Regulation Of Mitochondrial Translation And Oxidative Phosphorylation Through Reversible Acetylation
Open Access
- Author:
- Cimen, Huseyin
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 29, 2012
- Committee Members:
- Emine C Koc, Dissertation Advisor/Co-Advisor
Hasan Koc, Dissertation Advisor/Co-Advisor
Emine C Koc, Committee Chair/Co-Chair
Hasan Koc, Committee Chair/Co-Chair
Craig Eugene Cameron, Committee Member
Joseph C. Reese, Committee Member
Teh Hui Kao, Committee Member
Tae Hee Lee, Committee Member - Keywords:
- ribosome
mitochondria
acetylation
SIRT3
mitochondrial translation
oxidative phopshorylation
succinate dehydrogenase
TCA - Abstract:
- In a eukaryotic cell, mitochondria provide energy in the form of ATP through oxidative phosphorylation (OXPHOS). Human mitochondria have their own genome and transcription/translation system to synthesize mitochondrially encoded thirteen proteins of respiratory chain complexes. We identified mitochondrial ribosomal protein L10 (MRPL10) as the major acetylated ribosomal protein in mammalian mitochondria with two-dimensional gel electrophoresis followed by tandem mass spectrometry and immunoblotting analyses. In addition, we discovered that SIRT3, which is the main NAD+-dependent deacetylase localized into mitochondria, interacts with the ribosome and is responsible for the deacetylation of MRPL10. We employed SIRT3 knock-out mice in order to study the mechanism of reversible acetylation of MRPL10. The acetylation of MRPL10 resulted in increased MRPL12 binding to the L7/L12 stalk accompanied by enhanced protein synthesis in in vitro translation assays. Moreover, HIB1B, a brown adipocyte tissue cell line stably overexpressing SIRT3, demonstrated reduction in the acetylation status of MRPL10 and decreased MRPL12 binding to the L7/L12 stalk. By using [35S]-methionine pulse-labeling assays, we revealed that the mitochondrial protein synthesis was suppressed in these cells overexpressing SIRT3. Diminished synthesis of mitochondrial-encoded protein subunits of respiratory chain complexes resulted in reduced the activities of Complex I and IV and total ATP production. Overall, these findings define a possible mechanism by which SIRT3-dependent reversible acetylation of MRPL10 and MRPL12 binding to the ribosome regulates the mitochondrial protein synthesis and, therefore, modulates the OXPHOS and ATP production. Next, we identified and assessed the acetylation of the flavoprotein (SdhA) subunit of Complex II, succinate dehydrogenase, which resulted in reduced activity of Complex II in SIRT3 knock-out mice. Moreover, we identified three additional members of the mitochondrial ribosome; CHCHD1, AURKAIP1, and CRIF1 in our mass spectrometry-based proteomic analyses. We found that siRNA mediated knockdown of the newly identified ribosomal proteins to impair mitochondrial protein synthesis as determined by [35S]-methionine pulse-labeling assays. Given that the components of mitochondrial translation are crucial in the synthesis of respiratory chain subunits, the newly identified ribosomal proteins in addition to acetylation of MRPL10 and SdhA provide a more complete picture of mitochondrial translation and regulation of energy production in the cell.