The majority of the proteins detectable by Coomassie blue staining were not affected by trypsin treatment, indicating that cytoplasmic proteins were not exposed to proteolysis. Globomycin inhibited PhoA processing When pTAP transformant cells were grown with increasing concentrations of globomycin, cell growth was inhibited. A concentration of 25 μg globomycin/ml was the highest

to still allow growth of cells. Growth in 25 μg globomycin/ml resulted in an increase in the molecular weight of PhoA (Figure 3A, lane 25 μg/ml) compared to that seen in cells grown in the absence of globomycin (Figure 3A, lane 0 μg/ml). Figure 3 Lipoprotein processing of PhoA. A. Effect of globomycin on the processing of PhoA. Mycoplasma transformants were grown in broth without or with globomycin added, as indicated above each lane, and their Autophagy activity proteins separated on 10 % SDS-polyacrylamide gels, Western transferred and immunostained using a MAb to AP. In cells grown in globomycin (25 μg/ml), and thus in which signal peptidase II

was inhibited, a higher molecular weight band was seen, indicative of the presence of the prolipoprotein. In the absence of globomycin (0 μg/ml) the fully processed 47 kDa lipoprotein is seen. B. Radiolabelling of PhoA. M. gallisepticum cell proteins and pTAP transformed M. gallisepticum cells were radiolabelled OICR-9429 in vivo with [14 C]palmitate and separated on 10 % SDS-polyacrylamide gels. The polyacrylamide gels were stained with Coomassie brilliant blue and autoradiographed or

Western transferred and immunostained using a MAb to AP. Lanes 1, M. gallisepticum cells; 2, pTAP transformed cells. Panels CB, Coomassie brilliant blue stained; WB, Western transferred and immunostained; RL, radiolabelled and autoradiographed. The dark arrow indicates the 67 kDa VlhA protein and the open arrow indicates the 47 kDa protein. Radiolabelling of lipid modified proteins Lipoproteins of M. gallisepticum transformed with pTAP were radiolabelled with [14 C]palmitate, separated by SDS-PAGE gel and either stained with Coomassie brilliant blue (Figure 3B, CB) and autoradiographed (Figure 3B, RL) or Western transferred and immunostained (Figure 3B, WB). Following autoradiography, a band of 47 kDa, similar to the expected size of alkaline phosphatase, was detected in the pTAP transformed cells (Figure Oxymatrine 3B, RL, 2), suggesting that PhoA in pTAP transformed M. gallisepticum was a lipoprotein. A Western blot immunostained with a MAb to AP demonstrated the presence of a recombinant AP protein of similar size to that of the radiolabelled band in pTAP-transformed M. gallisepticum (Figure 3B, WB, 2). Two-dimensional gel electrophoresis and mass spectrometric analysis of PhoA proteins Following separation of Triton X-114 preparations of protein by 2-D gel electrophoresis, a spot corresponding to PhoA was excised, digested with trypsin and analysed by mass spectrometry.