At normal growth condition, cellular concentration of sigma-32 is

At normal growth condition, cellular concentration of sigma-32 is very low (10–30 copies/cell at 30°C) and increases up to 12–15 folds with the temperature up-shift [4]. Instead of heat, cytoplasmic accumulation of the membrane or periplasmic proteins elevates Sirolimus manufacturer the syntheses of hsps in E. coli. Any membrane or periplasmic protein of E. coli is known to be synthesized initially in cell cytoplasm as precursor form, which contains an N-terminal signal-sequence [5]. The signal sequence targets the precursor towards

the plasma membrane translocase that transports the precursor across the membrane [6]. The signal peptide is then cleaved by a signal peptidase, an integral membrane protein with active site facing the periplasm [7]. The matured protein is then positioned at its membrane or periplasmic location with functionally correct orientation. The PMF across E. coli plasma membrane acts as an energy source for protein translocation [8, 9]. The inhibition of translocation and consequent storage of membrane proteins in cell cytosol is found to induce

hsps in export deficient mutants (where the multi-subunit translocase is nonfunctional) [10, 11], in signal sequence mutants (where the precursor proteins cannot be targeted to the translocase) [12, 13], and in wild type cells treated with protonophores like CCCP or DNP [14, 15]. However, it is still obscure how the inhibition of protein translocation phenomenon is related to the induction of cellular heat-shock response at the molecular level. Therefore, in the present study, we target selleck kinase inhibitor to investigate 1) how the cellular level of the heat-shock regulator protein sigma-32 is modulated under the condition of inhibition of protein translocation by the protonophores like CCCP/DNP, 2) what is the final fate of the non-translocated

proteins, stored in cell cytoplasm and 3) how the induced hsps do interact with the non-translocated proteins. Methods Bacterial strains and plasmid The E. coli strain Mph42 [16], mostly used in this study, was a generous gift from Dr. Jonathan Beckwith, Interleukin-2 receptor Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, USA. The E. coli strains JT4000 (∇ lon-510) [17] and SG22159 (clpP:: kan) [17], mutants of the Lon and ClpP protease respectively, and their wild type strain SG20250 (MC4100, clp +, lon +) [17] were kindly gifted by Dr. Susan Gottesman, Laboratory of Molecular Biology, NCI, NIH Bethesda, USA. Sigma-32 was isolated from E. coli strain BB2012 (a His-tagged clone), a kind gift from Dr. Matthias P. Mayer, Institute for Biochemistry and Molecular Biology, University of Freidburg, Germany. The plasmid pET vector containing dnaK gene was obtained from Prof. C. K. Dasgupta, Department of Biophysics, Molecular Biology & Genetics, University of Calcutta, Kolkata, India.

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