Abstract 1312 Characterization of post-translational modifications of the mitochondrial transcription machinery

Mitochondria play a central role in energy production and cellular metabolism. The mitochondria also contain their own small genome and proteins that are encoded by mitochondrial DNA (mtDNA) play a role in the synthesis of ATP. The mitochondrial proteome is subject to post-translational modifications, including lysine acetylation and the phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription of mtDNA are subject to modification which suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation and/or phosphorylation controls the function of two nuclear-encoded mitochondrial proteins: mitochondrial RNA polymerase (POLRMT) and mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize POLRMT and promote transcription. Mass spectrometry was used to identify post-translational modifications on POLRMT and MRPL12. We identified multiple sites of acetylation and phosphorylation on POLRMT and MRPL12. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) which are found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phospho mimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system. MRPL12 acetylation was studied using acetyl modification mimics in vitro and in HeLa cells. MRPL12 modification mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT in vitro and mitochondrial transcript levels in cells. Our results suggest that MRPL12 and MRPL12 acetyl mimics do not affect the mtDNA promoter binding of POLRMT, mtDNA content, or mitochondrial transcript levels. The finding that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 suggests a potential role for dynamic acetylation controlling MRPL12 function in a role other than the control of gene expression. This material is based upon work supported by the National Science Foundation under Grant Nos. 2017708 (CHEM MRI) and 1814845 (MCB RUI). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.