To examine the putative association of YsxC with ribosomes, a co-purification experiment was carried out. Staphylococcal ribosomes were extracted from other cellular materials by several ultracentrifugation and washing steps, and core ribosomes were depleted of accessory ribosomal proteins by Talazoparib nmr ammonium chloride extraction. Equivalent samples from different stages of the purification process were separated by SDS-PAGE,

Western blotted and immuno-detected with anti-YsxC antibodies (Figure 4). YsxC is in the insoluble fraction following the initial ultracentrifugation of a total cell extract (lane 3) and remains in the insoluble fraction after solubilisation of the membranes with Triton X-100 (lane 5). When this insoluble fraction was resuspended in 1 M NH4Cl, YsxC was solubilised (lane 6). These results suggest that YsxC is associated with the ribosome but is not a core ribosomal protein. Figure 4 Subcellular localisation of YsxC. The ribosome-containing fraction of S. aureus SH1000 was made by ultracentrifugation after cell breakage HKI-272 molecular weight and removal of cellular debris. Lane: 1, pre-stained molecular mass markers; 2, supernatant after ultracentrifugation; 3, pellet resuspended in buffer, containing 0.5% (v/v) Triton X-100, equal to that of the original suspension; 4, supernatant after

the ultracentrifugation step was repeated; 5, pellet resuspended in buffer containing 1 M ammonium chloride (NH4Cl); 6, supernatant after further ultracentrifugation; 7, pellet resuspended in an equal amount of buffer containing 1 M NH4Cl. Samples were resolved by 12% (w/v) SDS-PAGE and A) Coomassie Blue stained, or B) Western blotted with antibodies against YsxC. Each lane contains the equivalent of 1 ml of original culture. Association of YsxC with specific ribosomal subunits In order to elucidate the nature of the YsxC-ribosome association, material from S. aureus SH1000 containing ribosomes was separated by ultracentrifugation in a sucrose gradient. This separates the ribosome

into its constituents, i.e., 30 Amylase S and 50 S subunits, as well as the whole 70 S ribosome. The association of YsxC with a particular ribosomal fraction was determined by Western blot immunodetection with anti-YsxC antibodies. As shown in Figure 5 the extract contained the three expected ribosomal fractions and YsxC was primarily located in samples 8-14 corresponding to the 50 S subunit. Figure 5 Association of YsxC with ribosomal subunits. A) A260 of a ribosome containing fraction of S. aureus SH1000 separated by a 10-30% (w/v) sucrose gradient centrifugation. 1 ml samples were taken and analysed for RNA content (A260). B) Western blot of gradient samples probed with anti-YsxC. Role of YsxC in the ribosome YsxC may play a role in ribosome assembly, activity or stability. Ribosome profiles of wild type and YsxC-depleted cultures were compared.