Nucleotide and Deduced Amino Acid Sequences. Ellen Klann, Serge Yelle2, and Alan B. Bennett*. Mann Laboratory, Department of Vegetable Crops, University ...
Plant Physiol. (1992) 99, 351-353
Received for publication October 21, 1991 Accepted December 16, 1991
0032-0889/92/99/0351/03/$01 .00/0
Plant Gene Register
Tomato Fruit Acid Invertase Complementary DNA1 Nucleotide and Deduced Amino Acid Sequences Ellen Klann, Serge Yelle2, and Alan B. Bennett* Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616 imately 25 amino acid residues N terminal to a characteristic n-region domain that is more commonly found at the extreme N terminus of secretory proteins. A structurally similar signal sequence has been identified at the N terminus of an endoplasmic reticulum-localized auxin-binding protein (2, 4). In addition, the predicted molecular mass of the mature polypeptide (60 kD) exceeds that determined by SDS gel electrophoresis and gel filtration (52 kD, ref. 7), suggesting that maturation of tomato acid invertase may involve additional proteolytic processing at the C terminus. C-terminal propeptides in other vacuolar proteins have been shown to function as vacuolar targeting signals (1, 6).
Acid invertase (EC 3.2.1.26) is responsible for the hydrolysis of sucrose to glucose and fructose. It has been found to be localized in either the cell wall or vacuole. Acid invertase is important in the metabolism of imported sucrose and has been proposed to play a central role in determining sink strength of developing plant organs, including tomato fruit (3). It has also been suggested that the absence of invertase in developing fruit contributes to the accumulation of sucrose, rather than hexoses, in a wild species of tomato and in sweet melons (5, 7, 9). In addition, the carrot acid invertase gene has been shown to be regulated by wounding and bacterial infection (8), suggesting a possible role in pathogen defense mechanisms. To critically assess the role of acid invertase in regulating carbohydrate metabolism in developing fruit, we previously purified and characterized tomato fruit acid invertase (9) and, using information derived from the invertase protein, isolated the corresponding cDNA. Here, we report the nucleotide and deduced amino acid sequence of the full-length tomato (Lycopersicon esculentum) fruit acid invertase cDNA. The tomato fruit acid invertase amino acid sequence is 40.5% identical with carrot (Daucus carota) extracellular acid invertase, the only other previously characterized plant acid invertase cDNA, and contains a domain (NDPNG, residues 150-154) highly conserved among sucrose hydrolases (8). The deduced amino acid sequence includes 92 amino acids preceding the start of the mature protein (Fig. 1, Table I). The 92-amino acid presequence appears to be comprised of a hydrophobic signal sequence with a predicted signal sequence cleavage site between residue 47 and residue 48 followed by an additional 45-amino acid domain (prosequence) that precedes the N terminus of the mature protein. The predicted 47-amino acid signal sequence is unusually long with approx-
LITERATURE CITED 1. Bednarek SY, Wilkins TA, Dombowski JE, Raikhel NV (1990) A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell 2: 1145-1155 2. Hesse T, Feldwisch J, Balshusemann D, Bauw G, Puype M, Vandekerckhove J, Lobler M, Klambt D, Schell J, Palme K (1989) Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin. EMBO J 8: 2453-2461 3. Ho LC (1984) Partitioning of assimilates in fruiting tomato plants. Plant Growth Regula 2: 277-285 4. Inohara N, Shimomura S, Fukui T, Futai M (1989) Auxinbinding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure. Proc Natl Acad Sci USA 86: 3564-3568 5. Lingle SE, Dunlap JR (1987) Sucrose metabolism in netted muskmelon fruit during development. Plant Physiol 84:
386-389 6. Neuhaus J-M, Sticher L, Meins F, Boller T (1991) A short Cterminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88: 10362-10366 7. Schaffer AA, Aloni B, Fogelman E (1987) Sucrose metabolism and accumulation in developing fruit of Cucumis. Phytochemistry 26: 1883-1887 8. Sturm A, Chrispeels M (1990) cDNA cloning of carrot extracellular f3-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell 2: 1107-1119 9. Yelle S, Chetelat RT, Dorais M, DeVerna JW, Bennett AB (1991) Sink metabolism in tomato fruit. IV. Genetic and biochemical analysis of sucrose accumulation. Plant Physiol 95: 1026-1035
Research supported by grant No. US-1872-90C from the Binational Agricultural Research and Development Fund, and a research gift from Campbell's Institute for Research and Technology. 2 Present address: Departement de Phytologie, Universite Laval, Quebec, Canada G 1K 7P4.
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GATTCCTCTATCTTCTATTATGGCCACTCAGTGTTATGACCCCGAAAACTCCGCCTCTCGTTACACATTACTCCCGGATCAACCCGATTCCGGCCACCGG M A T Q C Y D P E N S A S R Y T L L P D Q P D S G H R
100 27
AAGTCCCTTAAAATCATCTCCGGCATTTTCCTCTCCGTTTTCCTTTTGCTTTCTGTAGCCTTCTTTCCGATCCTCAACAACCAGTCACCGGACTTGCAAA K S L K I I S G I F L S V F L L L S V A F F P I L N N Q S P D L 0 t TCGACTCCCGTTCGCCGGCGCCGCCGTCAAGAGGTGTTTCTCAGGGAGTCTCCGATAAAACTTTTCGAGATGTAGCCGGTGCTAGTCACGTTTCTTATGC I D S R S P A P P S R G V S 0 G V S D K T F R D V A G A S H V S Y A
200 60
GTGGTCCAATGCTATGCTTAGCTGGCAAAGAACGGCTTACCATTTTCAACCTCAAAAAAATTGGATGAACGATCCTAATGGACCATTGTATCACAAGGGA W S N A M L S W Q R T A Y H F 0 P Q K N W M N D P N G P L Y H K G
400 127
TGGTACCACCTTTTTTATCAATACAATCCAGATTCAGCTATTTGGGGAAATATCACATGGGGCCATGCTGTATCCAAGGACTTGATCCACTGGCTCTACT W Y H L F Y Q Y N P D S A I W G N I T W G H A V S K D L I H W L Y
500
TGCCTTTTGCCATGGTTCCTGATCAATGGTATGATATTAACGGTGTCTGGACAGGGTCCGCTACCATCCTACCCGATGGTCAGATCATGATGCTTTATAC L P F A M V P D Q W Y D I N G V W T G S A T I L P D G Q I M M L Y T
600
CGGTGACACTGATGATTATGTGCAAGTGCAAAATCTTGCGTACCCCGCCAACTTATCTGATCCTCTCCTTCTAGACTGGGTCAAGTTCAAAGGCAACCCG G D T D D Y V Q V Q N L A Y P A N L S D P L L L D W V K F K G N P
700 227
GTTCTGGTTCCTCCACCCGGCATTGGTGTCAAGGACTTTAGAGACCCGACTACTGCTTGGACCGGACCACAAAATGGGCAATGGCTGTTAACAATCGGGT V L V P P P G I G V K D F R D P T T A W T G P Q N G Q W L L T I G
260
CTAAGATTGGTAAAACGGGTGTTGCACTTGTTTATGAAACTTCCAACTTCACAAGCTTTAAGCTATTGGATGGAGTGCTGCATGCGGTTCCGGGTACGGG S K I G K T G V A L V Y E T S N F T S F K L L D G V L H A V P G T G
900 294
TATGTGGGAGTGTGTGGACTTTTACCCGGTATCTACTAAAAAAACAAACGGGTTGGACACATCATATAACGGGCCGGGTGTAAAGCATGTGTTAAAAGCA M W E C V D F Y P V S T K K T N G L D T S Y N G P G V K H V L K A
1000 327
AGTTTAGATGACAATAAGCAAGATCATTATGCTATTGGTACGTATGACTTGGGAAAGAACAAATGGACACCCGATAACCCGGAATTGGATTGTGGAATTG S L D D N K Q D H Y A I G T Y D L G K N K W T P D N P E L D C G I
1100 360
GGTTGAGACTAGACTATGGGAAATATTATGCATCAAAGACTTTTTATGACCCGAAGAAAGAACGAAGAGTACTGTGGGGATGGATTGGGGAAACTGACAG L R L D Y G K Y Y A S K T F Y D P K K E R R V L W G W I G E T D S
1200 394
TGAATCTGCTGACCTGCAGAAGGGATGGGCATCTGTACAGAGTATTCCAAGGACAGTGCTTTACGACAAGAAGACAGGGACACATCTACTTCAGTGGCCA
1300 427
G
E
S
D
A
L
K
Q
G
A
W
S
V
P
I
S
Q
R
T
Y
L
V
D
K
K
T
G
T
H
L
Q
L
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GTGGAAGAAATTGAAAGCTTAAGAGTGGGTGATCCTACTGTTAAGCAAGTCGATCTTCAACCAGGCTCAATTGAGCTACTCCGTGTTGACTCAGCTGCAG E
V
E
I
E
S
L
R
G
V
D
P
T
K
V
Q
L
D
V
P
Q
S
G
E
I
L
L
R
V
D
S
A
A
AGTTGGATATAGAAGCCTCATTTGAAGTGGACAAAGTCGCGCTTCAGGGAATAATTGAAGCAGATCATGTAGGTTTCAGTTGCTCTACTAGTGGAGGTGC E L D I E A S F E V D K V A L Q G I I E A D H V G F S C S T S G G A TGCTAGCAGAGGCATTTTGGGACCATTTGGTGTCATAGTAATTGCTGATCAAACGCTATCTGAGCTAACGCCAGTTTACTTTTACATTTCTAAAGGAGCT A
R
S
G
I
G
L
F
P
G
I
V
V
A
I
D
Q
T
L
S
E
L
P
T
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Y
F
Y
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I
K
G
G
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T
S
S
E
A
G
P
V G
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Y
V
S
G
S
V
P
ACGGTGAAAAACATTCAATGAGATTATTGGTGGATCACTCAATTGTGGAGAGCTTTGCTCAAGGAGGAAGAACAGTCATAACATCGCGAATTTACCCAAC D
E
G
K
S
H
M
R
L
L
D
V
H
I
S
V
E
S
A
F
G
Q
G
R
T
I
V
S
T
I
R
P
Y
T
AAAGGCAGTAAATGGAGCAGCACGACTCTTTGTTTTCAACAATGCCACAGGGGCTAGCGTTACTGCCTCCGTCAAGATTTGGTCACTTGAGTCAGCTAAT K
A
V
N
G
A
A
R
L
F
V
F
N
N
A
T
G
A
S
V
T
A
S
V
K
I
W
S
L
E
S
A
194
800
1400 460 1500 494 1600
1700
560
L
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160
527
A
GATGGTCGTGCAGAGACTCACTTCTGTGCTGATCAAACTAGATCCTCTGAGGCTCCGGGAGTTGGTAAMCAAGTTTATGGTAGTTCAGTACCTGTGTTGG D
300 94
N
1800 594 1900 627
ATTCAATCCTTCCCTTTGCAAGACTTGTAATCTTCTTTATTTCGTTTTTTTTTTCTTTTTCATTTGAAGGTTATTTCACCGACGTCCCATCAAGAAAGGG I 0 S F P L Q D L 636
2000
AAGAGGGAGATCAATATATGTAGTGTTATTCGCCCTACCTTAGGATTAGATGTCATCTAGCAATGTCAAATCTAGTAGAGTATACAATGTATGGGTTCCT
2100
GGAAACCGAGTAGAGCTTACCTGGATTCTATGTAAACTAAGAAAGCTCAGCAAATATATGCACAAATAATTTACAGAAAAAAAAAAAAAAAAAAAAAAAA
2200
AAAAAAAAAAAAAAAAAAAAAAA
2223
Figure 1. Nucleotide and predicted amino acid sequence of the tomato fruit acid invertase cDNA. Underlined regions correspond to sequences confirmed by sequencing fragments of the protein. Predicted signal sequence cleavage site is indicated by an arrow, and the N-terminal tyrosine residue is indicated by *.
TOMATO ACID INVERTASE SEQUENCE
Table I. Characteristics of Invertase cDNA from Tomato Organism: Lycopersicon esculentum. Gene; Gene Product: TIVi; acid invertase (,B-fructosidase, EC 3.2.1.26). Techniques: cDNA isolation using oligonucleotide probe corresponding to Nterminal and internal protein sequence; 5' RACE polymerase chain reaction; complete dideoxy sequencing of both strands. Method of Identification: Presence of coding regions matching known protein sequences. Expression: Polyadenylated transcript of approximately 2200 bases. Expressed throughout tomato fruit development, with dramatic increases in mRNA abundance during ripening. Structural Features of the Gene: Translation start site at base 20. Stop site at base 1928. Codon Usage: All coding codons used. A slight preference for T in the third position. (G + C) Content: 46% in the coding region. Features of the Protein: Open reading frame 636 residues; 47-amino acid predicted signal sequence; 45-amino acid predicted prosequence. Potential glycosylation sites at amino acids 144, 211, 276, and 608. Predicted molecular weight of mature protein, 60, 166. Antibodies: None available. Subcellular localization: Probable localization vacuolar. GenBank Accession No.: M81 081
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