Investigation of components of the TIM23 complex involved in lateral insertion of mitochondrial inner membrane proteins

Abstract

Mitochondrial inner membrane (IM) proteins carrying a cleavable N-terminal presequence are encoded by nucleus and inserted into the IM by translocase of outer membrane (TOM complex) and translocase of inner membrane (TIM23 complex). In addition, the TIM23 complex mediates translocation of proteins into the matrix. How the TIM23 complex recognizes different localization signals of precursor proteins and sorts into the IM or matrix remains elusive. To better understand the mechanisms of the TIM23-mediated membrane protein insertion in mitochondria, I undertook two approaches. First, I attempted to identify the regions or residues in the TIM23 components critical for lateral insertion of proteins into the IM by genetic screening. This approach identified Tim17 mutant that allows enhanced membrane insertion of a hydrophobic segment containing Proline residue that normally translocates to the matrix. Sequencing analysis shows that this Tim17 mutant is truncated at the C-terminus, lacking the loop region in the matrix. Since this region is shown important for interaction with a motor component in the matrix, it is speculated that impaired interaction between the TIM23 complex and the matrix motor complex might cause decreased translocation efficiency of proteins into the matrix. Second approach focused on Mgr2 which is a newly identified subunit of the TIM23 complex. When Mgr2 is absent, interaction of the TIM23 core with Tim21 and respiratory chain complexes are shown impaired. Mgr2 is also shown to be involved in the TOM-TIM23 cooperation and protein import to the matrix. Another study revealed that membrane protein insertion efficiency was increased in the absence of Mgr2 whereas overexpression of Mgr2 delayed the insertion, so they reported Mgr2 as a lateral gatekeeper. To investigate whether Mgr2 has a general role in regulating lateral insertion of proteins, membrane insertion efficiency of Mgm1 variants with diverse sequence characteristics were determined in Mgr2 mutants. The results show that overexpression of Mgr2 increases hydrophobicity threshold for membrane insertion of a transmembrane domain whereas a lack of Mgr2 decreases. Mgm1 variants carrying alterations in flanking charged residues of the transmembrane domain were better inserted when Mgr2 was deleted. Insertion of a transmembrane domain containing Proline residues was decreased in the absence of Mgr2. So far these data suggest that Mgr2 is involved in sensing the determining features of the transmembrane domain and has a role in modulating membrane protein insertion.