Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post-translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single-site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

中文翻译：

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赖氨酸乙酰化在线粒体核糖体蛋白L12功能中的作用

线粒体在能量产生和细胞代谢中发挥着核心作用。线粒体含有自己的小基因组（线粒体 DNA，mtDNA），携带 ATP 合成所需蛋白质的遗传指令。线粒体蛋白质组，包括线粒体转录机制，受到翻译后修饰 (PTM) 的影响，包括乙酰化和磷酸化。我们着手确定与 mtDNA 相关的蛋白质 PTM 是否可以为线粒体基因表达的调节提供潜在机制。在这里，我们重点关注线粒体核糖体蛋白 L12 (MRPL12)，它被认为可以稳定线粒体 RNA 聚合酶 (POLRMT) 并促进转录。通过质谱法鉴定了 MRPL12 的许多乙酰化位点。我们使用 MRPL12 的乙酰化（赖氨酸到谷氨酰胺突变体）和去乙酰化（赖氨酸到精氨酸突变体）版本的氨基酸模拟物来探究赖氨酸乙酰化在体外转录起始和 HeLa 细胞中线粒体基因表达中的作用。纯化 MRPL12 乙酰基和脱乙酰基蛋白模拟物，并评估其影响 POLRMT 的 mtDNA 启动子结合的能力。我们分析了 MRPL12 乙酰基和脱乙酰基模拟物过度表达后 HeLa 细胞中的 mtDNA 含量和线粒体转录水平。我们的结果表明，MRPL12 单位点乙酰基模拟物不会改变 POLRMT 的 mtDNA 启动子结合能力或 HeLa 细胞中的 mtDNA 含量。单个乙酰基模拟物可能对线粒体转录水平有适度的影响。我们发现线粒体脱乙酰酶 Sirtuin 3 能够在体外使 MRPL12 脱乙酰化，这表明动态乙酰化在基因表达调控之外的作用中具有控制 MRPL12 功能的潜在作用。