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The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans., (© 2023 Gerber et al.)

Despite their simplicity compared to multicellular organisms, single-celled yeasts such as the baker's yeast Saccharomyces cerevisiae are widely recognized as model organisms for the study of eukaryotic cell biology. To gain deeper insights into the molecular mechanisms underlying cellular processes, it is of utmost interest to establish the interactome of distinct proteins and to thoroughly analyze the composition of individual protein complexes and their dynamics. Combining affinity purification of epitope-tagged proteins with high-resolution mass spectrometry and quantitative proteomics strategies, in particular stable isotope labeling by amino acids in cell culture (SILAC), represents an unbiased and powerful approach for a most accurate characterization of protein complexes. In this chapter, we provide detailed protocols for the generation of yeast strains (S. cerevisiae) amenable to SILAC-labeling, for epitope tagging of a protein of interest for affinity purification, and for the SILAC-based characterization of membrane protein complexes including the identification of stable core components and transient interaction partners.