Inactivation of the Neurospora crassa mitochondrial outer membrane proteinTOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology

Mitochondrial biogenesis requires the efficient import of hundreds of diffe rent cytosolically translated preproteins into existing organelles. Recogni tion and translocation of preproteins at the mitochondrial outer membrane i s achieved by the TOM complex (translocase of the outer mitochondrial membr ane). The largest component of this complex is TOM70, an integral outer mem brane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP). Mutant strains were ide ntified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70. Strains that lack TOM70 grow more slowly than wild-type s trains, conidiate poorly, and contain enlarged mitochondria. In vitro prepr otein import studies using TOM70-deficient mitochondria revealed a defect i n the uptake of the ADP/ATP carrier. Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondria l-processing peptidase (MPP) which was imported more efficiently by TOM70-d efrcient mitochondria. These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein imp ort. The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial mor phology.