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

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, Ra...

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Bibliographic Details
Published in:eLife 2017-10, Vol.6
Main Authors: Shin, Donghyuk, Na, Wooju, Lee, Ji-Hyung, Kim, Gyuhee, Baek, Jiseok, Park, Seok Hee, Choi, Cheol Yong, Lee, Sangho
Format: Article
Language:English
Subjects:
Analysis
Biochemistry
Biochemistry and Chemical Biology
Cell Line
DNA Mutational Analysis
Down-Regulation
Epidermal growth factors
G proteins
Gross domestic product
Guanine
Guanine Nucleotides - metabolism
Guanosine triphosphate
Humans
Hydrolysis
Immunoglobulins
Infection
Localization
Protein Binding
Protein Conformation
Proteins
Purines
Rab5
rab5 GTP-Binding Proteins - chemistry
rab5 GTP-Binding Proteins - genetics
rab5 GTP-Binding Proteins - metabolism
SAXS
Scattering, Small Angle
Structural Biology and Molecular Biophysics
Transfection
Ubiquitin
Ubiquitination
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Summary: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 mUbRab5 , whereas GDP release and GTP loading activities were altered in mUbRab5 . By contrast, mUbRab5 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.
ISSN:2050-084X
2050-084X
DOI:10.7554/elife.29154