Identification of beta-glucosidase aggregating factor (BGAF) and mapping of BGAF binding regions on Maize beta -glucosidase.
In certain maize genotypes (nulls), beta-glucosidase does not enter the gel and therefore cannot be detected on zymograms. Such genotypes were initially thought to be homozygous for a null allele at the glu1 gene. We have shown that a beta-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize beta-glucosidases and forms large insoluble aggregates. To understand the mechanism of the beta-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize beta-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum beta-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind. The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu(50)-Val(145)) and an extreme C-terminal region (Phe(466)-Ala(512)) together form the BGAF binding site on the enzyme surface. In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it in Escherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues. A data base search revealed that BGAF is a member of the jasmonite-induced protein family. Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice. Each of these repeat units is postulated to be involved in forming a site for binding to maize beta-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays.
Blanchard
D J
DJ
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406, USA.
Cicek
M
M
Chen
J
J
Esen
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000378:metabolism*
C426244:BGAF protein, Zea mays; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase