Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion

Abstract

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB

“…Proteins were concentrated to 10 mg/mL and stored for crystallization. PR-ubiquitinated Rtn4 peptide were synthesized by incubation with GST-SdeA FL , NAD, Ub in the reaction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) at 37 C for 2 h. To effectively modify the peptide, Ub were iteratively added to reaction mixture every 30 min (Shin et al, 2017). PR-ubiquitinated peptide were further purified with Ni-NTA agarose and size exclusion chromatography (Superdex 30 prep grade 16/60, GE Healthcare).…”

Ubiquitination of Rheb governs growth factor-induced mTORC1 activation

“…Ubiquitination is categorized into two major types termed mono-ubiquitination and polyubiquitination. Both mono-ubiquitination and polyubiquitination are involved in a wide variety of cellular functions 20–24 . Recent studies indicate that K63-linked polyubiquitination of RagA mediated by E3 ligases RNF152 and SKP2 strongly inhibits mTORC1 activation 25,26 .…”

Cracking the Monoubiquitin Code of Genetic Diseases

“…While monoubiquitination of the GTPase Ras-related protein RAB5 at K165 affects its GTPase activity, attachment of ubiquitin at K140 alters the ability of RAB5 to bind and activate its downstream effectors. Ubiquitin conjugation to specific lysines also differentially affects the activity and subcellular localization of the Rat sarcoma (RAS) proto-oncogenes [28]. Specifically, the monoubiquitination of RAS at K117 promotes GTP loading, whereas monoubiquitination at K147 impairs GTP hydrolysis activity, both resulting in RAS activation.…”