BIOGENESIS OF ISP6, A SMALL CARBOXYL-TERMINAL ANCHORED PROTEIN OF THERECEPTOR COMPLEX OF THE MITOCHONDRIAL OUTER-MEMBRANE

To study the biogenesis of ISP6, an outer membrane component of the mi tochondrial protein translocation complex, two fusion proteins have be en made by fusing ISP6 to either the carboxyl- or amino-terminal end o f the mouse dihydrofolate reductase (DHFR), In vitro import experiment s showed that when DHFR was placed at the carboxyl-terminal end of ISP 6, the resulting fusion protein 6-DHFR inserted into mitochondrial mem brane less efficiently than the other form of the fusion proteins. In vivo this fusion protein lost its ability to suppress the temperature- sensitive phenotype of an isp42 mutant, while the other fusion protein DHFR-6, which was found targeted correctly to mitochondria, suppresse d the mutant as well as the wild-type ISP6. Further analysis showed th at the binding and insertion of DHFR-6 to mitochondrial outer membrane was not affected by deletion of either of the two mitochondrial prote in receptors or by the predigestion of mitochondrial surface proteins prior to import. Additional data indicated that ISP42, which closely a ssociates with ISP6 in the translocation complex, does not likely play the role of a targeting partner for ISP6. In summary, these data sugg est that ISP6 may target to mitochondria by sequences at its carboxyl terminus and that the import process of ISP6 is most likely distinct f rom that of most other mitochondrial precursors, which are recognized by protein receptors on mitochondrial surface.