Expression, purification, and characterization of BsSco, an accessory protein involved in the assembly of cytochrome c oxidase in Bacillus subtilis.
The studies described here were performed to characterize further the plasma membrane associated protein BsSco, which is the product of the gene ypmQ, in Bacillus subtilis. BsSco is a member of the Sco family of proteins found in the inner mitochondrial membrane of yeast and humans and implicated as an accessory protein in the assembly of the Cu(A) site of cytochrome c oxidase. We have cloned the gene expressing BsSco, placed a six-histidine tag on its C-terminus, and over-expressed this protein in B. subtilis. Recombinant BsSco with the his-tag has been purified from Triton X-100-solubilized plasma membranes by nickel metal affinity chromatography. Mass spectral analysis of the purified protein is consistent with processing of BsSco by signal peptidase II removing an N-terminal putative transmembrane sequence to leave an acyl-glyceryl moiety at cysteine residue 19. Antibodies, raised against purified, recombinant BsSco, were used to characterize the timing of the level of native BsSco in batch cultures of wild-type B. subtilis. There is a marked lag in the level of native BsSco, but it does appear prior to cytochrome c oxidase, which is expressed in late stage growth. This work supports a role for BsSco in the assembly of the Cu(A) site of cytochrome c oxidase and its functional relationship to the Sco proteins found in eukaryotic cells.
Andrews
Diann
D
Department of Biochemistry and Center for Protein Function Discovery, Queen's University, Kingston, Ont., Canada K7L 3N6.
Rattenbury
Jennifer
J
Anand
Vijay
V
Mattatall
Neil R
NR
Hill
Bruce C
BC
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry / Q000379:methods
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C482909:Sco protein, Bacillus subtilis; D003576:Electron Transport Complex IV