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 isoen zymes of acid beta-fructofuranosidase (beta F) (invertase), which eith er accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). U sing antibodies against isoenzyme I of carrot soluble beta F, we isola ted several cDNA clones encoding polypeptides with sequences character istic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived p olypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F is oenzyme I. A second clone codes for a related polypeptide (63% identit y and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respecti vely) clearly different from the extracellular enzyme, which has a bas ic isoelectric point of 9.9. Marked differences among the three nucleo tide 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 c erevisiae. All three carrot acid beta Fs, are preproenzymes with signa l peptides and N-terminal propeptides. A comparison of the sequences o f the soluble enzymes with the sequence of the extracellular protein i dentified C-terminal extensions with short hydrophobic amino acid stre tches that may contain the information for vacuolar targeting.