes; Enzymes; Fractionation; Guanosine triphosphatases; Immunoprecipitation; Localization; Membranes; Plasmids; Post-translation; Protein transport; Proteins; Rab7; Recruitment; RNF167; Ubiquitin; Ubiquitin-protein ligase; Ubiquitination
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23147847

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7′s membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other’s lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot–Marie–Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.