TARGETING OF YERSINIA YOP PROTEINS INTO THE CYTOSOL OF HELA-CELLS - ONE-STEP TRANSLOCATION OF YOPE ACROSS BACTERIAL AND EUKARYOTIC MEMBRANES IS DEPENDENT ON SYCE CHAPERONE

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate abo ut whether YopE targeting proceeds as a direct translocation of polype ptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for trans location into the eukaryotic cytosol. Here, we used the selective solu bilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, Y opM and YopN were found in the eukaryotic cytosol but not in the extra cellular medium. When bound to SycE chaperone in the Yersinia cytoplas m, YopE residues 1-100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis f ailed to detect an extracellular intermediate of YopE targeting, sugge sting a one-step translocation mechanism.