Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation 7689-03-4 IC50 of respiratory chain biogenesis. INTRODUCTION Mitochondrial function requires selective proteolysis, which is 7689-03-4 IC50 carried out by a number of specific proteases, including processing peptidases, ATP-dependent proteases, and oligopeptidases (Koppen and Langer, 2007 ). YME1L was identified as the human orthologue of the yeast ATP-dependent protease Yme1 (Coppola oxidase (CcO) subunit 2 7689-03-4 IC50 (Cox2), and the Ups1 and Ups2 proteins, which are involved in mitochondrial phospholipid metabolism (Nakai mutant (Shah has been repeatedly shown to be related to cancer progression and has been identified as an MYC-responsive gene (Wan or mRNAs (Figure 2A), we concluded that the loss of YME1L leads to the selective stabilization of these polypeptides. Given that the dynamin-related GTPase OPA1 is involved in 7689-03-4 IC50 the control of mitochondrial fusion and cristae morphology and was previously identified as a substrate of human YME1L (Griparic also appeared to be significantly elevated after the pulse (Figure 7, A and C). In contrast, the 17-h chase revealed that newly synthesized ND1, ND2, and ND6 subunits and, to a lesser extent, the Cox2 subunit, were significantly stabilized in YME1L KD cells compared with Rabbit Polyclonal to OR1A1 controls (Figure 7, A and C). This result appeared to be consistent with the blue native immunoblotting data, which showed a marked increase in complex I subcomplexes containing ND1 subunit. The observed slight increase in newly synthesized Cox1 could be explained by a comparable increase in mRNA in these cells (Figure 7B). Consistently, the newly synthesized Cox1 in YME1L KD cells is likely to be stabilized by its assembly into CcO subcomplexes (Figure 4E). Similarly, most of the remaining mitochondrially encoded complex I subunits may be stabilized within the Ndufb6 and ND1 subcomplexes in YME1L KD cells. Given the fact that the quantification of the mtDNA copy number did not reveal any significant changes in YME1L KD cells (data not shown) and that the mRNA levels of other tested mitochondrial translation products were also not elevated (Figure 7B), our results indicate polypeptide-specific stabilization of mitochondrial translation products in YME1L KD cells. FIGURE 7: The involvement of YME1L in the proteolysis of a subset of mitochondrially encoded subunits of complex I. (A) The loss of YME1L leads to the polypeptide-specific stabilization of mitochondrial translation products. Cells were labeled with a [35S]methionine-cysteine … ND5, ND2, and ND6 are bona fide substrates of the human i-AAA protease Next we investigated whether some of the stabilized mitochondrial translation products are indeed proteolytic substrates of YME1L or whether their increased stability is a 7689-03-4 IC50 secondary effect of their assembly within protective protein complexes. We performed [35S]methionine labeling of mitochondrial translation products in YME1L KD cells that were previously transfected with empty expression vector, the wild-type YME1L-FLAG construct, or the YME1LE543Q-FLAG construct. The subsequent anti-FLAG coimmunoprecipitation showed that, of the nine mitochondrial translation products that could be detected on fluorographs of coimmunoprecipitation inputs, Cox2, ND6, ND2, and ND5 exhibited increased coimmunoprecipitation with the proteolytically inactive YME1LE543Q variant compared with the wild-type YME1L-FLAG protein. The highest pulldown efficiency was observed for Cox2 and ND6, followed by ND2 and ND5 (Figure 7D). It is surprising that despite their markedly increased levels, both Atp6 and cytochrome failed to coimmunoprecipitate with the proteolytically inactive YME1L variant (Figure 7D). Similarly, the otherwise increased ND1 subunit did not efficiently copurify with YME1LE543Q-FLAG (Figure 7D). Collectively, these results support the previous finding that YME1LE543Q-FLAG coimmunoprecipitates with Cox2 and suggest that human YME1L is directly involved in the proteolytic degradation of the ND5, ND2, and ND6 subunits of the membrane arm of complex I. DISCUSSION We used shRNA knockdown and expression studies in HEK293 cells to define the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast mitochondrial i-AAA.