Vol. 269, No. Issue March 4, pp. 6908-6917, 1994 in USA. the … · 2001. 6. 27. · Vol. 269, No. 9, Issue of March 4, pp. 6908-6917, 1994 Printed in USA. Deletion of the Carboxyl-Terminal

Vol. 269, No. 9, Issue of March 4, pp. 6908-6917, 1994 Printed in U S A .

Deletion of the Carboxyl-Terminal Region of 1-hinocyclopropane-l-carboxylic Acid Synthase, a Key Protein in the Biosynthesis of Ethylene, Results in Catalytically Hyperactive, Monomeric Enzyme*

(Received for publication, July 28, 1993, and in revised form, October 7, 1993)

Ning Li and Autar K. MattooS From the Plant ~ o l e c u ~ a r Biology Laboratory, United States Department of ~ i c u l ~ ~ r e - B e l t s v i l l e ~ i ~ ~ t u r a l Research Center mest), Beltsville, iwaryland 20705-2350

l-Aminocyclopropane-l-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon es- culentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC synthases. Deletion of the COOH terminus through Are2* results in complete inactivation of the enzyme. Deletion of 4 W 2 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non- conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

l-Aminocyclopropane-1-carboxylate (ACC)’ synthase is a key plant enzyme that regulates the biosynthesis of the hormone ethylene in higher plants (for review, see Mattoo and White, 1991). ACC synthase is a pyridoxal-5’-phosphate (PLPI- dependent enzyme that catalyzes the synthesis of ACC from S-adenosylmethionine (SAM); ACC thus produced is then con- verted to ethylene by ACC oxidase (Yang and Hoffman, 1984). The low abundance of ACC synthase, occurrence of several isofonns, and high degree of instability afker isolation have led to only limited characterization of this protein. The cloning and sequencing of genes encoding ACC synthase from a number of

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank-IEMBL Data Bank with accession numbeds) X62536.

$ To whom correspondence should be addressed: Plant Molecular Bi-

timore Ave, Beltsville, MD 20705-2350. Tel.: 301-504-5103; Fax: 301- ology Laboratory, Bldg. 006, Rm. 118, USDNARS/BARC-W, 10300 Bal-

The abbreviations used are: ACC, l-aminocyclopropane-l-carboxylic acid; PLP, pyridoxal-5’ phosphate; PMSF, phenylmethanesulfo~c acid; SAM, S-adenoaylme~o~ne; PAGE, polyacrylamide gel electrophoresis; IPTG, i sopropy l -~ -~~ ioga la~op~anos ide ; EPPS, N42-hydroxyethyl)piperazin~-~-3-propanesulfo~c acid; D’IT, dit~othrei~l.

504-5320.

model plant systems as well as crops of agronomic and horti- cultural importance (Sato and Theologis, 1989; Nakajima et al., 1990; Van der Straeten et al., 1990; Bailey et al., 1992; Bottela et al., 1992; Dong et al., 1992; Li et al., 199213; Liang et al., 1992; Park et al., 1992; Theologis, 1992; Van der Straeten et al., 1992; Liu et al., 1993) have provided a new dimension and impetus for molecular-genetic and finer biochemical studies of this enzyme. In this regard, the expression of functional zucchini (Sato and Theologis, 1989) and tomato (Rottmann et al., 1991; Li et al., 1992b) ACC synthases in heterologous systems provides an opportunity to explore overexpression and purification and anatysis of kinetic and structure-function aspects of each iso- form.

Nucleotide sequence analysis of ACC synthase cDNAs from a variety of plants reveals the deduced amino acid sequence to be 72.3-80.7% similar and 53.3-66.7% identical (Theologis, 1992). Seven distinct regions (Dong et al., 1991) as well as the putative active site (Yip et al., 1991) are highly conserved. The deduced, full-length amino acid sequences reveal diversity in the subunit molecular mass of the protein: 454 amino acids (49.9 kDa) in apple (Dong et al., 19911, 484 amino acids (53.2 kDa) in soybean (Liu et al., 19931,485 amino acids (53.4 kDaf in tomato (Van der Straeten et al., 19901, 491 amino acids (54.0 kDa) in tobacco (Bailey et al., 1993, 493 amino acids (54.2 kDa) in squash and zucchini (Nakajima et al., 1990; Sat0 et al., 19911, and 516 amino acids (56.8 kDa) in carnations (Park et aE., 1992). This diversity in size is also apparent from in vitro translation experiments in which the translation products fractionated on SDS-PAGE showed molecular masses of 48, 55, 56, and 58 kDa, respectively, for apple (Dong et al., 1991), zucchini (Sato et al., 1991), tomato (Van der Straeten et al., 19901, and squash (Nakajima et al., 1990). Furthermore, when one compares the size of purified versus in vitro translated ACC synthase, in several cases, the in vitro product is larger by 8-9 kDa (Nakajima et al., 1988; Edelman and Kende, 1990; Van der Straeten et ai., 19901, raising the possibility that the protein is processed. However, Sat0 et al. (1991) reported that the in vivo and the in vitro translated zucchini ACC synthase are of the same size (55 kDa). Discrepancies exist in the literature on whether the native enzyme exists as a monomer or a dimer. For instance, it has been suggested that the wound-induced tomato fruit ACC synthase might be a monomer (Bleecker et al., 19861, while the corresponding ZUC-

chini and squash ACC synthases might exist as homodimers (Nakajima et al., 1986; Sat0 et al., 1991).

The length and primary sequence at the COOH-terminal region of various ACC synthases sequenced thus far are hypervariable despite a high degree of similarity in the rest of the protein (Theologis, 1992; Park et al., 1992). However, this non- conserved domain is highly conserved in one respect, its net

6908

COOH Terminus Role in ACC Synthase Function TABLE I

Oligonucleotides used to introduce deletion and site-directed mutations into tomato ACC synthase

6909

The bases underlined were site-directed mutagenized to introduce new amino acids to replace the original ones coded for by oligomer C1* and c2+.

Oligomer name Oligonucleotide sequences

P3 5"TAT TTT GAT GGG TGG AAA GCA TAC GA-3' P4 5"CCA TTG TTG CTT CTT TTC CAT CGA-3' c1+ 5"CG AGG ATT CGG AGG TTC GTA GGT GTT GAG AAA AGT TAG-3' c1- 5"GATC CTA AGT TTT CTC AAC ACC TAC GAA CCT CCG AAT CCT CG-3' c2+ 5"CG AGG ATT CGG AGG TTC TAA TAG-3' c2- 5"GATC CTA TTA GAA CCT CCG AAT CCT CG-3' c3+ 5"CG AGG ATT CGG TAA TAG-3' c3- 5"GATC CTA TTA CCG AAT CCT CG-3' c4+ 5"CG AGG TAA TAG-3' c4- 5"GATC CTA TTA CCT CG-3' c5+ 5"CG TAG-3' c5- 5"GATC CTA CG-3' C6+ C6- c7+ c7- 5"GATC CTA ACG TTT CCG TTT CTT CCG GAA CCT CCG AAT CCT CG-3'

5"CT GCG ATT GCA GCG TTC TAA TAG-3' 5"GATC CTA TTA GA.4 CGC TGC AAT CGC CG-3' 5"CG AGG ATT CGG AGG TTC CGG AAG AAA CGG AAA CGT TAG-3'

positive charge. Previously, we expressed functional tomato fruit ACC synthase in Escherichia coli, retaining biochemical features such as substrate-dependent inactivation of the native tomato enzyme (Li et al., 199213). The expression of ACC synthase in E. coli with both pCRlOOO and pETll vectors en- abled us to analyze the role of the COOH terminus in the conformation, size, and biochemical parameters of highly purified full-length as well as truncated (COOH terminus deleted) proteins. We demonstrate here that the wild-type ACC synthase expressed in E. coli is a dimer while selective truncation of its COOH terminus results in a monomer which is enzymatically more active and efficient than the former. We conclude that non-conserved COOH terminus of ACC synthase not only affects its enzymatic function but also its dimerization.

EXPERIMENTAL PROCEDURES Construction of Deletion and Site-directed Mutants of ACC

Synthase-A 1.6-kilobase pair cDNA clone, pTACC-B1 (representing PRTOMACCSl), encoding a wound- and ripening-induced tomato ACC synthase was obtained via RNA-based polymerase chain reaction (Li et al., 1992a, 1992b). The chimeric plasmid was simultaneously digested with two restriction enzymes, BamHI and NruI, to produce 0.3- and 3.3-kilobase pair fragments. The 3.3-kilobase pair fragment containing most of the coding region of ACC synthase was purified from low melt- ing point agarose gel and ligated overnight at 12 "C to a group of double-stranded oligonucleotides prepared by annealing two comple- mentary single-stranded synthetic oligonucleotides as listed in Table I. These double-stranded oligonucleotides had one end blunted and the other end equivalent to half of the BamHI site. The pCRlOOO recombinants harboring deletion and site-directed mutants of ACC synthase were transformed into competent E. coli DH5clF' cells. The recombinant plasmid DNA from these transformants was isolated and sequenced to ensure that the deletion mutants created were as expected. Then, the DNA from these mutants as well as the wild-type (from which the mutants were generated) were digested with NcoI and BamHI, the resulting DNA fragments gel purified and finally ligated to the gel-purified expression vector pETlld that had been previously digested with the same restriction enzymes. The series of pETlldACC synthase recombinants thus produced were retransformed into the DH5aF' cells to obtain sufficient recombinant plasmid DNA for sequencing. The clones with the correct sequences were retransformed into E. coli BL21(DE3) pLysS cells possessing the T7 RNA polymerase expression system specially designed for overexpression of foreign proteins in E. coli (Novagen, Madison, WI). Because these cells lack both soluble as well as membrane proteases, the recombinant proteins produced have a better chance of remaining stable in the heterologous system. The pET/ACS plasmid construction strategy is shown in Fig. 1.

Ouerproduction of Wild-type and Mutant ACC Synthuses in E. coli-A higher level of expression of ACC synthase and its mutants was achieved by modifying the protocols described by Studier et al. (1990). A single colony containing either the wild-type ACC synthase or the

individual deletion mutants was grown on agar plates made with M9LB medium, pH 7.2, in the presence of 100 pg/ml ampicillin and 25 pg/ml of chloramphenical. These colonies were then transferred to 20 ml of M9LB medium containing 200 pg/ml ampicillin and incubated at 37 "C with constant shaking until the cell cultures reached Am nm of 0.6. Isopropyl-p-o-thiogalactopyranoside (IPTG), the inducer of T7 RNA polymerase in the BL21(DE3) pLysS system, and PLP, the coen- zyme of ACC synthase, were then added to each culture to a final concentration of 1 m and 5 w, respectively. Cell cultures were then allowed to grow for l h at 25 "C before rifampicin (200 pg/ml), a host RNA polymerase inhibitor, was added. Incubation was continued for additional 2 h at 25 "C. Cells were pelleted by centrifugation at 5,000 x g for 5 min, washed once in half of the original volume with a buffer containing 20 m N-(2-hydroxyethyl)piperazine-N"3-propanesulfonic acid (EPPS), pH 7.5, 20 m EDTA, 2 m DIT, 5 w PLP, and 100 m NaCl, and stored at -70 "C until used for protein extraction. ACC re- leased in the medium by each clone in the presence and absence of IPTG was measured (Fig. 2). For large scale E. coli protein preparations, a single colony was first inoculated into 100 ml of M9LB medium, incubated until Am ~~ was 0.6, then transferred into 5-10 liters of the same medium containing 200 pdml ampicillin, and induced under the same conditions. Cells were harvested by centrifugation at 1,500 x g for 20 min at 4 "C.

Preparation of Soluble Protein Extract-The frozen cell pellets were suspended in one-twentieth the original volume in the extraction buffer (Mehta et al., 1988) containing 100 m EPPS, pH 7.5,4 m Dm, 10 pm PLP, 10 m EDTA, 1 m PMSF, 10 pg/ml aprotinin, 10 pg/ml pepstatin, and 10 pg/d leupeptin. The cells were lysed by sonication. Aliquots of each cell extract were fractionated on SDS-PAGE, immunoblotted, and ACC synthase protein detected by a polyclonal antibody (Rottmann et al., 1991). The intensity ofACC synthase bands on the immunoblot was used to estimate the amount of ACC synthase expressed in E. coli (Fig. 3). The remaining cell extracts were gel-filtered on 20-ml Sephadex G-25 columns and then used for enzyme purification. The ACC synthase activity was determined as previously described (Mehta et al., 1988).

Purification of Wild-type and Mutant ACC Synthases-Cell extract (200 ml) obtained afbr sonication of 5 liters of cell cultures transformed with wild-type ACC synthase were mixed with polyethyleneimine solution (50%, Eastman Kodak) at a final concentration of 0.4%, incubated on ice for 30 min, and then centrifuged at 26,000 x g for 20 min. The supernatant was fractionated on a 1-liter Sephadex G-25 column. Frac- tions (12 ml) eluted with the binding buffer (10 m EPPS, pH 8.3,l II~M DIT, 1 m EDTA, 20 nm NaC1, 1 pg/ml leupeptin, 1 pg/ml aprotinin, and 0.1 m PMSF) at a flow rate of 5 d m i n and containing higher ACC synthase activity were pooled. A total of 400-ml of protein extract was collected and concentrated to 210 ml using centriprep-10 filter (Amicon, Beverly, MA). The concentrate was then clarified using a 0.22" filter and divided into 6 aliquots. Each aliquot (160 mg of protein) was loaded onto a MonoQ column. The crude extracts from cultures transformed with del-1 and del-2 mutants were first precipitated with 30-95% ammonium sulfate instead of polyethyleneimine and then desalted on a Sephadex G-150 column. The desalted and filtered wild-type and mutant ACC synthases were then separately bound to a MonoQ column (HR 10/10) at 10 nm NaCl, pH 8.3. Elution was effected with NaCl and

6910 COOH Terminus Role in ACC Synthase Function

0' 1) Nru I / Barn Hldigestlon (Lac2

Barn HI yN: iarn HI

pCRACS-61 4.5 Kb

\

<,, /.' Gel purtficatlon of 4.2 Kb &A'\ 4.2 Kb

pCRACS-Bl.5

L.

c 1 -.-.-...-.. c 2 .""". '

c5 .-.

c 7 .""_. C6 __Y_

1) Nco I l Barn HIdouble dlgestlon 2) Gel puriltcation of DNA fragments encoding

wlld type (ACS) and mutant (del) ACC synthases. 1 Nco I

Ncol ' barn^/

del-1 del-2 deb3 del-4 del-5 del-6 ". del-7 ACS

-.

Llgatlon

FIG. 1. The strategy employed for the preparation of various ACC synthase constructs for expression in E. coli. pCRACS-B1, previously called pTACC-Bl (Li et al., 1992b), is the wound-inducible tomato fruit ACC synthase clone isolated using RNA polymerase chain reaction. pCR is an abbreviation for a TA cloning vector, pCRlOOO (Invitrogen Corp, San Diego, CA). ACS represents ACC synthase. Lacl and Lac2 represent the repressor for Lac operon and P-galactosidase gene, respectively. pETl ld is the overexpression vector (Novagen, Madison, WI). Dashed and dotted horizontal bars labeled C 1 4 7 represent the double-stranded oligonucleotides. The horizontal bars on the left indicate the wild-type (ACS) and mutant (de l -141-7 ) ACC synthases.

pH gradients of 0.02 M to 0.3 (or 0.5) M NaCl and pH 8.3-7.3. Each fraction (3 ml) was collected a t a flow rate of 1.5 ml/min. Aliquots (5-20 pl) were assayed for ACC synthase activity (Fig. 4). The peak activity fractions were collected and refractionated on the MonoQ column under the same conditions. The fractions containing higher ACC synthase activity were pooled and loaded onto two 5-ml hydroxylapatite columns in series (Econo-Pac HTP Cartridges, Bio-Rad). Approximately 25 mg of protein was bound to the HTP column using 0.02 M KH,PO,, pH 6.9. Protein was eluted with 0.42 M phosphate buffer, pH 8.0, a t a flow rate of 0.8 mumin. Aliquots (5 or 20 pl) were assayed for ACC synthase activity (Fig. 5). The fractions containing peak ACC synthase activity were pooled, dialyzed against gel-filtration buffer, and finally concentrated to 0.6-0.8 ml. The concentrate was loaded (0.2 rnUrun) onto two consecutively linked Superose-12 columns to determine the native size of the active wild-type and mutant ACC synthases. The purification of the wild-type, del-1 and del-2 mutant ACC synthases is summarized in Tables 11, 111, and N, respectively.

Determination of Kinetic Parameters for ACC Synthase-The purified ACC synthase proteins were assayed for enzyme activity in 4-10-pl aliquots containing 8-220 ng of protein as described previously (Mehta et al., 1988). The reaction assay contained 50 mM EPPS, pH 8.2, 10 p~ PLP, 2 mM D'IT and different concentrations of SAM. Four to 12 replicates were performed a t each SAM concentration. The kinetic parameters, K,, V,,,, and K,, were determined from substrate saturation kinetics data using a combination of Sigmaplot curve-fitting software (Jandel Scientific, Corte Madera, CA) and Slidewriter curve-fitting software (Advanced Graphic Software, Inc., Qunnyvale, CA). The plots were drawn according to Haldane's substrate inhibition equation u =

1 + IPTG - IPTG

J wt del-1 del-2 del-3 del-4 del-5 del-6 del-7

FIG. 2. Quantification of extracellular ACC in E. coli harboring the wild-type (wt) or muta ted constructs (del-l&l-7). The open and dashed bars indicate IPTG-induced and uninduced cultures, respectively. pET represents the control without the insert.

Stain lmmunoblot PET WT del-1 del-2 PET WT del-1 del-2

IPTG - + - + " + - + - + - + - + - + "" ""

kDa

97.4 -

67 -

43 - - -""" - - """

29 -

FIG. 3. SDSPAGE and immunoblot anaiysis of the wild-type (WT), del-1 (de l -1 ), and del-2 (del-2) ACC synthases expressed in E. coli. Lane 1, standard molecular weight markers; lanes 2.3, and 10, 11, proteins from the control pETll vector/BL21(DE3) plysS; lanes 4 , 5 , and 12, 13, proteins from E. coli with WTACC synthase; lanes 6, 7 , and

del-2 mutant. Even numbered lunes represent the uninduced cultures, 14, 15. proteins from del-1 mutant; lanes 8,9, and 16, 17, proteins from

whereas the odd numbered lanes from lane 3 onward represent the IPTG-induced cultures. Cell-free extracts (20 pg of protein equivalentl lane) of E. coli cultures harboring the indicated ACC synthase constructs were fractionated by SDS-PAGE, the gels were then either stained with Coomassie Blue (Stain) or immunoblotted and reacted with the anti-ACC synthase antibody (Immunoblot).

V,,.J(l+ KJs + s/Ki) (Haldane, 1965) using the curve-fitting program of Slidewriter software. The protein concentration used in each assay was predetermined by the Rose Bengal method (Elliott, 1978) or by fractionating the proteins on 10% SDS-polyacrylamide gels together with bovine serum albumin (50500 ng) followed by staining with Coo- massie Blue (Marder et al., 1986).

Molecular Weight Determination by Size Exclusion Chroma- tography--Two Superose-12 (HR 10/30) columns linked by a plastic tube were used to determine the native size of the wild-type and mutant ACC synthases expressed in E. coli. The total bed volume (V,) ofthe columns was 48 ml, and the flow rate was 0.3 mumin with a backpressure of 2.2 MPa (22 bar, 319 psi). Each sample containing 0.015-2.4 mg of protein in 200 p1 was loaded per run using a buffer containing 50 mM K2HP04, pH 7.0, 150 mM NaCI, 2 m~ D m , 5 p~ PLP, 0.25 mM EDTA, 1 pg/ml leupeptin, 1 pg/ml aprotinin, and 0.1 m~ PMSF. Forty-five fractions of 400 pl each were collected for each run. The column was calibrated using blue dextran (2,000,000 kDa), aldolase (158 kDa), bovine serum albumin (67 m a ) , ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and cytochrome c (12.4 kDa). A molecular weight standard curve for the

COOH Terminus Role in ACC Synthase Function

1 .o

0.8

I o.6

i 0.4 a

0.2

0.0

1 5 10 25 30

1 5 10

7

.

15 20 25 30

.

0.0

4

1 5 10 15 20 25 30

1 .o

0.8 1 0.6 ~ 'E

2 - 3 2 t 2

0.4

0.2 * w

0.0

Fraction Number FIG. 4. MonoQ chromatography of wild-type (WT) and del-1

and del-2 ACC synthases expressed in E. coli. Soluble proteins (8CL160 mg) after gel-filtration were loaded onto a MonoQ column (HR 10/10) at a flow rate of 1.5 ml/min. Elution was effected with a salt gradient (indicated by dashed line), fractions (3 ml) were collected and analyzed for protein content (Azao ,,m) and ACC synthase activity (indicated by a cross within a circle). One unit ( U ) of enzyme activity is defined as the formation of 1 pmol of ACC in 1 h at 30 "C.

determination ofACC synthase native size was established using a 4th degree polynomial curve fitting equation, MW, = exp[exp (exp(2.005exp(-5.902*K.,)))1, with an estimated standard deviation of 3.4 kDa (Fig. M). In contrast, the estimated standard deviation of these molecular standards on the semilog curve was 26 kDa (Fig. 6B) . There- fore, the exponential equation established here between Kav and molecular weight gives a more precise estimation of the native ACC syn-

10 - 0 0

WT 0 0 1 8.0-

0 0

0 0

0 - r L E 6.0-

2. z P B 4.0-

c 2.0-

W

0.0 - 50 e 40

li 30 E 2.

F

- Q 20

: 10

F 4

W

r

0.0

6911

0.42

I I

0.34 ! - z 0.26 n, 0'

t Y

0.16

0.1 0

0.42

I

0.34 I I I

E. a,

0.26 6 I Y

0.16

0.1 0

- - L E 0.9 - 2. - i 0.6- 4

p 0.3 - E W ,

0

0.0 - 10 20 30 40 50

0.10

Fnctlon Number

(WT) and del-1 and del-2 ACC synthases. Peak ACC synthase frac- FIG. 5. Hydroxylapatite column chromatography of wild-type

tions from the MonoQ column containing 1.2-7 mg of protein were loaded onto a hydroxylapatite column at a flow rate of 0.8 ml/min, and elution was effected using a phosphate gradient (indicated by the dashed line). A total of 50 fractions (1 ml each) were collected and analyzed for ACC synthase activity (solid line).

thase molecular weight than the semilog method which tends to either over- or underestimate the value.

Gel Electrophoresis and Immunoblotting-Proteins were fractionated by SDS-PAGE on 10% polyacrylamide gels according to Laemmli (1970). Half of each gel was stained with silver (Wray, 1981) and the other half electrophoretically transferred to nitrocellulose paper. Immu- nodetection with antibody to ACC synthase was done using the alkaline phosphatase-conjugated goat anti-rabbit I g G (H+L) as secondary antibody and the protein quantified as described previously (Callahan et al., 1989).

RESULTS

Deletion Analysis of ACC Synthase-Optimum alignment of deduced amino acid sequences of various ACC synthases shows high conservation of roughly the 430 NH2-terminal amino acids but a distinct, hypervariable region of 18-85 amino acids in the COOH-terminal domain (Park et al., 1992; O'Neill et al., 1993). Because of this high variability of the COOH terminus and the observations showing processed forms of ACC synthase, we investigated whether this region is required for the protein to be fully functional and if it is prone to proteolysis. When we

6912 COOH Terminus Role in ACC Synthase Function

Purification of wild-type ACC synthase expressed in E. coli TABLE I1

The abbreviations used are: PPPE,,polyethyleneimine precipitated protein; G-25, Sephadex G-25; HTP, Hydroxylapatite column.

Purification S k P

Total volume Total protein Total activity Specific activity Purification Yield

rnl mg unitD unit lmg" -fold %

120 3316 725.580 845.400

100

180.640 116.5 24.9 23.3

0.48 93.600 195.000 890 12.9

PPPE

MonoQ 40 17.8 HTP Superose 12

G-25 200

33 12

0.219 1 0.890 4 10.148 46

10.7 168.828 15.778 72

950

a unit = 1 pmol of ACC formedh at 30 "C.

TABLE I11 Purification of del-1 mutant ACC synthase expressed in E. coli

Tht abbreviations used are: (NH4),S0,, 30-95% ammonium-sulfate precipitated protein from crude extract; G-150, Sephadex 6-150; HTP, hydroxylapatite column.

Purification SkP Total volume Total protein Total activity Specific activity Purification Yield

rnl rng unit" unitlmg" -fold %

(NH4)2SO, 270 1478.25 1 100 11796.30 7.98 7222.00 8.78

28.00 1436.05 G-150 575

35 10.5 6.60 1175.48 91.00 11.40

822.25 MonoQ HTP Superose 12 a 1 unit = 1 m o l ofACC formedh at 30 "C.

1.10 61.22 51.28 6.42 12.17

9.96 3.6 2.00 385.00 192.22 24.09 3.2

TABLE IV Purification of del-2 mutant ACC synthase expressed in E. coli

The abbreviations used are: (NH4)2S04, 3&95% ammonium sulfate-precipitated protein from cell extract; G-150, Sephadex G-150; HTP batch, hydroxylapatite solution; HTP, hydroxylapatite column.

~ ~

Purification step Total volume Total protein Total activity Specific activity Purification Yield

rnl rng unit" (NH4)2SO, 150 HTP Batch 300 MonoQ 18 2.394 HTP 8 0.064 10.4 Superose 12 3.6 0.006 1.4

994.500 191.25 462.000 120.00

18.00

unit I mg' -fold %

0.19 1 100 0.26 1.37 62.75 7.52 39.58

162.50 9.41

855.26 233.33 1228.05

5.43 0.73

a 1 unit = 1 m o l ofACC formed/h at 30 "C.

constructed a mutant clone (pTACC-B1A) in which 57 deduced amino acids from the COOH terminus were deleted and expressed it in E. coli, we were surprised to find that the protein produced is enzymatically inactive (Mattoo et al., 1993). Recon- struction of the full-length COOH terminus in this mutant restored the enzymatic activity to the protein (Mattoo et al., 1993). We therefore further explored the role of the COOH terminus in ACC synthase function. Deletions were made between Ala428 and Ser439 to delineate the region(s) at the COOH terminus essential for restoration of biological activity to the protein. These deletion mutant genes, labeled as del-1, del-2, del-3, del-4, del-5, del-6, and del-7 (Fig. 7A) as well as the wild-type gene were then expressed in E. coli and assayed for ACC synthase activity. Enzyme activity was normalized to the amount of ACC synthase protein detected on the immunoblots (Fig. 7B). The specific activity of ACC synthase encoded by del-1 was found to be 1.6-fold higher than the wild-type enzyme. The COOH-terminal residue of del-1 was Ser439 and the protein lacked 46 amino acids, which was reflected in the increased mobility of the protein identified on immunoblots (Fig. 7B, del-1) . Deletion of additional residues VGVEKS to obtain a protein with Phe433 as the COOH terminus further enhanced the specific activity of the expressed truncated protein (Fig. 7, del-2), which was respectively 2.5- and 4-fold higher than the del-1 mutant and wild-type ACC synthases. When VGVEKS in del-1 mutant was replaced entirely with positively charged Lys- Arg residues, encompassing and Ser439 (comprising del-7 mutant) (Fig. 7B, del-1 versus del-71, the specific enzyme activity increased considerably, being, respectively, 2- and 3.3-fold

higher than the del-1 and wild-type enzymes. These results suggested that the positive residues located within this domain of ACC synthase are beneficial for maintaining a higher activity of the expressed enzyme. Likewise, replacement of the COOH terminus segment RIRRF in del-2 mutant with AIAAF' (constituting the del-6 mutant) reduced the specific activity by approximately 45-fold (Fig. 7A, compare del-2 with del-6). To- gether, the results with del-1 versus del-7, and del-2 versus del-6, suggest that positively charged residues impart a higher specific activity to ACC synthase. Additional deletions toward the NH2 terminus caused a dramatic reduction in the enzymatic activity of the protein expressed in E. coli, maximum loss occurring when Ile430 and were deleted (del-3 to del-4). When was deleted, the protein expressed was non-functional (Fig. 7A, del-5).

Immunoblot analysis of the lysates from these different cultures was carried out to ascertain the expression level and size of the expressed ACC synthase protein. The unaltered, wild- type ACC synthase was expressed as a major, full-size product of M, 54,000 (Fig. 7B, wt) with two additional, but minor protein bands with M, 52,000 and 48,000. All the truncated forms of ACC synthase were relatively stable and did not show the presence of minor, smaller M, fragments as did the wild-type enzyme (Fig. 7B). Compared with the others, the deletions represented by del-1 and del-7 mutants produced products with lower electrophoretic mobility, consistent with the fact that they have 6-11 more amino acids at the COOH terminus. Ex- pression levels of the constructs varied only slightly. The size of the smallest, processed form of wild-type ACC synthase was 1

COOH Terminus Role in

tm 140

t 120 d

20

0 1 1 0.20 0.25 0.30 0.35 0.40 0.45

a 0 t E - 3

.” 0.20 0.25 0.30 0.35 0.40 0.45

Kav FIG. 6. Standard curves used for the molecular weight deter

mination of ACC synthases gel-filtered on two Superose 12 col- u m n s in series. A, the indicated equation was formulated to fit the exponential curve relating molecular weight to Kav. The asterisk in the equation represents the multiplication factor used in the Slidewriter software program (Advanced Graphic Software, Inc., Qunnyvale, CA) to draw the curves. erp stands for the base of natural logarithm (e). B , conventional semilog curve relating molecular weight to K,, as per the equation: ICav = (V. - VJ/(V, - Vel; V,, elution volume for a protein; V,, bed volume (48 ml); V,, void volume.

kDa larger than the 47,000-dalton del-1-encoded enzyme (Fig. 7B 1. These results suggested that the proteolytic processing at the COOH terminus is most likely located beyond, but close to, Phe433. Progressive deletions, demarcated by Phe433 as the COOH terminus, resulted in a gradual increase in specific enzyme activity, suggesting that these domains somehow interact with either the substrate or the substrate-binding domain or both to keep the enzyme in a less active state.

Kinetic Parameters of ACC Synthase and Its Mutants- Because the del-1 and del-2 mutant ACC synthases had higher specific activity than the wild-type enzyme, we surmised that the respective deletions might result in changed kinetic prop- erties of the enzyme. To eliminate the potential interference from other E. coli proteins in the determination of these biochemical parameters, the full-length ACC synthase and the mutant del-1 and del-2 enzymes were purified to homogeneity (see “Experimental Procedures”). The purified wild-type, del-1, and del-2 proteins were resolved on SDS-PAGE (Fig. 8) into silver-stainable bands of M, 54,000,47,000, and 46,000, respectively. These M, values are in close agreement to the respective molecular masses of 53,400 (wt), 48,300 ( d e l - I ) , and 47,600 (del-2) derived from the deduced amino acid sequences. All three bands reacted with the polyclonal antibody made against the wound-inducible ACC synthase (Rottmann et al., 1991). The purified preparations of the wild-type, del-1, and del-2 ACC synthases fractionated on SDS-PAGE and stained with silver were estimated to be 99% pure.

The substrate saturation kinetics of the three purified ACC synthase preparations are presented in Fig. 9. The wild-type enzyme exhibited high substrate inhibition at SAM concentrations higher than 0.13 mM. The best fit of the data points

ACC Synthase Function 6913

represented in double-reciprocal plots (Fig. 9) was obtained using the Haldane (1965) equation for substrate-inhibition kinetics. The wild-type ACC synthase had a K,,, of 22 p~ for SAM and a V,, of 96 ( p o l h” mg-l) (Table V). The del-1-truncated form of ACC synthase showed lesser substrate inhibition than the wild-type enzyme and the kinetic parameters were, sur- prisingly, vastly different, with a K , >12-fold higher and a V,, >4-fold higher than the wild-type enzyme (Fig. 9B and Table VI. However, additional deletion of 6 residues, VGVEKS, giving rise to the del-2 enzyme, resulted in restoration of sensitivity to high substrate concentrations as well as the K , value to nearly the levels seen with the full-length ACC synthase. But the V,,, of the del-2 enzyme was, respectively, nine and two times higher than the wild-type and del-1 enzymes (Fig. 9C and Table V). The Ki for SAM was similar for both wild-type and del-2 enzymes, whereas the del-1 enzyme had a KirsMl which was more than twice that of the other two. Thus, a domain in the COOH-terminal52 amino acids of this wound-inducible tomato ACC synthase keeps the enzyme in an “inhibited” state whereas at least two different domains separated by the inter- nal sequence VGVEKS might influence enzyme catalysis. The close correlation between higher substrate affinity and greater substrate inhibition, and vice versa, suggest that the two may be interlinked. Alternatively, the unusual catalytic character- istics of del-1 ACC synthase may be related to a dramatic conformational change (see below).

Is the COOH Terminus Involved in the Oligomerization of ACC Synthase?-The differential kinetic behavior of del-1 uersus del-2 mutant proteins raised the possibility that the COOH terminus might be involved in influencing the native structure ofACC synthase. Therefore, the purified wild-type and mutant ACC synthase proteins were subjected to gel filtration under non-denaturing, native conditions using two Superose 12 columns linked together to enhance the resolution of proteins in the range of M, 67,000 and 158,000. It was deemed essential to have a highly resolving gel-filtration apparatus. The 2-column system allowed the R, between aldolase (158 kDa) and bovine serum albumin (67 kDa) to be 0.094 (four fractions apart) instead of 0.037 (only 1.6 fractions apart) with one column. The native molecular weight of the wild-type enzyme was found to be 97,600 2 4,000 while on SDS-PAGE an apparent M, of 53,350 was obtained (Figs. 8 and lo), suggesting that the full- length ACC synthase expressed in E. coli is a homodimer (Table VI). The native M, of the del-2 enzyme was 52,000 2 1,800, close to 46,000 determined by SDS-PAGE (Figs. 8 and lo), suggesting it exists as a monomer. On the other hand, the del-1 enzyme was determined to have a native M, of 60,000 5 2,300 while on SDS-PAGE it was 47,000 (Figs. 8 and 10). The gel-filtration and SDS-PAGE data show the wild-type and del-2 mutant enzyme to be, respectively, dimer and monomer. However, the behavior of the del-1 enzyme was relatively complex because the subunit M, of 47,000 is indicative of a monomer and yet the native M, was much higher, 60,000. If the del-1 is a monomer like the del-2 enzyme, then much higher native M, of del-1 could be due to a conformational change resulting from the presence of VGVEKS at the COOH terminus. Thus, it would seem that the COOH terminus of ACC synthase influences not only the kinetics of the enzyme but also its conformation as well as the ability to dimerize.

DISCUSSION

We have demonstrated that the COOH terminus of a tomato ACC synthase induced by wounding and during fruit ripening plays an important bifunctional role by affecting both enzyme catalysis as well as the structure of the native protein. Thus, while the deletion of 52 amino acid residues results in a dramatic increase in the specific activity of the trun-

del-1 -ALARIRRNGVEKS

del-2 -ALARIRRF

307.6

785.8

del4 -ALARIR 97.7

del4 -ALAR 3.8

0.0

17.6

627.7

wt del-1 del-2 del-3 del4 del-5 del-6 del-7 FIG. 7. COOH-terminal deletions of ACC synthase and their expression in E. coli. A, deletion mutants (del-141-5) and site-directed

mutants (del-6 and del-7) of ACC synthase were constructed via recombination of a series of double-stranded oligonucleotides (see 'Experimental Procedures") with pETlld expression vector. The first NH,-terminal Met of ACC synthase deduced from its open reading frame was used as the translation initiation site. No fusion protein was found among these mutants. The del-1, -2, -3, -4, and -5 mutants represent respective deletions

arrow on the upper side of wild-type ( w t ) ACC synthase indicates the last Phe4s3 which is highly conserved across all ACC synthases sequenced of 46, 52,54,56, and 57 amino acids. The boxed amino acids in del-6 and del-7 indicate the site-directed mutagenized amino acid sequence. The

thus far. The specific activity of the wild-type and COOH-terminal deleted ACC synthases expressed in E. coli is listed on the right. The enzyme activity was measured with 400 p~ SAM. B, immunoblot analysis of wild-type and deletion mutants ofACC synthase. Proteins of transformed E. coli were fractionated on 10% SDSPAGE and then electrotransferred to nitrocellulose. The blot was incubated overnight with the polyclonal antibody (1:3000 dilution) against LE-ACC2 at 4 "C on a shaker and then treated with alkaline phosphatase conjugated to goat anti-rabbit antibody.

cated protein, further deletions cause inactivation of the proteins expressed in E. coli. Deletion encompassing the highly conserved Are29 completely abolished the enzyme activity; thus conservation of Arez9 is important for the suste- nance of some enzyme activity. When one compares the catalytic efficiency, denoted by the ratio V,,,JK,,,, of the wild-type and truncated forms of ACC synthase, del-2 enzyme (created by the deletion of the 52 amino acid residues from COOH terminus) was the most efficient. The preponderance of positively charged amino acids, arginindlysine, at the COOH terminus seems to impart an important character to this domain. One example of this role was illustrated when the sequence V434GvEKS439 in del-1 mutant was replaced with R434KKRKR439. The resulting del-7 mutant was twice as active as the parent del-1 enzyme. Similarly, when the arginine residues at the COOH terminus of the del-2 mutant were replaced with alanine residues, the resultant del-6 mutant enzyme had little enzyme activity. The fact that the substrate affinity of the full-length, wild-type ACC synthase is comparable to the del-2 enzyme suggests that at least two domains within the COOH terminus interact to maintain a high affinity for SAM. The purified wild-type ACC synthase expressed in E. coli was resolved into the full-length protein and two distinct processed polypeptides of 52 and 48 kDa, despite the fact that during the purification procedure protease inhibitors such as aprotinin, leupeptin, and PMSF were present throughout to prevent proteolysis. It is possible that the nature of the COOH terminus of ACC synthase makes it prone to cleavage. The 48- kDa processed ACC synthase is 1 kDa longer than the del-1 enzyme, which corresponds to it being 8 amino acids larger than the del-1 protein. The cleavage of the wild-type enzyme to produce the 48-kDa fragment is therefore thought to occur within a few residues of the G l ~ ~ ~ - L y s ~ ~ ~ - L y s ~ ~ sequence. Using a similar estimation, the 52-kDa fragment may arise by

cleavage of the parent protein within or around the sequence G l ~ ~ ~ - S e f i ~ ~ - V a l ~ ~ ~ . ACC synthase from tomato (Edelman and Kende, 1990; Van der Straeten et al., 19901, zucchini (Sato et al., 1991), and winter squash (Nakajima et al., 1990) fruit pericarp tissues is often found associated with processed fragments that are 8-9 kDa smaller than the full-length protein. This processing seems to occur invariably when the tissue is wounded or when cells are homogenized. This phenom- enon may be attributed to the release of proteases upon wounding and during tissue senescence. These data raise the possibility that the native tomato fruit ACC synthase might be cleaved a t the COOH terminus within the sequence encom- passed by amino acid residues 446-470, generating -50- and -48-kDa processed fragments.

The deletions of the COOH terminus resulting in del-1 and del-2 truncated but active ACC synthases also affected the behavior of these proteins on gel filtration uersus SDS-PAGE. While the wild-type, full size enzyme expressed in E. coli is a dimer, the del-1 and del-2 proteins are monomers. These results suggest that the COOH terminus in ACC synthase is involved in the oligomerization of the protein. The sensitivity of the COOH terminus to cleavage, and the resultant effect on oligomer formation without loss of enzyme function, under- scores the necessity to determine the purity of the protein prior to the unambiguous determination of its molecular mass. "his is particularly important because, in the literature, there is evidence for both monomeric and dimeric forms of ACC synthase (see Satoh et al., 1993). Since in previous studies little attention was paid to the extent of protein processing, it is diflicult to conclude whether the protein was a dimer, monomer, or both. Our results exemplify that monomeric, truncated forms are more active than the dimeric, full- length enzyme.

Both ACC synthase and aspartate (and tyrosine) amino-

COOH Terminus Role in ACC Synthase Function 6915

wt del-I del-2 stain blot stain blot stain blot

200 -

97.4 -

66.2 -

45 -

31 -

1 2 3 4 5 6 FIG. 8. SDS-PAGE and immunoblot analysis of the purified

wild-type and truncated ACC synthases. Wild-type (wt), del-1, and del-2 ACC synthases were purified (see "Experimental Procedures") and fractionated on 10% SDS-PAGE and silver-stained. A duplicate gel was electroblotted onto nitrocellulose paper and probed with anti-ACC synthase antibody. Lams 1, 3, and 5 represent stained portions of the purified wild-type (290 ng), del-1 (440 ng), and del-2 (110 ng) ACC synthases, respectively, while lanes 2, 4, and 6 are the corresponding immunoblots. The positions of standard protein size markers are indicated.

transferases share sequence similarity at the binding site for PLP (Nakajima et al., 1990; Van der Straeten et al., 1990; Huang et al., 1991). Based on this, an attempt has been made to relate the two proteins in structural and evolutionary aspects. In this regard, we note that alanime amino acid transferase is active only as a dimeric form because dissociation of the dimer into monomer inactivates the enzyme activity (Her- old, 1990). However, ACC synthase can exist (for instance as the del-2 enzyme) in monomeric form which is more active than the full-length dimeric ACC synthase. Our data suggest that the conformation ofACC synthase as related to function may be quite different from that of alanime amino acid transferase. The primary sequence homology found between alanime amino acid transferase and ACC synthase may have, therefore, arisen as a product of convergent evolution.

It is possible that limited proteolysis of ACC synthase oc- curs under natural situations. Signals generated by wounding of plant tissues (Pearce et al., 1991; Pena-Cortes et al., 1989) or by elicitors (Chalutz et al., 1984) result in a burst of ethylene biosynthesis. The target enzyme in ethylene biosynthesis for such a trigger has generally been the ACC synthase. The mechanism of this activation of ACC synthase is a matter of conjecture. However, wounding is known to cause activation of proteases (Mehta, 1993). Also, PMSF and soybean trypsin inhibitor, which are inhibitors of protease action, have been shown to inhibit the development of ACC synthase during wound induction (Mattoo and Anderson, 1984) and following elicitor treatment (Anderson et al., 1982). These results sug-

0.00 0.25 0.50 0 75 1.00

0.00 [SAM]. mM

0 40 80 120 160 200 240

l /[SAM], mM-'

0 10 20 30 40 50 60

l/[SAM], mM-'

o'olz//

del-2 0.01 0

0.008

0.00 0.25 0.50 0.75 1.00

0.000 [SAM]. mM

0 40 80 120 160 200 240

I/[SAM], mM-l FIG. 9. Determination of kinetic constants of wild-type, del-1,

and del-2 ACC synthases. ACC synthase activity was measured a t

taining 50 m~ EPPS, pH 8.2,lO p~ PLP and 2 m~ Dm. The amount of different SAM concentrations as indicated in a reaction mixture con-

protein used for each reaction was 21, 220, and 8 ng, respectively, for wild-type (wt, A), del-1 ( B ) , and del-2 (C). Each point represents an average value of four replicates. Both substrate saturation curves (in- sets) and the Lineweaver-Burk plots are shown. The curves were drawn using Slidewriter software based on Haldane's high substrate inhibition equation (see "Experimental Procedures").

gested that specific proteolytic activity in vivo may be associated with the ethylene induction processes (Mattoo and Anderson, 1984). In view of our findings, it is now tempting to

6916 COOH Terminus Role in ACC Synthase Function TABLE V

Biochemical parameters of purified wild-type (wt), del-I, and del-2 ACC synthases expressed in E. coli

Enzyme K,,, V,, Ki V d L

mM pmol h-I m g - I mM pmol h" mg" mK' wt 0.022 96 0.690 4,364 del-1 0.280 420 1.533 1,500 del-2 0.042 880 0.620 20,952

Fraction number 13 14 15 16 17 18 19 20 21 22 23 24 25 26

wt

del-1

del4

20,""

15 7 I h

wt

del-1

0 -

I C e a b c d I\

0.00-1. , . , . , . , . , . . . , . . . I . , . , . , . , . , - , . , . , . , . , . , . , . r 2 4 6 8 101214161820222426283032343638404244

Fraction number FIG. 10. Determination of the size of native wild-type, del-1.

and del-2 ACC synthases by gel filtration. The peak ACC synthase activity fractions from the hydroxylapatite column were pooled and passed through Superosel2 column. Wild-type (in three batches at 0.2, 0.24, and 2.4 mg of protein), and del-1 and del-2, both in four batches at protein concentrations from 0.1 to 0.45 mg, ACC synthases were analyzed. Each run was followed with one or two calibrations with standard protein markers. The shift in the absorbance peak at 280 nm did not exceed 0.1 fraction volume (40 pl) among the separations. Aliquots (100 pl) from each fraction were mixed with 50 pl of loading buffer, and 45 pl from this solution were fractionated on SDS-PAGE and analyzed by immunoblots. A, immunoblot analysis of wild-type (wt), del-1, and del-2 ACC synthases fractionated on Superose 12. Fractions from B which were immunoblotted are listed on the top in A. B, elution profiles of wild-type (closed circles), del-1 (closed squares), and del-2 (closed tri- angles) ACC synthase activity from Superose 12 column. Each data point represents an average of three separate assays from each indicated fraction indicated at the bottom of C. C, a representative elution profile for standard protein markers on the Superose 12 column. The molecular mass markers used were: aldolase, 158 kDa (a); bovine serum albumin, 67 kDa (b); ovalbumin, 43 kDa (c) ; carbonic anhydrase, 29 kDa (d); cytochrome c, 12.4 kDa (e).

TABLE VI The native and subunit molecular weights of wild-type, del-1, and del-2 ACC synthases determined respectively by Superose 12 gel

filtration and SDS-PAGE

Enzyme Mass on Superose 12 Mass on SDS-PAGE Enzyme conformation

kDa wt 96 f 5.7 54 del-1 60 e 2.3 47 del-2 52 e 1.8 46

Dimer Monomer Monomer

speculate that one of the mechanisms by which certain endog- enous or exogenous stimuli cause activation of ACC synthase may involve specific processing at the COOH terminus, form- ing monomeric ACC synthase having 4-fold or higher specific enzyme activity than the unprocessed full-length enzyme. This enhancement would also be reflected in an enzyme form with better catalytic efficiency (V,,,JK,,,), as demonstrated here for the del-2 ACC synthase.

Acknowledgments-We thank Dr. Anasthasios Theologis for the kind gift of the polyclonal antibody against ACC synthase. The valuable assistance of Dr. Teng Li in formulating the equation used in gel filtration analysis is gratefully acknowledged. We also thank Drs. Marvin Edelman, Jeffrey Suttle, Mark Tucker, and Mark Swegle for construc- tive critiques on the original version of the manuscript, Michael Reinsel for preparation and purification of oligonucleotides and for technical assistance, and Dingbo Zhou for assistance with the preparation of Fig. 9.

REFERENCES

Anderson. J. D., Mattoo, A. K., and Lieberman, M. (1982) Biochem. Eiophys. Res.

Bailey, B. A., Ami, A,, Li, N., Mattoo, A. IC, and Anderson, J. D. (1992) Plant Commun. 107.588594

Bleecker, A. B. (1987) Cur,: Ibp. Plant Biochem. Physiol. 6.15-24 Bottela. J. R.. Arteca, J. M., Schlagenhaufer, C. D., Arteca, R. N., and Phillips. A.

Callahan, F. E., Wergin, W. P., Nelson, N., Edelman, M., and Mattoo, A. K. (1989)

Chalutz, E., Mattoo, A. K, Solornos, T., and Anderson, J. D. (1984) Plant Physiol.

Dong, J. G., Kim, W. T., Yip, W. K, Thompson, G. A., Li, L., Bennett, A. B., and

Edelman, L., and Kende, H. (1990) Planta 182,635-638 Elliot, J. I., and Brewer, J. M. (1978) Arch. Biochem. Biophys. 190,351-357 Haldane, J. B. S. (1965) Enzymes, Massachusetts Institute of Technology Press,

Herold, M., and Kasper, K (1990) Biochemistry 29,1907-1913 Huanp, P. L.. Parks, J. E., Fbttmann, W. H., and Theologis, A. (1991) Proc. Natl.

Physiol. 100, 1615-1616

T. (1992) Plant Mol. Biol. 18, 793-797

Plant Physiol. 91, 629-635

74,99-103

Yang, S. F. (1991) Planta 185.38-45

Cambridge

Laemmli, U. K. (1970) Nature 227,680-685 Li, N., Parsons, B. L., Liu, D., and Mattoo, A. K (1992a) Plant Mol. Biol. 18,

Ac&. Sci. U. S. A. 88,7021-7025

Li, N., Wiesman, Z., Liu, D and Mattoo, A. K (199213) FEES Lett. 306,103-107 Liang, X. W., Steffen, A., Keller, J. A,, Shen, N. F., and Theologis, A. (1992) Proc.

Liu. D.. Li. N.. Dube. S.. Kalinski. A,. Herman. E., and Mattoo, A. K (1993) Plant

477487

Natl. Acad. Sci. U. S. A. 89, 11046-11050

Cell'PhysioZ. 34, hi-1157 . .

Marder. J. B., Mattoo, A. K., and Edelman, M. (1986) Methods Enzymol. 118,

Mattoo, A. K, and Anderson, J. D. (1984) in Ethylene: Biochemical, Physwl@cal 384-396

and Applied Aspects (Fuchs, Y., and Chalutz, E., eds) pp. 139-147, Martinus Nijhoff Publishers, The Netherlands

Mattoo, A. K, and White, B. W. (1991) in The Plant Hormone Ethylene (Mattoo, A. K, and Suttle, J. C., eds) pp. 21-42, CRC Press, B o a Raton, FL

Mattoo, A. K., Li, N., and Liu. D. R. (1993) in Cellular and MokcularAspects ofthe Plant Hormone Ethylene (Pech, J. C., Latche. A., and Balague, C.. eds) pp.

Mehta, R. (1993) Molecular Aspects ofRipening and Stress-related Protein Metabo- 223-231, Kluwer Academic Publishers, Dordecht

lism in Higher Plants, Ph. D. dissertation, University of Maryland at Baltimore County

Mehta, A. M., Jordan, R. L., Anderson, J. D., and Mattoo, A. K (1988) Proc. Natl. Acad. Sei. U. S. A. 86,8810-8814

Nakajima, N., and Imaseki, H. (1986) Plant Cell Physwl. 27,969-980 Nakajima, N., Nakagawa, N., and Imaseki, H. (1988) Plant Cell Physiol. 29,989-

Nakajima, N., Mori, H., Yamazaki, K, and Imaseki. H. (1990) Plant Cell Physwl.

O'Neill, S. D., Nadeau, J. A,, Zhang, X. S., Bui,A. Q., and Halevy,A. H. (1993) Plant

Park, K. Y., Drory, A., and Woodson, W. R. (1992) Plant Mol. Bwl. 18,377486 Pearce, G., Strydom, D., Johnson, S., and Ryan, C. A. (1991) Science 253,895-898 Pena-Cortes, H., Sanchez-Serrano, J. J., Mertens, R., Willmitzer, L., and Prat. S.

998

31,1021-1029

Cell 5,419432

(1989) Proc. Natl. Acad. Sei. U. S. A. 86,9851-9855

COOH Terminus Role in ACC Synthase Function 69 17 Rottmann, W. E., Peter, G. F., Oeller, P. W., Keller, J. A,, Shen, N. F., Nagy, B. P.,

Taylor, L. P., Campell, A. D., and Theologis, A. (1991) J. Mol. Biol. 222,937-961 Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U. S. A 68,66214625 Sato, T., Oeller, P. W., and Theologis, A. (1991) J. Biol. Chem. 266, 3752-3759 Satoh, S., Mori, H., and Imaseki, H. (1993) Plant Cell Physiol. 34, 753-760 Studier, F. W., Rosenberg,A. H., Dunn, J. J., and Dubendor& J. W. (1990) Methods

Theologis, A. (1992) Cell 70, 181-184 Van der Straeten, D. Van Wiemeersh, L., Goodman, H. M., and Van Montagu, M.

Enzymol. 166,60-89

Van der Straeten, D., Rodrigues-Pousada, R. A,, Wllarroel, R. Hanley, S., Goodman, (1990) Proc. Natl. Acad. Sci. U. S. A 87,48594663

9973 H. M., and Van Montagu, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9969-

Wray, W., Boulikes, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118. 197-203

Yang, S . F., and HofFman, N. E. (1984)Ann. Reu. Plant Physiol. 36, 155-189 Yip, W. K, Dong, J. G., Kenny, J. W., Thompson, G. A., and Yang, S . F. (1990) Proc.

Natl. Acad. Sci. U. S. A. 87, 7930-7934

Documents

Vol. 269, No. Issue March 4, pp. 6908-6917, 1994 in USA. the … · 2001. 6. 27. · Vol. 269, No. 9, Issue of March 4, pp. 6908-6917, 1994 Printed in USA. Deletion of the Carboxyl-Terminal