ROLE OF THE NEGATIVE CHARGES IN THE CYTOSOLIC DOMAIN OF TOM22 IN THE IMPORT OF PRECURSOR PROTEINS INTO MITOCHONDRIA

TOM22 is an essential mitochondrial outer membrane protein required fo r the import of precursor proteins into the organelles. The amino-term inal 84 amino acids of TOM22 extend into the cytosol and include 19 ne gatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitoch ondrial preproteins, presumably via electrostatic interactions. We con structed a series of mutant derivatives of TOM22 in which 2 to 15 of t he negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transform ed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hy gromycin R disruption in one nucleus, All constructs restored viabilit y to the disruption-carrying nucleus and gave rise to homokaryotic str ains containing mutant tom22 alleles, Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutr alized residues (strain 861), imported precursor proteins at efficienc ies comparable to those for wild-type organelles. Precursor binding st udies with mitochondrial outer membrane vesicles from several of the m utant 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 t o the disruption-containing nucleus, but mutants lacking the entire re gion cannot. Taken together, these data suggest that an abundance of n egative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, ot her features in the domain are required.