Segregation of molecules at cell division reveals native protein localization

Abstract

The authors compare segregation of a protein into two daughter cells for the wild-type protein and a fluorescently tagged version, by assessing protein activity in the two cells; differences in segregation between the two protein versions indicate mislocalization artifacts caused by the fluorescent tag. Using this system they identify widespread artifacts in the localization of bacterial proteases. We introduce a nonintrusive method exploiting single-cell variability after cell division to validate protein localization. We found that Clp proteases, widely reported to form biologically relevant foci, were uniformly distributed in Escherichia coli cells, and that many commonly used fluorescent proteins caused severe mislocalization when fused to homo-oligomers. Retagging five other reportedly foci-forming proteins with the most monomeric fluorescent protein tested suggests that the foci were caused by the fluorescent tags.