The conformation of mature human alpha-amylase conditions its secretion from yeast.
The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.
Sato
T
T
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Uemura
H
H
Izumoto
Y
Y
Nakao
J
J
Nakamura
Y
Y
Matsubara
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D005796:Genes; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012469:Salivary Glands / Q000378:metabolism; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA; D000516:alpha-Amylases