Comparison of barley malt alpha-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast.
Germinating barley produces two alpha-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMY1) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMY1 is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMY1::AMY2 hybrid.
Juge
N
N
Laboratoire BBMN, Faculté des Sciences et Techniques de Saint-Jérôme, Université d'Aix-Marseille III, France.
Søgaard
M
M
Chaix
J C
JC
Martin-Eauclaire
M F
MF
Svensson
B
B
Marchis-Mouren
G
G
Guo
X J
XJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D001467:Hordeum / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics*
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D004247:DNA; D000516:alpha-Amylases