FIGURE 2.

Neither post-translational nor cotranslational signal peptides direct efficient secretion of glycolytic enzymes. MC4100 were transformed with a plasmid that encodes Pgk (A), GapA (B), or Eno (C) fused to the indicated signal sequence and subjected to pulse-chase labeling. A C-terminal HA tag was attached to each protein during cloning. Because of the difficulty of resolving the precursor (p) and mature (m) forms of the proteins, in some cases cells were converted to spheroplasts after a 1-min chase and treated with proteinase K (PK). Precursor and mature forms of each enzyme were immunoprecipitated with an anti-HA antiserum. In one case the cytoplasmic protein Ffh was immunoprecipitated to demonstrate the integrity of the IM.