Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner
Membrane Protein Export
Machinery |
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Authors: | Takashi Sato and Katsuyoshi Mihara |
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Institution: | Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812, Japan |
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Abstract: | Oxa1 is a mitochondrial inner membrane protein with a predicted
five-transmembrane segment (TM1~5) 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.Most mitochondrial proteins are nuclear DNA-coded, and their import into
mitochondrial compartments, that is, the mitochondrial outer membrane
(MOM),3 mitochondrial
inner membrane (MIM), intermembrane space (IMS), and matrix, is mediated by
five protein translocation systems: translocase of the outer membrane (TOM
complex), sorting and assembly machinery of MOM (SAM/TOB), translocases of the
inner membrane (TIM23 complex and TIM22 complex), and a fifth system in the
MIM that mediates integration of proteins from the matrix into the MIM
(1,
2). The last system, which has
been analyzed in detail in yeast, requires a membrane potential across the MIM
and matrix ATP and mediates MIM integration of the mtDNA-encoded proteins as
well as the integration of certain nuclear DNA-encoded proteins considered to
be of bacterial origin, such as cytochrome c oxidase subunit II,
F1Fo-ATPase subunit 9, and Oxa1
(3–5).
Translocation efficiency is affected by the charge difference across the
transmembrane (TM) in accordance with the positive-inside rule
(5). Furthermore, the
matrix-exposed C-terminal segment of Oxa1 is essential for binding
mitochondrial ribosomes during cotranslational integration of mtDNA-encoded
proteins (6,
7). Recent reports further
demonstrated that the MIM protein Mba1, as a ribosome receptor, cooperates
with the C-terminal ribosome binding segment of Oxa1
(8). The machinery and the
underlying mechanisms of MIM insertion from the matrix must be further
analyzed.Oxa1 protein, originally identified in yeast, is a component of the
matrix-to-MIM export system conserved from prokaryote to eukaryote and is
involved in Oxa1 biogenesis
(9–14).
YidC, a bacterial homologue of Oxa1, is involved in the biogenesis of inner
membrane proteins in a Sec-dependent or Sec-independent manner
(15,
16). In yeast, IMS export from
the matrix of the Oxa1 N-terminal segment emerging from the Tim23 channel
requires a membrane potential
(4,
17), and the export is
compromised in mitochondria isolated from a temperature-sensitive
Oxa1-expressing strain at a non-permissive temperature
(12). Herrmann and Bonnefoy
(18) reported that Oxa1
protein functions in the export of a single hydrophilic loop region that was
artificially produced by ligating the C-terminal region of cytochrome
b with cytochrome c oxidase subunit II and placed between TM
segments. Direct interaction of Oxa1 with an immature subunit in complex V was
observed during its biogenesis
(19). So far, these studies
have only been performed in yeast, and no information is available on the
mechanism of topogenesis in mammals with regard to how Oxa1 is involved in the
export of multiple regions in a protein molecule. Our in vivo study
revealed that the correct topogenesis of Oxa1 in the MIM proceeds as a result
of the cooperation of all five TMs and that the cooperation of TM1 and the
species-conserved six-residue segment (GLPWWG) in the N-terminal flanking
region is essential for export from the matrix of both the N-terminal segment
and hydrophilic L2 into the IMS. |
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Keywords: | |
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