coli. However, hydrophobicity Panobinostat price profile analysis revealed that the N-terminus of the A domain has a putative transmembrane segment. The N-terminus of the A domain might act as an integral membrane anchor, indispensable for FtsY membrane association (Bibi et al., 2001). When this putative transmembrane segment was fused to the E. coli NG domain, the chimera construct was capable

of rescuing wild-type EcFtsY depletion in a conditional FtsY-deletion mutant of E. coli. In contrast, the E. coli NG domain alone could not fully rescue wild-type FtsY depletion (Maeda et al., 2008). These results suggest that the N-terminus of S. coelicolor FtsY (ScFtsY) has a functional role. The ScFtsY N-terminus may contribute to the membrane targeting of FtsY, but there is no direct evidence. In this study, the membrane-targeting ability of the N-terminal hydrophobic segment of the ScFtsY A domain was assessed by membrane protein extraction and Mal-PEG

labeling experiments. Results show that this part of the ScFtsY A domain might form a membrane insertion structure that can anchor ScFtsY to the membrane. The S. coelicolor strains used in this study are listed in Table 1. The E. coli strain ET12567 (MacNeil et al., 1992), which contains the plasmid pUZ8002, was used for plasmid introduction by conjugation into S. coelicolor M145 (Kieser et al., 2000). http://www.selleckchem.com/products/Romidepsin-FK228.html All S. coelicolor strains were grown at 30 °C, 220 r.p.m. min−1 in TSB liquid media for protein expression. Apramycin (50 μg mL−1) was added when necessary. All the plasmids used in this study are listed in Table 2. All primers are listed in Supporting Information, Appendix S1, and the detailed protocol for plasmid construction and protein expression is provided in Appendix S2. Subcellular fractions were isolated as described in the study by de Leeuw et al. (1997). Cells were suspended in lysis buffer (Mao et al., 2009) and lysed by freezing and short ultrasonic treatment. The cellular debris was removed from

the lysate by sedimentation (12 000 g for 15 min); the supernatant was then subjected to ultracentrifugation (356 000 g for 45 min), and the membrane pellet fraction [precipitant (‘P’)] was separated from the soluble fraction GPX6 [supernatant (‘S’)]. The supernatant was precipitated with 1 vol 10% TCA and resuspended in SDS-loading buffer, whereas the pellet fraction was directly dissolved in the same amount of SDS-loading buffer. The same amount of ‘P’ and ‘S’ samples was loaded onto an SDS-PAGE gel. The EGFP mutants in the samples were detected using the EGFP antibody. The protein content in ‘P’ and ‘S’ was calculated using the Quantity One software (Bio-Rad™). For carbonate extraction, the membrane pellet fraction, ‘P’, was incubated with 0.2 M Na2CO3 for 30 min at 4 °C and subsequently ultracentrifuged for 45 min at 356 000 g; the precipitant was the membrane pellet fraction (‘P′’), and the supernatant was the soluble fraction (‘S′’).