Correspondence to J. F. Martín, Area of Microbiology, Faculty of Biology, Department of Ecology, Genetics and Microbiology, University of León, E-24071 León, Spain
Biochemical characterization of the SecA protein of Streptomyces lividans
Interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein
Article first published online: 25 DEC 2001
European Journal of Biochemistry
Volume 257, Issue 2, pages 472–478, October (II) 1998
How to Cite
Blanco, J., Driessen, A. J. M., Coque, J. J. R. and Martin, J. F. (1998), Biochemical characterization of the SecA protein of Streptomyces lividans. European Journal of Biochemistry, 257: 472–478. doi: 10.1046/j.1432-1327.1998.2570472.x
Fax: +34 987 291505.
Abbreviations. YEMES, yeast extract, malt extract, sucrose medium; NBS, nucleotide-binding site.
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- (Received 19 May/18 June 1998)
- Cited By
- protein secretion;
- membrane−protein interaction;
- nucleotide binding;
- SecA−SecB interaction.
The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61−65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [α-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 μM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.