Mitochondrial carriers constitute a large family of proteins in the inner mitochondrial membrane. Due to their hydrophobic nature, they employ a distinct translocation pathway through the TIM22 complex. However, how carriers are inserted and folded in their correct orientation remains poorly understood, and factors that facilitate the biogenesis of carriers have not been identified so far. Therefore, I wondered whether newly imported carrier proteins engage with other proteins at the site of the inner mitochondrial membrane, in addition to the established import proteins. To find interaction partners of carriers, I developed a proximity labeling approach using the TurboID successor DestniID in yeast. Proximity labeling with a fusion protein consisting of DestniID and Odc1, an oxodicarboxylate carrier, combined with mass spectrometric analysis, revealed Aim11 to be spatially close carriers. Using fluorescence imaging, in vitro imports, swelling and carbonate extraction assays, I could validate that Aim11 is a mitochondrial inner membrane protein. Co-immunoprecipitation revealed that Aim11 is part of a complex comprising three additional rather uncharacterized proteins – Gep7, Iai11, and Mtc3. I employed an inducible CRISPR interference (CRISPRi) system to deplete PHB1 and could show that prohibitin depletion had a synthetic negative effect in combination with a deletion of AIM11. Loss of both complexes led to the decrease of many inner membrane proteins in the whole cell proteome, but mainly affected proteins involved in transmembrane transport and fatty acid metabolism. Besides the classical in vitro import, I employed a carrier cleavage assay, which I developed. I could demonstrate that Aim11 loss not only hampered the import, but also the insertion of carriers into the inner membrane. The absence of Aim11 resulted in a partial mislocalization of carriers into the intermembrane space (IMS), both in vitro and in vivo. Using co-immunoprecipitation, I could show that Aim11 co-eluted with newly synthesized proteins, but only in an intermediate import state, in which the precursors were still susceptible to degradation from the cytosol. Proteins involved in the mitochondria-associated ubiquitin-proteasome system (UPS) also showed negative genetic interaction with the temperature-sensitive tim22-14 mutant, and thus imply that constitutive degradation of carriers at the site of the TOM complex might influence the biogenesis of carriers. The cohibitin complex might prevent carrier degradation and thereby facilitate carrier biogenesis by localizing them close to the inner mitochondrial membrane.
Büsra Kizmaz (Thu,) studied this question.