PhoA acticities (Upper) and GFP fluorescence-emission intensities at 508 nm (Lower) for the 12 E. coli inner-membrane protein GFP/PhoA fusions studied here. Note that the PhoA activity for YdgE (13.100 units) and the GFP fluorescence intensity for YgjV (65.500 units) are off scale, and that the YgjV/PhoA fusion (indicated by *) was not made. Horizontal lines indicate the thresholds used to decide on the periplasmic or cytoplasmic location of the different fusion proteins.

Test of GFP as a marker for C-terminal membrane protein topology. (A) GFP fluorescence emission spectra of cells expressing Lep-inv/GFP and Lep/GFP grown at 37°C. (B) GFP fluorescence-emission intensities at 508 nm for GFP fusions to test proteins with known topology. Only Lep/GFP has a periplasmically located C terminus. (C) Immunoblots using Lep (Upper) and GFP (Lower) antisera of whole-cell lysates prepared from cells overexpressing Lep-inv/GFP and Lep/GFP. Lane 1, Lep/GFP, uninduced; lane 2, Lep/GFP, induced at 25°C; lane 3, Lep/GFP, induced at 37°C; lane 4, Lep-inv/GFP, uninduced; lane 5, Lep-inv/GFP, induced at 25°C; lane 6, Lep-inv/GFP, induced at 37°C.

Western blots of GFP fusions decorated with GFP antibody. Lane 1, YgjV/GFP (theoretical Mw = 44 kDa); lane 2, YcbM/GFP (51 kDa); lane 3, YiaB/GFP (36 kDa); lane 4, TatC/GFP (54 kDa); lane 5, YedZ/GFP (48 kDa); lane 6, YddQ/GFP (58 kDa); lane 7, PstA/GFP (58 kDa); lane 8, YdgE/GFP (37 kDa); lane 9, YnfA/GFP (37 kDa); lane 10, YigG/GFP (40 kDa); lane 11, MarC/GFP (50 kDa); lane 12, YaeL/GFP (75 kDa). GFP itself has a molecular mass of 25 kDa and is detected as a proteolytic fragment for YgjV, YddQ, PstA, and MarC. Lanes 1–7 and 11 show whole-cell fractions (the fusions in lanes 1–7 all have high GFP fluorescence), and lanes 8–10 and 12 show purified membrane fractions of fusions with low GFP fluorescence (see Materials and Methods). For the latter, blots of whole-cell lysates do not show detectable signals (data not shown).