cDNA Cloning of Carrot (Daucus carota) Soluble Acid [beta]-Fructofuranosidases and Comparison with the Cell Wall Isoenzyme

Abstract

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid 8-fructofuranosidase (BF) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular BF). Using antibodies against isoenzyme I of carrot soluble BF, we isolated several cDNA clones encoding polypeptides with sequences characteristic of BFs, from bacteria, yeast, and plants. l h e cDNA-derived polypeptide of one of the clones contains all partia1 peptide sequences of the purified isoenzyme I and thus codes for soluble acid BF isoenzyme 1. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. lhese two soluble BFs, 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 BF 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 BFs, are preproenzymes with signal peptides and Nterminal 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.

Invertases. Primary Structures, Functions, and Roles in Plant Development and Sucrose Partitioning

“…A 70-kD monomeric form of vacuolar invertase of mung bean hypocotyls was found to be split into a 30-kD N-terminal and a 38-kD C-terminal fragment (Arai et al, 1991). Likewise, the 68-kD monomer of isoform I of vacuolar invertase from carrot was fragmented into N-and C-terminal polypeptides of 43 and 25 kD, respectively (Unger et al, 1992(Unger et al, , 1994. Under native conditions, these fragments appear to be tightly associated and, in a complex, possess enzyme activity.…”

“…1) (Sturm and Chrispeels, 1990;Unger et al, 1994). The function of the putative propeptides is not clear, but in analogy to other "preproenzymes," they may play a role in protein folding (Klionsky et al, 1988), protein targeting (Klionsky et al, 1988, or in the control of enzyme activity (Hasilik and Tanner, 1987).…”

“…1) (Unger et al, 1994). The corresponding domains of isoform I and II of vacuolar invertase of carrot contain short hydrophobic amino acid stretches, which, in analogy to studies on barley lectin and tobacco chitinase, may form the critical cores of vacuolar sorting signals (Bednarek and Raikhel, 1992).…”

“…Four amino acid residues (NDPN) of the pentapeptide NDPNG, which is a hallmark Figure 1. Schematic comparison of the amino acid sequences deduced from cDNAs of carrot cell wall invertase and isoenzymes I and II of carrot vacuolar invertase (Unger et al, 1994). The mature proteins share 64% similarity and 46% identity.…”

Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors

“…Both invertase types exist in different isoforms [3][4][5]. Sequence comparisons indicate a common evolutionary origin [4].…”