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Li, Haoran

University of British Columbia

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.

Text

2022-09-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/79075

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/