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Mol Cell Biol, June 1998, p. 3173-3181, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

Frank E. Nargang,1,* Doron Rapaport,2 R. Gary Ritzel,1 Walter Neupert,2 and Roland Lill2,dagger

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9,1 and Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, 80336 Munich, Germany2

Received 18 November 1997/Returned for modification 6 January 1998/Accepted 17 March 1998

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Phone: (403) 492-5375. Fax: (403) 492-1903. E-mail: frank.nargang{at}ualberta.ca.

dagger Present address: Institut für Zytobiologie der Philipps-Universität Marburg, 35033 Marburg, Germany.

Mol Cell Biol, June 1998, p. 3173-3181, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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