Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.
|Number of pages||11|
|Journal||International Journal of Biological Macromolecules|
|Publication status||Published - 2022 Jan 1|
Bibliographical noteFunding Information:
We thank the staff members at beamline 4C of Pohang Accelerator Laboratory for technical assistance in SAXS data collection. This work was supported by the Basic Science Research Program ( 2021R1F1A1050449 ), the Bio & Medical Technology Development Program ( 2021M3A9I4022936 and 2020M3A9E4039217 ) and the Science Research Center Program ( 2017R1A5A1014560 ) through the National Research Foundation of Korea (NRF) grants and a grant from the Next-Generation BioGreen 21 Program ( PJ015672 ), Rural Development Administration , Republic of Korea.
© 2021 Elsevier B.V.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology
- Economics and Econometrics