SUBCELLULAR LOCATIONS OF MOD5 PROTEINS - MAPPING OF SEQUENCES SUFFICIENT FOR TARGETING TO MITOCHONDRIA AND DEMONSTRATION THAT MITOCHONDRIALAND NUCLEAR ISOFORMS COMMINGLE IN THE CYTOSOL

MOD5, a gene responsible for the modification of A37 to isopentenyl A3 7 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Ini tiation of translation at the first AUG of the MOD5 open reading frame generates DELTA2-isopentenyl pyrophospbate:tRNA isopentanyl transfera se I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In the se studies, we fused MOD5 sequences encoding N-terminal regions to gen es encoding passenger proteins, pseudomature COXIV and dihydrofolate r eductase, and studied the ability of these chimeric proteins to be imp orted into mitochondria both in vivo and in vitro. We found that the s equences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluor escence and cell fractionation to locate the MOD5 isozymes in yeast. I PPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuc lei and the cytosol. The nuclear location of this protein is surprisin g because the A37-isopentenyl A37 modification had been predicted to o ccur in the cytoplasm. MOD5 is one of the first genes reported to enco de isozymes found in three subcellular compartments.