The targeting of bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N‐terminus mature part

The vacuolar sorting domain of sporamin transports GUS, but not levansucrase, to the plant vacuole

“…Alternatively, the sporamin-levansucrase protein could be retained in the secretory pathway. In yeast, the signal sequence of a-amylase is able to translocate the levansucrase protein across the endoplasmic reticulum, but the levansucrase activity is retained within the cell (Scotti et al, 1996). A retention signal present in the levansucrase protein might, therefore, interfere with protein secretion both in plant cells and in yeast.…”

“…determining the affinity for concanavalin A (Con A), which binds sugar residues present on glycosylated proteins. The levansucrase protein contains six potential glycosylation sites and it was shown by Scotti et al (1996) that it was glycosylated when targeted to the ER of yeast by an a-amylase signal sequence. The non-glycosylated bacterial levansucrase protein isolated from B. subtilis, and the glycosylated a-amylase targeted levansucrase protein, isolated from yeast (Scotti et al 1996), were used as controls.…”

“…The levansucrase protein contains six potential glycosylation sites and it was shown by Scotti et al (1996) that it was glycosylated when targeted to the ER of yeast by an a-amylase signal sequence. The non-glycosylated bacterial levansucrase protein isolated from B. subtilis, and the glycosylated a-amylase targeted levansucrase protein, isolated from yeast (Scotti et al 1996), were used as controls. Con A affinity chromatography revealed that the yeast-derived glycosylated levansucrase protein was retained by the column while the non-glycosylated protein did not bind (Table 2).…”

“…By this procedure, only 30% of the non-glycosylated protein was detected in the unbound material. It was found that, using a B. subtilis levansucrase-specific antibody in a dot blot analysis (Scotti et al, 1996), the remaining 69 % of the levansucrase protein could be detected in the wash solution, indicating that the levansucrase protein exhibits some non-specific affinity for the Con A column (results not shown). Extracts were prepared from tobacco protoplasts transformed with the sporamin-levansucrase construct.…”

“…Levansucrase activity, however, is not affected by glycosylation. Scotti et al (1996) used endo H to deglycosylate yeast-derived a-amylase-levansucrase and found no difference in activity between the glycosylated and non-glycosylated sporamin-levansucrase protein. Inhibiting glycosylation, by the addition of tunicamycin, also had no effect on the activity of the levansucrase in a tobacco protoplast transformation assay (M. Ebskamp, unpublished).…”