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

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.

RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association.

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

Functional interaction of ubiquitin ligase RNF167 with UBE2D1 and UBE2N promotes ubiquitination of AMPA receptor.

Protein ubiquitination has been historically associated with protein degradation, but recent studies have demonstrated other cellular functions associated with substrate ubiquitination. Among the RING-type ubiquitin E3 ligase enzymes present in the human genome, RNF167 is a transmembrane protein located in endosomes and lysosomes and is implicated in controlling the endolysosomal pathway. Substrates of RNF167 have been identified, but the ubiquitin-conjugating E2 enzymes involved in the mechanism remain unknown. In this study, we describe the interaction between RNF167 and conjugating E2 enzymes. By means of in vitro autoubiquitination and binding assays, we show that RNF167 functionally interacts with many conjugating E2s, while fluorescence microscopy illustrates that these interactions occur in endosomes and lysosomes. Kinetic analyses of the interaction between RNF167 and selected conjugating E2 enzymes reveal submicromolar dissociation constants. The computed model of interaction between the RING domain of RNF167 and conjugating enzymes gives us insights on how RNF167 could interact with conjugating E2 enzymes. Furthermore, the results reveal that in vitro polyubiquitination of the AMPA-type glutamate receptor subunit GluA2, one of the RNF167's known substrates, is possible by the conjugating E2 enzyme UBE2N only after GluA2 has been primed by ubiquitin subsequent to the action of an initiating conjugating E2 enzyme functionally binding RNF167. Pharmacological inhibition of UBE2N in cultured hippocampal neurons diminishes AMPA-induced GluA2 ubiquitination. This study characterizes interacting partners of RNF167 and constitutes an initial step toward the identification of functional pairs assembled from RNF167 and ubiquitin-conjugating E2 enzymes involved in the ubiquitination of RNF167's substrate.