TIM29 is a core subunit from the carrier translocase and requires oxidized TIM22 to become built-into the mature TIM22 complex. with neurodegenerative disorders. As the TIM22 complicated is well examined in baker’s fungus, very little is well known about the mammalian TIM22 complicated. Using immunoprecipitation, we purified the individual carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is usually a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase. carrier translocase consists of the four integral membrane proteins, Tim22, Tim54, Tim18, and Sdh3 14, 15, 16, 17, 18. Tim22 is the central, pore\forming, subunit of the complex 16, 19. Tim54, Tim18, and Sdh3, are currently thought to support assembly and stability of the complex 15, 17. Sdh3, which is related to Tim18, is also a major component of the succinate dehydrogenase complex 18. The human TIM22 displays 40% homology with the yeast Tim22 20, 21. Human and yeast Tim22 possess cysteine residues in the oxidized state, which are important for TIM22 complex assembly 20, 22, 23. In the case of the human protein, four cysteine residues are oxidized, likely forming disulfide bonds 20. Clear homologs of Tim54 or Tim18 have not been identified in mammals and there is no evidence that this Sdh3 homolog, SDHC, interacts with the TIM22 complex. In yeast, the small Tim proteins, Tim9, Tim10, and Tim12 participate in the transport of metabolite carriers into the inner membrane 11, 24, 25. Three molecules of Tim9 and three molecules of Tim10 form a soluble 70 kDa complex 26, 27, 28. The peripheral module of the TIM22 complex is formed when one molecule of Tim10 is Senegenin usually substituted by one molecule of Tim12 29 which associates with the membrane integral module through the intermembrane space domain name of Senegenin Tim54 30. The family of small Tim proteins comprises of two further proteins, Tim8 and Tim13 31. These proteins have been shown to effect the import of certain noncarrier proteins into the inner membrane, such as Tim23 32, 33, 34. In mammals, six members of the family of small Tims Senegenin are expressed. One homolog each of Tim9 and Tim13 (human TIM9 and TIM13) and two homologs each of Tim8 (DDP1 and DDP2) and Tim10 (TIM10A and TIM10B) have been described in 21, 35. TIM10B Senegenin was further found to act as a functional homolog of yeast Tim12 36. Mutations in TIM8 (DDP1, the deafness\dystonia peptide) cause the MohrCTranebjaerg syndrome, a progressive neurodegenerative disorder characterized by sensorineural hearing loss, dystonia, mental retardation, and blindness 31, 37. In summary, despite the conservation of the small Tim proteins in mammals, the membrane module of the carrier translocate is not well conserved. We speculate that, besides the channel forming subunit TIM22, constituents of the human TIM22 KLHL1 antibody complex have been substituted by mammalian specific subunits during evolution. In order to identify novel subunits of the human TIM22 complex, we used immunoprecipitation studies, combined with a quantitative mass spectrometric approach. We found TIM29 (C19orf52) to be a specific binding partner of TIM22. TIM29 is usually a constituent of the 440 kDa human TIM22 complex. Depletion of TIM29 affects carrier protein translocation and concomitantly causes a significant decrease in cell growth. Thus, the human carrier translocase displays a composition that differs from its yeast counterpart. We conclude that adaptation of the translocation machinery to the more complex physiology of human mitochondria required novel translocase constituents to facilitate protein transport and membrane insertion. Materials and methods Cell culture Human embryonic kidney cell lines (HEK293T\Flp\In? T\Rex?) were cultured in DMEM, supplemented with 10% (v/v) heat\inactivated fetal bovine serum (Biochrom, Berlin, Germany) and 2 mm l\glutamine and incubated at 37 C with 5% CO2. For SILAC analysis, cells were produced.