Structural mechanism for regulation of Rab7 by site-specific monoubiquitination

Jung, Jaeeun; Baek, Jiseok; Tae, Kun; Shin, Dongsuk; Han, Seungsu; Yang, Wonjin; Yu, Wookyung; Jung, Sung-Min; Park, Seok Hee; Choi, Cheol Yong; Lee, Sang-Ho

doi:10.1016/j.ijbiomac.2021.11.074

Cited by 4 publications

(8 citation statements)

References 57 publications

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“…Recent studies identified three major sites of ubiquitination within the Rab7 protein [ 20 , 21 , 22 ]. Thus, we tested the location of RNF167-mediated Rab7 protein ubiquitination in HEK293T cells by co-transfecting plasmids encoding RNF167 with either GFP-Rab7 WT, K38R or 2KR, which combines the lysine-to-arginine substitution of both K191 and K194, as per prior studies performed by others [ 20 , 21 , 22 ]. Immunoblot of UB-modified Rab7 was performed after IP of GFP-Rab7 from cell lysates.…”

Section: Resultsmentioning

confidence: 99%

“…Late endosome to trans Golgi network (TGN) trafficking and late endosome and lysosome trafficking are regulated by Rab7A (named Rab7 hereafter) and Rab9 proteins [ 13 , 14 , 15 , 16 ]. The RAB7A gene encodes a protein that is found in eukaryotes throughout evolution [ 17 ]; recent studies have demonstrated that Rab7 protein regulation can be monitored by post-translational modifications, such as phosphorylation and ubiquitination [ 18 , 19 , 20 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

“…Major ubiquitination sites of Rab7 protein include K38, K191 and K194 [ 20 , 21 , 22 ]. K38 ubiquitination of Rab7 protein is mediated by the UB ligase (UBE3) Parkin.…”

Section: Introductionmentioning

confidence: 99%

“…Rab7 recruitment of RILP is also involved in dynein interaction, leading to retrograde transport on microtubules [ 34 ]. Single lysine to arginine residue substitution of K191 or K194 within Rab7 protein leads to the same phenotype in the cell [ 20 , 22 ], suggesting that both ubiquitination sites can affect Rab7 function similarly. However, divergence exists in the literature regarding how those two ubiquitination sites are involved in Rab7 membrane association.…”

Section: Introductionmentioning

confidence: 99%

“…The study concluded that Rab7 ubiquitination retained the GTPase on the membrane, whereas its removal by the deubiquitinase USP32 was important for Rab7 to leave the membrane [ 21 ]. Another study, using computational models where both lysines were simultaneously ubiquitinated, indicated that K191 and K194 ubiquitination prevents Rab7 membrane association [ 22 ]. Although a deubiquitinase was found for the K191 ubiquitination site, no UBE3 was shown to be specific for either the K191 or K194 site.…”

Section: Introductionmentioning

confidence: 99%

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

Ghilarducci

Cabana

Harake

et al. 2022

IJMS

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Major ubiquitination sites of Rab7 protein include K38, K191 and K194 [ 20 , 21 , 22 ]. K38 ubiquitination of Rab7 protein is mediated by the UB ligase (UBE3) Parkin.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Ghilarducci

Cabana

Harake

et al. 2022

IJMS

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Engineering a Cell-Based Orthogonal Ubiquitin Transfer Cascade for Profiling the Substrates of RBR E3 Parkin

Fang,

Zhou,

Chen

et al. 2024

Preprint

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The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate the transfer of UB to its substrates to regulate diverse cellular functions, including mitochondrial quality control, cell cycle progression, metabolism programming, and the establishment of synaptic functions. Mutations affecting the E3 ligase activity of Parkin are associated with cancer and Parkinson's disease (PD). An essential role of Parkin is to synthesize UB chains on the surface of damaged mitochondria to initiate mitophagy. Still, it is not clear how Parkin carries out other biological functions through the ubiquitination of its downstream targets in the cell. We hypothesized that a comprehensive substrate profile of Parkin would facilitate the discovery of ubiquitination pathways underpinning its multifaceted roles in cell regulation and reveal mechanistic linkages between Parkin malfunction and disease development. Here, we used phage display to assemble an orthogonal ubiquitin transfer (OUT) cascade of Parkin that can exclusively deliver an engineered UB mutant (xUB) to Parkin and its substrates in living cells. We then generated a substrate profile of Parkin by purifying xUB-conjugated proteins from cells and identifying them by proteomics. The OUT screen identified Parkin substrates involved in DNA replication, protein translation, intracellular protein transport, and rhythmic regulation. Based on previous literature implicating alterations in membrane vesicle trafficking in PD, we verified Parkin-catalyzed ubiquitination of Rab GTPases (Rab1a, Rab5a, Rab5c, Rab7a, Rab8a, Rab10, an Rab13) as well as CDK5, with reconstituted ubiquitination reactions in vitro and in cells. We also found chemical-induced stimulation of mitophagy enhanced Parkin-mediated ubiquitination of Rab proteins. These findings demonstrate that the OUT cascade of Parkin can serve as an empowering tool for identifying Parkin substrates to elucidate its cellular functions.

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Ubiquitin and its relatives as wizards of the endolysosomal system

Berlin

Sapmaz

Stévenin

et al. 2023

Journal of Cell Science

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The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.

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Structural mechanism for regulation of Rab7 by site-specific monoubiquitination

Cited by 4 publications

References 57 publications

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

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

Engineering a Cell-Based Orthogonal Ubiquitin Transfer Cascade for Profiling the Substrates of RBR E3 Parkin

Ubiquitin and its relatives as wizards of the endolysosomal system

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