Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991, Russia.
Biochemistry (Mosc). 2015 Mar;80(3):296-309. doi: 10.1134/S0006297915030050.
The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the β- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, β, γ, δ, ε, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, β-, α- and ε-subunits. The β-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.
我们研究了弗氏链霉菌 ATCC19609 反向膜囊泡中的蛋白质磷酸化模式，以阐明细菌膜结合 FoF1-ATP 合酶的调节机制。我们首次通过二维凝胶电泳和质谱法发现 FoF1-ATP 合酶复合物的β-和 b-亚基发生磷酸化；鉴定出 20 种具有已知功能的蛋白质。FoF1-ATP 合酶的所有 8 个亚基，即α、β、γ、δ、ε、a、b 和 c，都被克隆到大肠杆菌中，并作为重组蛋白表达。使用丝氨酸/苏氨酸蛋白激酶的粗制剂，我们证明了重组γ-、β-、α-和ε-亚基的磷酸化。β-亚基在重组蛋白和囊泡中均发生磷酸化。膜结合蛋白和重组蛋白的差异磷酸化可归因于每种制剂中不同的蛋白激酶池；此外，FoF1-ATP 合酶组装和功能的某些步骤可能伴随着特定的磷酸化模式。鉴定了包含所有亚基和调节蛋白 I 的操纵子的结构。研究了弗氏链霉菌 ATCC19609 的 FoF1-ATP 合酶与腐生菌和致病菌（包括结核分枝杆菌）中相应蛋白的系统发育相似性。因此，细菌丝氨酸/苏氨酸蛋白激酶对于 FoF1-ATP 合酶的调节很重要。从实际的角度来看，我们的结果为设计靶向抗菌药物提供了基础。
