Puromycin resistant biosynthesis of a specific outer membrane lipoprotein of Escherichia coli

Abstract

The reported puromycin resistance of the in vivo biosynthesis of a specific outer membrane lipoprotein of E. coli was further investigated. The biosynthetic machinery making the lipoprotein was made more accessible to puromycin by disruption of the cell structure using ethylenediaminetetraacetate or toluene, and finally in an in vitro protein biosynthesis system using polyribosomes. Puromycin sensitivity of overall protein synthesis increased by about 10 fold for each method of disruption of the cell structure; 50% inhibitions were obtained at 330, 35, 2.7, and 0.22 μg of puromycin per ml for intact cells, ethylenediamine tetraacetate treated cells, toluene treated cells, and the polyribosome system, respectively. However, the lipoprotein biosynthesis remained more resistant to puromycin than the biosynthesis of other proteins in all systems tested. These results strongly suggest that puromycin resistance of the lipoprotein biosynthesis is due to an intrinsic property of the lipoprotein biosynthetic machinery.