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

Donghyuk Shin; Wooju Na; Ji Hyung Lee; Gyuhee Kim; Jiseok Baek; Seok Hee Park; Cheol Yong Choi; Sangho Lee

doi:10.7554/eLife.29154.001

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

Donghyuk Shin, Wooju Na, Ji Hyung Lee, Gyuhee Kim, Jiseok Baek, Seok Hee Park, Cheol Yong Choi, Sangho Lee

Division of Life Sciences

Research output: Contribution to journal › Article › peer-review

23 Citations (Scopus)

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 cotransfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGFinduced 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_K140, whereas GDP release and GTP loading activities were altered in mUbRab5_K165. By contrast, mUbRab5_K116 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.

Original language	English
Article number	e29154
Journal	eLife
Volume	6
DOIs	https://doi.org/10.7554/eLife.29154.001
Publication status	Published - 2017 Oct 2

Bibliographical note

Publisher Copyright:
© Shin et al.

All Science Journal Classification (ASJC) codes

General Neuroscience
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

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10.7554/eLife.29154.001

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title = "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 cotransfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGFinduced 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.",

author = "Donghyuk Shin and Wooju Na and Lee, {Ji Hyung} and Gyuhee Kim and Jiseok Baek and Park, {Seok Hee} and Choi, {Cheol Yong} and Sangho Lee",

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AU - Shin, Donghyuk

AU - Na, Wooju

AU - Lee, Ji Hyung

AU - Kim, Gyuhee

AU - Baek, Jiseok

AU - Park, Seok Hee

AU - Choi, Cheol Yong

AU - Lee, Sangho

PY - 2017/10/2

Y1 - 2017/10/2

N2 - 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 cotransfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGFinduced 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.

AB - 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 cotransfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGFinduced 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.

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

Abstract

Bibliographical note

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