Characterization of Rat TOM40, a Central Component of the Preprotein Translocase of the Mitochondrial Outer Membrane

Abstract

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable ␣-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a ␤-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitoninsolubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a ϳ400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.Most mitochondrial proteins are synthesized in the cytosol as preproteins, delivered to the mitochondrial surface by cytosolic factors such as hsp70 and mitochondrial import-stimulating factor, and transported to the intramitochondrial compartments by the preprotein import machinery of the outer and the inner membranes (the TOM 1 and TIM complexes, respectively)(1-4). The Saccharomyces cerevisiae TOM complex is composed of at least nine proteins (Tom71, -70, -40, -37, -22, -20, -7, -6, and -5) (5, 6). Tom40, the central component of the translocation channel, stably associates with the Tom22 receptor and small Tom components, Tom7, -6, and -5, and forms a ϳ400-kDa general insertion pore complex in yeast (5). Composition of the Neurospora crassa TOM complex is similar to that of S. cerevisiae, but Tom5 has yet to be identified in N. crassa. The mitochondrial inner membrane has two separate import machineries (7): the Tim23-Tim17 system and the Tim54-Tim22-Tim18 system. The Tim23-Tim17 system functions in the translocation of preproteins across the inner membrane in conjunction with Tim44, mhsp70, and GrpE (8 -10), whereas the Tim54-Tim22-Tim18 system functions in collaboration with the intermembrane space proteins Tim13, Tim12, Tim10, Tim9, and Tim8, in the import of proteins without a cleavable presequence, such as the phosphate carrier, the ADP/ATP carrier, and several Tim proteins (Tim23, Tim22, and Tim17) (10 -16).Although the fundamental mechanisms of mitochondrial protein import seem to be conserved from lower eukaryotes to mammals, only limited information is available for higher eukaryotic systems. Several mammalian counterparts have...

An Internal EELD Domain Facilitates Mitochondrial Targeting of Mcl-1 via a Tom70-dependent Pathway

“…As shown in Figure 2C, the mitochondrial inner membrane protein, Cox subunit IV, was still detectable in the trypsin-treated mitochondria, i.e., the P fraction shown in Figure 2C (compare lanes 1 and 3), whereas such treatment nearly eliminated the detection of Mcl-1 in the P fraction by the Mcl-1 S-19 antibody ( Figure 2C), suggesting that the bulk portion of the Mcl-1 protein was exposed on the cytosolic side of MOM. Of note, under the same conditions, Tom70 was cleaved by trypsin, and a cytoplasmic fragment of ϳ58 -60 kDa was released into the S fraction, a result that is very similar to that reported for the rat homologue of Tom70 (Suzuki et al, 2002). Furthermore, like Bcl-2 and Tom40, Tom70 was resistant to alkali extraction (see Materials and Methods) after it had integrated into mitochondria (Figure 2, D and E), suggesting that it is also stably integrated into MOM.…”

“…The in vitro mitochondrial import assay was then carried out using freshly isolated mitochondria and 35 S-labeled proteins (synthesized by the TNT-coupled reticulocyte lysate system; Promega, Madison, WI) essentially as described by Suzuki et al (2002). The imported (pellet; P) and unimported fractions (supernatant; S) were subjected to SDS-PAGE, and specific signals were visualized by fluorography and quantified using a Bioimage Analyzer FLA5000 (Fuji, Tokyo, Japan).…”

Tom70 serves as a molecular switch to determine pathological cardiac hypertrophy

“…Tom70 also functions as a docking site for cytosolic chaperones, such as Hsp70 and Hsp90, to receive mitochondrial proteins [20], implying a distinct biological feature of Tom70 in cellular functions. Recently, mammalian counterparts of Tom70 have been identified [21], but their functions in the heart remain unknown. Herein, we identified a marked reduction in Tom70 among the Tom complex proteins in pathological hypertrophic cardiomyocytes from different species and discovered that Tom70 knockdown induced the pathological hypertrophic growth of mammalian primary cardiomyocytes and zebrafish hearts without affecting fibroblast transdifferentiation into cardiomyocytes (Supplementary information, Figure S1) or the expression of early cardiogenesis markers (Supplementary information, Figure S2F).…”

Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import

“…Tom70, the alternate receptor, is thought to mediate the import of preproteins that contain targeting information within their mature sequences (Koehler, 2004;Rehling et al, 2004). Although much of our knowledge of import derives from work in Saccharomyces cerevisiae and Neurospora crassa, the biological functions of the human Tom70 and Tom20 receptors seem to be largely conserved (Iwahashi et al, 1997;Schleiff et al, 1997;Yano et al, 1997;Suzuki et al, 2002).…”