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

Abstract

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.

Suspension culture promoted the expansion of NK‐92 cells ex vivo by enhancing the expression of IL‐2 receptor

“…The endocytic vesicle of the IL‐2‐IL‐2R complex usually fuses with a membrane‐bound structure called the early endosome (molecular marker EEA1), then a part of receptors binds to the recycling endosomes (molecular marker Rab11) and recycled back to the cell surface 28 . While the other part of receptors and overall IL‐2 binds to the late endosomes (molecular marker Rab7) and then enters the lysosome (molecular marker Lamp1) to be degraded 29 . To measure the cycle and degradation of IL‐2‐IL‐2R complex during endocytosis, the Pearsons's correlation coefficient of EEA1, Rab7, Rab11, and Lamp1 with IL‐2Rβ were analyzed respectively to determine the colocalization of key proteins and IL‐2Rβ during endocytosis.…”

“…28 While the other part of receptors and overall IL-2 binds to the late endosomes (molecular marker Rab7) and then enters the lysosome (molecular marker Lamp1) to be degraded. 29 cells was also significantly increased, indicating that the aggregation of Rab11 formed in the cells was also considerably enhanced in suspension culture (Figure 3L), suggesting that more IL-2Rβ was returned to the membrane through the recycling endosome for reuse. Then, the fluorescence intensity of late endosome, autolysosome, and their colocalization coefficients with IL-2Rβ were analyzed.…”

Regulation of Ubiquitin Family Signaling in Disease

“…Ghilarducci et al [ 6 ] identify RNF167 as a novel ubiquitin ligase for Rab7, a GTPase that plays a significant role in regulating late endosome and lysosome trafficking. In vitro assays demonstrate that Rab7 is a direct substrate of RNF167 and that RNF167 activity maintains Rab7’s membrane localization.…”