Green fluorescent protein functions as a reporter for protein localizationin Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein locali zation in Escherichia coli was explored by creating gene Fusions between ma lE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid protei ns composed of GFP Fused to the carboxy-terminal end of MBP. Fluorescence w as not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluoresce nce was observed. Cell fractionation studies showed that the fluorescent MB P-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluores cent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gen e fusions in different sec mutants, as well as signal sequence processing a ssays, confirmed that the periplasmically localized hybrid proteins were ex ported by the sec-dependent pathway. The distinction between fluorescent an d nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins compris ed of full-length MBP did not result in overproduction lethality characteri stic of some exported beta-galactosidase hybrid proteins, synthesis of shor ter, exported hybrid proteins was toxic to the cells. Purification of MBP-G FP hybrid protein from the different cellular compartments indicated that G FP is improperly folded when localized outside of the cytoplasm. These resu lts suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.