4a, lane 4), barely delivered in the supernatant and was in an insoluble form in the membrane fractions. When a set of eight amino acid residues was added in frame at the carboxy-terminal end of PsaA without PsaBC (pYA4796), PsaA was not detected (data not shown). These results indicate that PsaB and PsaC are essential for the processing and translocation of PsaA from the cytoplasm to the cell surface in Salmonella. Deletion of the first 26 amino acids of PsaA amino-terminal region (pYA3711) prevented the translocation of PsaA from the cytoplasm to the cell surface. The unprocessed PsaA form was not observed and the mature 15-kDa protein was decreased in the total extract,
find more cytoplasm and membrane fractions (Fig. 4b, lane 1). Deletion of the last nine amino acids of PsaA at the carboxy-terminal region (pYA4800), from threonine at position 155 to phenylalanine at position 163, drastically decreased its expression and was barely detectable as a ∼13.5-kDa product in supernatant and membrane fraction (Fig. 4a, lane 9). These results indicate that the amino-terminal region is necessary to secrete PsaA and that the carboxy-terminal region is required for its stability. Deletion of the PsaA A31 (pYA4374) or S32 (pYA4375),
which forms part of the SPase-I cleavage site, did not affect the synthesis or secretion of PsaA in any subcellular fraction (Fig. 4b, lanes 7 and 8), check details but with the ΔA31–ΔS32 double deletion (pYA4376), the unprocessed 18-kDa product was not detected in the total extract and barely observed in the membrane fraction (Fig. 4b, lane 9). In contrast, when the amino acids involved in the PsaA predicted SPase-II cleavage site,
cysteine at position 26 changed to valine (pYA3708) and the glycine at position 27 replaced by serine (pYA3709), the PsaA synthesis was not affected (Fig. 4b, lanes 2 and 5). To determine whether the cysteine residues at positions 10 and 26 play stiripentol a role in the PsaA biogenesis and stability, the cysteine10 (pYA3707) was replaced with valine and either cysteine was changed to valine (pYA3706). None of these mutations affected PsaA synthesis or secretion (Fig. 4b, lanes 4 and 6). We observed the same expression profile when the RASV strain containing each of the previously described plasmids was grown with either 0.2% or 0.02% arabinose in the culture medium (data not shown). The amino acid substitution of the putative glycosylation site, asparagine 30 to leucine (pYA3710) produced a shorter unprocessed ∼17-kDa PsaA (Fig. 4b, lane 3). These results indicate that in the absence of either A31or S32, other amino acids flanking this SPase-I cleavage site can generate alternative cleavage sites, but deletion of both A31 and S32 produces a new cleavage site, which is processed more efficiently than the original.