Investigating molecular clustering of Rv1747 in M. smegmatis
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Li, Haoran
issued: 2022-09-30T07:00:00Z
issued: 2021
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: Phase separation is involved in the organization and regulation of membraneless cellular components and their functions. We showed previously that the cytoplasmic regulatory module of the ABC transporter Rv1747 of Mycobacterium tuberculosis, which is involved in cell wall biosynthesis, is able to phase separate in vitro. Here we demonstrate that this regulatory module forms static foci and full length Rv1747 mobile ones dependent on the presence of the regulatory module in Mycobacterium smegmatis. Disrupting the key interactions by the FHA domains in this regulatory module, in particular FHA-1, and phosphorylated threonines in the linker between the FHA domains demolish the resulting foci in both constructs. Specifically, we show that the S47A single mutant and the S47A, S248A double mutant exhibit no foci formation while the S248A single mutant exhibits a mixed phenotype dominated by diminished clustering. Complementarily, the regulatory module of Rv1747 carrying phosphor-ablative mutations of all Thr/Ser to Ala in the linker also shows no sign of foci formation. In addition, charges of the intrinsically disordered linker also contribute to foci formation by the Rv1747 regulatory module, as the mutant carrying Arg/Lys to Ala mutations displays no foci in M. smegmatis. Collectively, these data strongly suggest that Rv1747 phase separates in M. smegmatis and highlight the role of phosphorylation and non-specific charge-charge interactions by the FHA domains and the ID linker in regulating phase separation of Rv1747 in M. smegmatis.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/79075
note: INVESTIGATING MOLECULAR CLUSTERING OF RV1747 IN M. SMEGMATIS by HAORAN LI B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2021 © Haoran Li, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Investigating molecular clustering of Rv1747 in M. smegmatis submitted by Haoran Li in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Dr. Jörg Gsponer, Professor, Biochemistry and Molecular Biology, UBC Supervisor Dr. Lawrence McIntosh, Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Yossef Av-Gay, Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Thibault Mayor, Professor, Biochemistry and Molecular Biology, UBC Additional Examiner iii Abstract Phase separation is involved in the organization and regulation of membraneless cellular components and their functions. We showed previously that the cytoplasmic regulatory module of the ABC transporter Rv1747 of Mycobacterium tuberculosis, which is involved in cell wall biosynthesis, is able to phase separate in vitro. Here we demonstrate that this regulatory module forms static foci and full length Rv1747 mobile ones dependent on the presence of the regulatory module in Mycobacterium smegmatis. Disrupting the key interactions by the FHA domains in this regulatory module, in particular FHA-1, and phosphorylated threonines in the linker between the FHA domains demolish the resulting foci in both constructs. Specifically, we show that the S47A single mutant and the S47A, S248A double mutant exhibit no foci formation while the S248A single mut…
has type: Thesis/Dissertation
date issued: 2021-11
is shown at: 10.14288/1.0400596
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Gsponer, Jörg
type: Text
identifier URI: http://hdl.handle.net/2429/79075