GxxxG Motifs - The Glue That Holds the TIM23 Complex

About 99% of the mitochondrial proteome is nucleus-encoded, synthesized in the cytosol and subsequently imported into and sorted to the correct compartment in the organelle. The TIM23 complex (Translocase of the Inner Mitochondrial Membrane) is the major protein translocase of the inner membrane responsible for translocation of proteins across and their insertion into the inner membrane. Tim23 and Tim17 are central components of this complex that form the core complex. Tim23 forms the import channel and Tim17 serves as a structural unit that keeps the complex intact. A high-resolution structure of the membrane embedded core complex is still missing, and structural elements important for its function are unknown. In the present work, we analyzed the importance of the highly abundant GxxxG motifs in the transmembrane segments of Tim23 and Tim17 for the structural integrity of the TIM23 complex. Introducing a bulky residue instead of the small residues of these motifs resulted in growth phenotypes in seven out of sixteen mutants in Tim17, and in Tim23, six out of ten mutants exhibited a growth phenotype. The severity of growth impairment correlated with the destabilization of the TIM23 complex. We show that the Tim23-Tim17 interaction is mediated through the first and second helices of Tim17 and Tim23 via the GxxxG motifs. Furthermore, we show that once this interaction is destabilized the import motor is not recruited to the complex. We conclude that the GxxxG motifs found in the first and second transmembrane segments of Tim17 and Tim23 are crucial for the structural integrity of the TIM23 complex.