Topogenesis of mammalian Oxa1, a component of the mitochondrial inner membrane protein export machinery.
Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1 approximately 5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.
Sato
Takashi
T
Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812, Japan.
Mihara
Katsuyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013367:Submitochondrial Particles / Q000378:metabolism
D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C090858:OXA1 protein; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003576:Electron Transport Complex IV