Msp1 in Trypanosoma brucei mitochondrial quality control

Gerber, Markus Daniel (2024). Msp1 in Trypanosoma brucei mitochondrial quality control (Unpublished). (Dissertation, Department of Chemistry, Biochemistry and Pharmaceutical Sciences, Faculty of Science)

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Mitochondria are vital organelles, prominently known for their role in ATP production. Even though mitochondria contain their own DNA, almost all mitochondrial proteins are encoded in the nucleus. These nuclear encoded mitochondrial proteins are translated in the cytosol and subsequently imported into the organelle. Disturbance of mitochondrial proteostasis by mislocalized or destabilized proteins, whose accumulation can lead to mitochondrial dysfunction, is an issue that demands immediate resolution. Therefore, eukaryotes contain a wide range of pathways to prevent and restore dysfunctional mitochondria. The ATPase-associated with diverse cellular activities (AAA) Msp1 contributes to mitochondrial proteostasis by extracting mislocalised tail-anchored proteins from the mitochondrial outer membrane (OM). Yeast Msp1 can extract substrate proteins from a lipid bilayer independently from associated proteins and substrate modifications. However, little is known about Msp1 orthologs outside the eukaryotic supergroup of the Opisthokonts, which includes animals and fungi. Trypanosoma brucei is a well-established eukaryotic model organism which belongs to the Discoba group and thus is essentially unrelated to Opisthokonts. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins, such as proteins of the atypical protein translocase of the outer mitochondrial membrane (ATOM), into their respective complexes. Inhibition of ATOM complex assembly via RNAi knockdown of pATOM36 triggers a pathway that results in the degradation of unassembled ATOM subunits by the cytosolic proteasome.

Here we have investigated this novel trypanosomal mitochondria-associated degradation pathway. We show that the trypanosomal Msp1 homolog (TbMsp1) and the trypanosomal homolog of the AAA-ATPase VCP (TbVCP) are involved in this quality control pathway. The RNAi knockdown of pATOM36 in combination with either TbMsp1 RNAi or TbVCP RNAi does not affect the pathway as pATOM36 substrates are still being degraded by the cytosolic proteasome. However, the simultaneous knockdown of TbMsp1 and TbVCP in the pATOM36 RNAi background prevents the removal of pATOM36 substrates from the OM despite the ablation of pATOM36. This suggests that there is some redundancy between TbMsp1 and TbVCP in this pathway. Furthermore, we show by in situ tagging, coimmunoprecipitation and mass spectrometry that TbMsp1 localises to both, glycosomes and the OM. Additionally, we demonstrate by reciprocal coimmunoprecipitations that TbMsp1 forms a stable complex with the four OM proteins POMP19, POMP31, TbJ31 and TbTsc13. Interestingly, upon pATOM36 and TbVCP ablation, POMP31, TbJ31 and TbTsc13 are required for efficient proteasomal degradation of pATOM36 substrates, suggesting these three TbMsp1-interacting proteins assist TbMsp1 in extracting the destabilized OM proteins. pATOM36 is a functional analogue of the yeast OM MIM complex and likely of the animal-specific OM protein MTCH2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans. The molecular details underlaying the interactions within the TbMsp1-containing complex, and the role of the individual TbMsp1-associated proteins in the extraction of pATOM36 substrates have yet to be elucidated. Additionally, it is unclear whether these interaction partners are required for the extraction of all TbMsp1 substrates. It is possible that their activity might be limited to a specific subset of TbMsp1 substrates which includes the pATOM36 substrates. Other TbMsp1 substrates than those extracted upon pATOM36 and TbVCP ablation could not yet be identified. Furthermore, it also remains unclear whether TbMsp1 functions independently of ubiquitin, as has been suggested for yeast Msp1.

Item Type:	Thesis (Dissertation)
Division/Institute:	08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)
Graduate School:	Graduate School for Cellular and Biomedical Sciences (GCB)
UniBE Contributor:	Gerber, Markus Daniel
Subjects:	500 Science 500 Science > 540 Chemistry 500 Science > 570 Life sciences; biology
Language:	English
Submitter:	Christina Schüpbach
Date Deposited:	03 May 2024 09:30
Last Modified:	03 May 2024 09:30
URI:	https://boris.unibe.ch/id/eprint/196465