CHARACTERIZATION OF MAMMALIAN TRANSLOCASE OF INNER MITOCHONDRIAL-MEMBRANE (TIM44) ISOLATED FROM DIABETIC NEWBORN MOUSE KIDNEY

Mammalian translocase of mitochondrial inner membrane (mTim44) was iso lated during representational difference analysis of cDNA from diabeti c mouse kidney. Streptozotocin-induced diabetic mouse kidney cDNA was prepared and subtracted by normal mouse kidney cDNA. By using one of t he isolated cDNA fragments as a screening probe, full length cDNA of m Tim44 was isolated from lambda ZAP kidney cDNA library. At the nucleot ide level, mTim44 did not exhibit significant homology with any known genes; however, at the amino acid level, it had 50% similarity and 29% identity with yeast Tim44. C-terminal FLAG epitope tagged mTim44 fusi on protein was transiently expressed in COS7 cells. By using anti-FLAG epitope M2 monoclonal antibody, mTim44 was found to have its subcellu lar localization associated with mitochondria. By immunoelectron micro scopy, mTim44 was seen in the paracrystalline structures within the mi tochondria, as well as in their cristae. Mitochondrial import assay of in vitro translated mTim44 indicated that its precursor product (appr oximate to 50 kDa) was imported and proteolytically processed to a mat ure approximate to 44-kDa protein, and its translocation was inner mem brane potential (Delta Psi)-dependent. Imported mTim44 was protected f rom protease digestion in which outer membranes were selectively perme abilized with digitonin. The mature form of mTim44 could be recovered in the supernatant of sonicated mitochondrial membrane fraction treate d with 0.1 M Na2CO3, pH 11.5. The data indicate that mTim44 is a mitoc hondrial inner membrane protein, one of the members of the mammalian T IM complex and up-regulated in hyperglycemic states.