Journal: bioRxiv

Article Title: Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid

doi: 10.1101/2023.11.06.565743

Figure Lengend Snippet: t-2-hex targets the mitochondrial TOM complex. (A) Hfd1 co-purifies with Tom70. Constitutively Hfd1-HA expressing cells were used in co-immunoprecipitation experiments from yeast whole cell extracts in the presence or not of endogenously expressed Tom22- or Tom70-Tap. (B) A chemoproteomic screen with t-2-hex alkyne identifies the TOM and Tim23 complexes as lipidation targets. Purified mitochondrial fractions from yeast wild type cells were treated or not with the clickable analogue t-2- hex-Alkyne (100μM). After addition of biotin to the modified proteins and purification with Streptavidin agarose pull-down, the protein identities of the t-2-hex targets were determined by mass spectrometry. Cysteine containing subunits of the TOM and Tim23 complexes are depicted as direct lipidation targets. The tables show the spectral reads for selected subunits in the chemoproteomic analysis for mock treated (Ctrl) and t-2-hex treated mitochondria (n = 2). (C) Tom40 is lipidated in vitro by t-2-hex. Upper panel: t-2-hex-Alkyne was used in the indicated concentrations to lipidate proteins in enriched mitochondrial preparations from Tom40-HA expressing cells or control cells. After t-2-hex addition, input samples were generated directly, while pull-down samples were treated with click chemistry for covalent biotin linkage and subsequent Streptavidin purification. Tom40-HA was detected in all samples by anti-HA western blot. Lower panel: Competition of Tom40 t-2-hex lipidation by free thiol groups. t-2- hex-Alkyne was used at 100μM to lipidate Tom40-HA in mitochondrial preparations in the presence or absence of 2mM DTT. (D) Model of the pro-apoptotic function of t-2- hex and the anti-apoptotic function of Hfd1 at the mitochondrial Tom complex. Left panel: Under normal conditions, the Hfd1 lipid aldehyde dehydrogenase located at the Tom complex safeguards mitochondrial protein import by t-2-hex degradation. Right panel: Upon severe stress conditions or in the absence of Hfd1 function, an excess of t- 2-hex directly lipidates Tom subunits such as Tom40 and thus inhibits mitochondrial pre-protein transport across the outer mitochondrial membrane. The resulting proteostatic imbalance in the cytosol, if not sufficiently repaired or counteracted by the heat shock response, proteasomal clearance or diminished de novo protein synthesis, can induce apoptotic cell death.

Article Snippet: Protein mixtures were incubated with 40μl PBST washed Streptavidin agarose resin (50% slurry, 69203-3 Merck) and incubated for 2h at room temperature on a roller.

Techniques: Expressing, Immunoprecipitation, Purification, Modification, Mass Spectrometry, In Vitro, Generated, Western Blot, Membrane