AN IMPORT SIGNAL IN THE CYTOSOLIC DOMAIN OF THE NEUROSPORA MITOCHONDRIAL OUTER-MEMBRANE PROTEIN TOM22

TOM22 is an integral component of the preprotein translocase of the mi tochondrial outer membrane (TOM complex). The protein is anchored to t he lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal po rtion to the intermembrane space. Here, we describe the sequence requi rements for the targeting and correct insertion of Neurospora TOM22 in to the outer membrane. The orientation of the protein is not influence d by the charges flanking its trans-membrane segment, in contrast to o bservations regarding proteins of other membranes. In vitro import stu dies utilizing TOM22 preproteins harboring deletions or mutations in t he cytosolic domain revealed that the combination of the trans-membran e segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment o f the cytosolic domain was found to be essential for the import and as sembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in whic h the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 c ontains physically separate import and membrane anchor sequences.