Recognition and Sorting of Mitochondrial Inner Membrane Proteins by the TIM23 Complex and the m-AAA Protease

Abstract

The TIM23 complex mediates translocation of proteins into two subcompartments of mitochondria

the inner membrane (IM) and the matrix, but how this complex is arranged in the IM and how it distinguishes targeted location of incoming polypeptides and opens the pore either transversally or laterally still remain elusive. Through site-directed mutagenesis of specific residues within the channel forming subunits, Tim17p and Tim23p, and translocation assay with various substrates of the TIM23 translocon, we attempted to elucidate their function in protein sorting. The mutagenesis study demonstrates that the second transmembrane domain (TMD2) of Tim23p plays a pivotal role. Particularly, mutation at the matrix side of the TMD2 affects the membrane insertion of proteins. The m-AAA protease participates in a quality control system of mitochondria and processing of specific proteins. However, it is yet to be revealed how it recognizes proteins for degradation and processing. To survey the characteristics of proteins that the m-AAA complex senses and subsequently exerts its ATPase activity, we utilized a set of Mgm1p variants with diverse sequence contexts. Our results show that depending on where the proline or charged residues are positioned within the TMD, the m-AAA protease differentially recognizes and dislocates the segments.