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Molecular and Cellular Biology, October 2000, p. 7205-7213, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification and Functional Analysis of Human
Tom22 for Protein Import into Mitochondria
Masato
Yano,1
Nicholas
Hoogenraad,2
Kazutoyo
Terada,1 and
Masataka
Mori1,*
Department of Molecular Genetics, Kumamoto
University School of Medicine, Kumamoto 860-0811, Japan,1 and Department of
Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia2
Received 25 May 2000/Returned for modification 20 June
2000/Accepted 17 July 2000
Mitochondria have a receptor complex in the outer membrane which
recognizes and translocates mitochondrial proteins synthesized in the
cytosol. We report here the identification and functional analysis of
human Tom22 (hTom22). hTom22 has an N-terminal negatively charged
region exposed to the cytosol, a putative transmembrane region, and a
C-terminal intermembrane space region with little negative charge.
Tom22 forms a complex with Tom20, and its cytosolic domain functions as
an import receptor as in fungi. An import inhibition assay, using
pre-ornithine transcarbamylase (pOTC) derivatives and a series of
hTom22 deletion mutants, showed that the C-terminal segment of the
cytosolic domain is important for presequence binding, whereas the
N-terminal domain is important for binding to the mature portion of
pOTC. No evidence for pOTC interaction with the Tom22 intermembrane
space domain was obtained. Binding studies revealed that the
presequence is critical for pOTC binding to Tom20, whereas both the
presequence and mature portion are important for binding to Tom22. A
cell-free immunoprecipitation assay indicated that an internal segment
of the Tom22 cytosolic domain is important for interaction with Tom20.
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Kumamoto University School of Medicine, Honjo
2-2-1, Kumamoto 860-0811, Japan. Phone: 81-96-373-5140. Fax:
81-96-373-5145. E-mail: masa{at}gpo.kumamoto-u.ac.jp.
Molecular and Cellular Biology, October 2000, p. 7205-7213, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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