Figure 2.

Effect of mutations on membrane insertion. (Top) Cartoon showing the expected topology of AqpZ. The color of the helices indicates the expected color of translational fusions ending after this helix using the pKTop system. The arrow heads indicate the last residues of the various fragments of AqpZ inserted into the pKTop reporter system. The numbers within the helices indicate their first and last residues, as determined by PyMOL. The positions of the mutations are indicated in various helices. The NPA (Asparagine Proline Alanine) labels indicate the incomplete helices that contain the conserved NPA motif. (Bottom) Spots (7 μL) of overnight bacterial cultures diluted to an OD_600nm of 0.5 were placed on indicator plates containing Red-Gal and BCIP and developed (see Materials and Methods). The numbers along the top edge indicate the last (C-terminal) helix in the construction, all constructions started from the N-terminal of the protein. Blue color indicates a periplasmic location of the C-terminal of the construction inserted into the plasmid pKTop, whereas red color indicates a cytoplasmic location. The pKTop control shows the result with no insert into the plasmid.