Investigation of the TIM23 complex-mediated sorting pathways of mitochondrial inner membrane proteins

Abstract

Nucleus encoded mitochondrial inner membrane (MIM) proteins carrying a cleavable presequence in Saccharomyces cerevisiae are inserted into the inner membrane medicated by translocases of outer and inner membrane (TOM-TIM23 complex). A hydrophobic sorting signal within transmembrane (TM) domain of the protein is recognized and laterally transferred into the membrane from the TIM23 complex (stop-transfer pathway). Otherwise, entire protein is first imported into the matrix and exported into the inner membrane (conservative sorting pathway). In addition, a multi-spanning mitochondrial protein utilizes both pathways for its membrane integration. While mitochondrial inner membrane is the protein-richest membrane harboring various complexes for critical functions in the cell, only small number of MIM proteins insertion mechanisms have been characterized owing to lack of robust experimental tools. Here, we established Mgm1 fusion protein (MFP) approach, which takes advantage of the rhomboid cleavage region in the C-terminal domain of Mgm1p, to elucidate the sorting pathways of MIM proteins in vivo. We validated this method with a set of proteins whose sorting pathways are well characterized and determined the membrane insertion mode of single or multi-spanning MIM proteins of which integration pathways were unknown. Our results suggested that Yta10p is inserted into the membrane not from matrix, but from intermembrane space (IMS). In addition, we found that a subunit of succinate dehydrogenase, Sdh4p and a mitochondrial inner membrane half-type ABC transporter, Mdl2p, are also integrated into the membrane via two different pathways. Furthermore, analyzing the sorting pattern of Mgm1 fusion proteins in various growth conditions and at different yeast mutant strains, we showed that insertion of proteins bearing moderately hydrophobic TM segments is more sensitive to intrinsic and extrinsic cellular factors and the presequence translocase-associated import motor (PAM complex) is necessary for the translocation of Cox18p, Mdl1p, and Mdl2p.