Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Abstract

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol as precursor proteins, and imported into mitochondria. The mitochondrial targeting signal is, in many cases, contained in the presequence, which is an N-terminal extension of the precursor protein and cleaved off by matrix-processing peptidase (MPP) after import into the matrix (1). The presequence has a potential to form a positively charged amphiphilic helix, which represents the mitochondrial targeting signal. Translocation of mitochondrial precursor proteins across the mitochondrial membranes requires coordinated actions of translocation machineries in the outer and the inner membranes, TOM (translocase of the outer membrane) and TIM (translocase of the inner membrane) complexes, respectively (2-5).In the past, many components of the TOM and TIM complexes have been identified. The TOM complex of yeast Saccharomyces cerevisiae consists of at least eight TOM proteins: Tom70, Tom37, Tom20, Tom22, Tom40, Tom5, Tom6, and Tom7. Among these TOM proteins, only Tom40 and Tom22 are essential proteins for cell growth (6-8). Tom40 is an integral membrane protein and forms a protein-conducting channel in the outer membrane (9, 10). Tom22 is anchored to the outer membrane by a central trans-membrane segment. The N-terminal cytosolic domain is highly acidic, and the C-terminal intermembrane space (IMS) domain contains some acidic residues, too. The roles of the cytosolic domain and the IMS domain of Tom22 in protein import have been a matter of debate (11-15).The question, what drives protein translocation across the outer and inner membranes in mitochondria, is one of the key issues in the protein import into mitochondria. The outer membrane lacks any transmembrane electrochemical potential or ATP-driven chaperones on the trans side of the membrane. A model was proposed in which the mitochondrial outer membrane has two binding sites for the presequence, the cis site on the cytosolic side and the trans site on the IMS side of the outer membrane (16). The transfer of the presequence from the cis site to the trans site was suggested to be coupled with unfolding of the mature domain and to drive vectorial translocation of the precursor protein across the outer membrane. Characterization has been made for the translocat...

Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles

“…This transfer of the presequence across the membrane is mediated by an array of presequence-binding sites on the TOM complex that are spatially arranged in order of affinity for the presequence (Schatz, 1997;Komiya et al, 1998). Indeed, the TOM complex contains presequencebinding sites on both sides of the membrane, and presequences can spontaneously go through the TOM channel to reach the trans site on the IMS side (Rapaport et al, 1997;Rapaport et al, 1998;Kanamori et al, 1999).…”

“…In this intermediate, the presequence reaches the presequence-binding site on the IMS side of the TOM complex (the 'trans site'), which involves Tom40 (Fig. 3A) (Rapaport et al, 1997;Kanamori et al, 1999 Mitochondria containing mutant Tim23 that lacks the Nterminal 50 residues have defects in protein import, but mitoplasts in which the outer membrane is selectively ruptured do not (Donzeau et al, 2000). This suggests that the N-terminal domain of Tim23 also facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex.…”

Reinvestigation of the Requirement of Cytosolic ATP for Mitochondrial Protein Import

“…The precursor proteins we tested are precursors of alcohol dehydrogenase III (pADH), the ␤-subunit of F 1 -ATPase (pF 1 ␤), cytochrome oxidase subunit IV (pCoxIV), mitochondrial Hsp60 (pHsp60), and a fusion protein joining cytochrome oxidase subunit IV with mouse dihydrofolate reductase (pCoxIV-DHFR). Previously, pADH, pF 1 ␤, and AAC were reported to require external ATP for their import into isolated mitochondria, whereas pHsp60, pCoxIV, and pCoxIV-DHFR were imported in the absence of external ATP (18,(35)(36)(37). We synthesized the radiolabeled precursor proteins in reticulocyte lysate and divided them into two.…”

“…After synthesis of 35 S-labeled precursor proteins with reticulocyte lysate, translation was stopped by the addition of 10 g/ml RNase A and 1 mg/ml cold Met, and the translation products were depleted of ATP by treatment with apyrase or gel filtration. For apyrase treatment, the reticulocyte lysate was incubated with 25 units/ml apyrase (grade III; Sigma), which was pretreated with 1 mM phenylmethylsulfonyl fluoride, for the indicated times at 10°C.…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…These receptors mediate the transfer of the presequence to the pore forming protein Tom40 (translocon) that channels the preprotein across the outer mitochondrial membrane in a linear mode from the N-to the C-terminus (N→C) (Model et al, 2008). This transfer is probably driven by the increasing affinity of the presequence to different components of the TOM-complex (acid chain hypothesis), which also involves a domain of Tom22 in the intermembrane space (Komiya et al, 1998;Kanamori et al, 1999). In the intermembrane space, the presequence interacts with the translocon of the inner mitochondrial membrane complex (TIM complex;Tim23,50,17,21).…”