Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition.
The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse-chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.
Morosoli
R
R
Centre de Recherche en Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, Université du Québec, Ville de Laval, Québec, Canada. rolf_morosoli@iaf.uquebec.ca
Dupont
C
C
D043364:Endo-1,4-beta Xylanases; D004792:Enzyme Precursors / Q000378:metabolism; D010957:Plasmids; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology*; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D019810:Sodium Azide; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases