Cellular localization of FtsY in vitro and in vivo. (A) Escherichia coli cells were grown on LB medium up to mid-exponential phase (OD600 1.2). Cell breakage was performed using a French pressure cell in the presence of protease inhibitors. Unbroken cells and large cell fragments were removed by centrifugation and the supernatant of this centrifugation was then separated by ultracentrifugation into the soluble fraction (S) and the pellet fraction (P). Conditions indicate the centrifugation time in a Ti50.2 rotor at 45,000 rpm. After western transfer, the different fractions were analysed using antibodies against FtsY, against the integral membrane protein YidC and against the soluble protein Hsp60 (GroEL). (B) The functionality of the FtsY-green fluorescent protein (GFP) constructs was analysed by expressing plasmid-borne copies in the conditional FtsY depletion strain IY28. IY 28 containing either no plasmid (IY28) or the indicated plasmids was grown on LB-plates in the presence or absence of arabinose. Wt FtsY corresponds to untagged FtsY, wt FtsY-GFP corresponds to full length FtsY fused C-terminally to GFP. FtsY(B3)-GFP and FtsY(NG+1)-GFP correspond to GFP-tagged FtsY mutants which exhibit reduced activity due to impaired membrane binding. The growth experiments were performed in the absence of IPTG for preventing high-level expression of the plasmid-borne FtsY derivatives. (C) Western blot analyses of IY28 cells containing either no or the indicated plasmids. Cells were grown in the presence of arabinose or fructose but in the absence of IPTG.
Mircheva et al. BMC Biology 2009 7:76 doi:10.1186/1741-7007-7-76