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Characterization of mitochondrial Lace1 ATPase (LB168)

Lace1 (Lactation elevated 1) is a human homolog of yeast Afg1 (ATPase family gene 1) ATPase with sequence identity ranging from 41,5 % H. sapiens vs. S. cerevisiae to 88,9 % H. sapiens vs. M. musculus. The protein consists of ATP/GTP binding P‐loop motif and a common five domain structure. The aim of our work was to perform detailed cell biological characterization of human Lace1 utilizing stable shRNA‐based RNA interference approach, proteomics and expression analyses using human embryonic kidney cell line (HEK293). We found that human Lace1 is a mitochondrially localized integral membrane protein with an apparent molecular weight of 50 kDa. The prepared stable shRNA LACE1 knockdown HEK293 cell line showed markedly reduced Lace1 protein levels (<10% of controls). SDS‐PAGE western blotting demonstrated markedly elevated levels of subunit SDHA of respiratory complex II, Cox2, Cox3, Cox4, Cox5a and Cox6aL subunits of respiratory complex IV, and ATP d subunit of complex V. On the other hand, the amount of the antiapoptotic factor Bcl‐2 was found to be decreased in these cells. Furthermore, slightly elevated levels of the tumor suppressor p53 were found in LACE1 knockdown cells. Quantitative 2D‐PAGE analysis coupled to mass spectrometric identification (MS) showed elevated accumulation of chaperonine 10 and ATPase F1‐alpha. Our work have thus far identified Lace1 as a mitochondrial factor playing a role in protein turnover of subunits of the oxidative phosphorylation system, possibly confirming its predicted function as an adaptor protein of mitochondrial proteases. Future experiments will involve short term downregulation of Lace1 using Stealth siRNA approach as well as co‐immunoprecipitation analyses coupled to MS identification with P‐loop mutant Lace1 protein.