cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.
Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.
Unger
C
C
Friedrich Miescher-Institut, Basel, Switzerland.
Hardegger
M
M
Lienhard
S
S
Sturm
A
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000032:analysis; D017343:Genes, Plant; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014675:Vegetables / Q000201:enzymology* / Q000235:genetics; D043324:beta-Fructofuranosidase
D018076:DNA, Complementary; D007527:Isoenzymes; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase