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Biochemistry (Moscow)

, Volume 80, Issue 3, pp 296–309 | Cite as

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: Structural, biochemical, and functional characterization

  • M. G. Alekseeva
  • T. A. Mironcheva
  • D. A. Mavletova
  • S. M. Elizarov
  • N. V. Zakharevich
  • V. N. DanilenkoEmail author
Article

Abstract

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, ɛ, 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 ɛ-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.

Key words

FoF1-ATP synthase Ser/Thr protein kinase inverted membrane vesicles Streptomycetes 

Abbreviations

a.a.

amino acid residue

Bis-I

bis-indolylmaleimide I

bp

base pairs

DTT

dithiothreitol

KN-62

inhibitor of Ca2+-dependent protein kinases

PMSF

phenylmethylsulfonyl fluoride

STPK

serine/threonine protein kinases

TCA

trichloroacetic acid

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Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • M. G. Alekseeva
    • 1
  • T. A. Mironcheva
    • 1
  • D. A. Mavletova
    • 1
  • S. M. Elizarov
    • 2
  • N. V. Zakharevich
    • 1
  • V. N. Danilenko
    • 1
    Email author
  1. 1.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia
  2. 2.Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia

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