Figure 3

Expression and secretion of site-specifically mutated Peb1. The amino acid sequence of mature Peb1 shows the TAR1 and TAR2 regions in pink and yellow, respectively. Dots indicate sites for Ala point substitutions (encoded by plasmids pTAR1P and pTAR2P), the underlined sequences show sites for longer Ala and Gly substitutions (pTAR1A, pTAR1G, pTAR2A and pTAR2G), and the arrow indicates the site of a pentaglycine insertion (pTAR15G). Every tenth residue is indicated by a vertical line. The bottom panel shows whole cells (C) and cell-free culture supernatants (S) from strain MKS12 carrying the plasmids indicated above the panels analyzed by Western blotting using anti-Peb1 antibodies (see Fig. 2A and Table 1 for details regarding the plasmids). Wt Peb1 is encoded by the plasmid pLA25. Samples correspond to 100 μl of culture. Molecular weight markers in kDa are indicated on the left.