Characterization of the Novel Mitochondrial Protein Import Component, Tom34, in Mammalian Cells

Abstract

Tom34 is a newly-found component of the mitochondrial protein import machinery in mammalian cells with no apparent counterpart in fungi. RNA blot and immunoblot analyses showed that the expression of Tom34 varies among tissues and differs from that of the core translocase component Tom20. In contrast to a previous report [Nuttal, S.D. et al. (1997) DNA Cell Biol. 16, 1067-1074], the present study using a newly-prepared anti-Tom34 antibody with a high titer showed that Tom34 is present largely in the cytosolic fraction and partly in the mitochondrial and membrane fractions after fractionation of tissues and cells, and that the membrane-associated form is largely extractable with 0.1 M sodium carbonate. The in vitro import of preproteins into isolated rat mitochondria was strongly inhibited by DeltahTom34 which lacks the NH2-terminal hydrophobic region of human Tom34 (hTom34). Import was also strongly inhibited by anti-hTom34. In pulse-chase experiments using COS-7 cells, pre-ornithine transcarbamylase (pOTC) was rapidly processed to the mature form. Coexpression of hTom34 resulted in a stimulation of pOTC processing, whereas the coexpression of hTom34 antisense RNA caused inhibition. The results confirm that Tom34 plays a role in mitochondrial protein import in mammals, and suggest it to be an ancillary component of the translocation machinery in mammalian cells.

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

“…As shown previously, overexpression of Tom20 in COS-7 cells also inhibited mitochondrial import of pOTC, whereas the cytosolic domain of Tom20 had little effect (26). Among the other mammalian transport factors, overexpression of Tom34 stimulated the import of pOTC (20), and that of HSDJ (hdj-2/dj2), a cytosolic DnaJ homologue which participates in mitochondrial protein import, inhibited the pOTC import (21). Overexpression of an unrelated cytosolic protein, phenylalanine hydroxylase, had no effect (21).…”

“…Proteins were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Antisera against human OTC (20), gelly fish GFP (22), human arginase II (29), hTom20 (22), human Tom22 (30), Tom40, and human metaxin were used as primary antibodies. Immunodetection was performed by the use of the chemiluminescence kit (ECL kit, Amersham).…”

“…The NMR structure of rat Tom20 in a complex of a presequence peptide was recently solved and showed that presequence recognition is mainly due to interactions between the hydrophobic face of the signal peptide and the receptor domain (18). Another novel component, human Tom34 was shown to be involved in mitochondrial protein import in mammalian cells (19,20). Tom34 is largely cytosolic and partly associated with the mitochondria and has no apparent counterpart in fungi.…”

The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance

“…Anti-Tom-complex antiserum was kindly provided by Dr. M. Mori of the Department of Molecular Genetics, Kumamoto University School of Medicine, Japan. Anti-human Tom20 (Yano et al, 2000), Tom22C (C-terminal region) (Yano et al, 2000) and Tom34 (Chewawiwat et al, 1999) antiserum were prepared as described. Antiserum against Tom70 was prepared using a histidine-tagged soluble domain (region 60-608) of human Tom70, which was expressed in Escherichia coli and purified using metal chelation chromatography.…”

“…Tom34 functions as a chaperone-like protein and not just as a receptor (Chewawiwat et al, 1999). The glycosylated s-tpis protein was expressed in the cytosol of ovaries, but not in spermatozoa ( Fig.…”