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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 ~ Biology ar Laboratory, United States Department Center mest), Beltsville, iwaryland 20705-2350

of

~ i c u l ~ ~ r e - B e l t s v~ i l li e ~ ~ t u Research r a l

l-Aminocyclopropane-l-carboxylic acid (ACC) syn- model plant systems as well as crops of agronomic and hortithase is a key enzyme regulating biosynthesis of the cultural importance (Sato and Theologis, 1989; Nakajima et al., an enzymati- 1990; Van der Straeten et al., 1990; Baileyet al., 1992; Bottela plant hormone ethylene. The expression of cally active, wound-inducible tomato (Lycopersicon es- et al.,1992;Dong et al., 1992; Li et al., 199213;Liang et al., 1992; .cv Pik-Red) ACC synthase (485 amino acids Park et al.,1992; Theologis, 1992; culentum L Van der Straeten et al., 1992; long) in a heterologous Escherichia coli system allowed Liu et al., 1993) have provided a new dimension and impetus us to study the importance o f hypervariable COOH ter- for molecular-geneticand finer biochemical studies of this enminus in enzymatic activity and protein conformation. zyme. In this regard, the expression of functional zucchini (Sato W e constructed several deletion mutants of the gene, and Theologis, 1989) and tomato (Rottmann et al., 1991; Li et expressed thesein E. coli, purified the protein productsal., 1992b)ACC synthases in heterologous systems provides an to apparent homogeneity, and analyzed both conformaopportunity to exploreoverexpression and purification and tion and enzyme kinetic parameters of the wild-type andanatysis of kinetic and structure-function aspects of each isotruncated ACC synthases. Deletion o f the COOH termi- form. nus through results in complete inactivation of Nucleotide sequence analysis of ACC synthase cDNAs from of 4 W 2 aminoacidsfromthe theenzyme.Deletion a variety of plants reveals the deduced amino acid sequenceto COOH terminus results in an enzyme that has nine times higher affinityfor the substrate S-adenosylmethionine be72.3-80.7% similar and 53.3-66.7% identical (Theologis, 1992). Sevendistinct regions (Donget al., 1991) as well as the than the wild-type enzyme. The highly efficient, trunputative active site (Yip et al., 1991) are highly conserved.The cated ACC synthase was found to be a monomer of 52 deduced, full-length amino acid sequences reveal diversity in 1 . 8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar condi- the subunit molecular mass of the protein: 454 amino acids tions, is a dimer. These results demonstrate that nonthe(49.9 kDa) in apple (Dong et al., 19911, 484 amino acids (53.2 n soybean (Liu et al., 19931,485 amino acids (53.4 kDaf conserved COOH terminus of ACC synthase affects its kDa) i in tomato (Van der Straeten et al., 19901, 491 amino acids enzymatic functionas well as dimerization.

Are2*

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 isa pyridoxal-5-phosphate(PLPIdependent enzyme that catalyzes the synthesis ofACC from ACC thus produced is then conS-adenosylmethionine (SAM); verted to ethylene by ACC oxidase (Yang and Hoffman, 1984). The low abundance ofACC 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 be hereby marked payment of page charges. This article must therefore aduertisementin accordancewith 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 shouldbe addressed: Plant Molecular Biology Laboratory, Bldg. 006,Rm. 118,USDNARS/BARC-W, 10300 BalFax: 301timore Ave, Beltsville, MD 20705-2350.Tel.: 301-504-5103;
504-5320.

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 s o p r o p y l - ~ - ~ ~ i o g a l a ~ o p ~ a n o EPPS, s i d e N42-hydroxy;

ethyl)piperazin~-~-3-propanesulfo~c acid; DIT, dit~othrei~l.

(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 a i . , 19901, raising the possibility that theprotein is processed. However,Sat0 et al. (1991) n vitro translated zucchini reported that the in vivo and the i ACC synthase are of the same size (55 kDa). Discrepancies exon whether the native enzyme exists as a ist in the literature 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 ZUCchini 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 this nonprotein (Theologis, 1992; Park et al., 1992). However, conserved domain is highly conserved in one respect, its net

6908

COOH Terminus Role in ACC Synthase Function

6909

TABLE I Oligonucleotides usedto introduce deletion and site-directed mutations into tomato ACC synthase The bases underlined were site-directed mutagenized to introduce new amino acids to replace the original ones coded for by oligomer C1* and c2+.
Oligonucleotide
Oligomer name

P3 P4

c1+ c1c2+ c2c3c4+ c4c5+ c5C6+ C6c7+ c7-

c3+
CG-3'

5"TAT T T T GAT GGG TGG AAA GCA TAC GA-3' TTC CTT CTT 5"CCA TTG TTG CAT CGA-3' 5"CG AGG ATT CGG AGG TTC GTA GGT GTT GAG AAA AGT TAG-3' 5"GATC CTA AGT T T T CTC ACC AAC TAC GAA CCT CCG AATCG-3' CCT AGG 5"CG ATT CGG AGG TTC TAA TAG-3' 5"GATC CTA TTA GAA CCT CCG AAT CG-3' CCT 5"CG AGG ATT CGG TAA TAG-3' 5"GATC TTA CTA CCG CG-3' AAT CCT 5"CG AGG TAA TAG-3' CCT TTA 5"GATC CTA 5"CG TAG-3' CG-3' 5"GATC CTA 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' 5"GATC CTA ACG T T T CCGCTT TTT CCG GAA CCT CCG AATCG-3' CCT

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 enabled 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.
EXPERIMENTALPROCEDURES 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 polymerasechain 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-kilobasepair fragments. The 3.3-kilobase pair fragment containing most of the coding regionof ACC synthase was purified from low melting point agarose gel and ligated overnight at 12 "C to a group of double-stranded oligonucleotides prepared by annealing two complementary 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 ofACC synthase were transformed into competent E. coli DH5clF' cells. The recombinant plasmid DNA from these transformants was isolated and sequenced to ensure that thedeletion 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 producedwere 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 strategyis shown in Fig. 1. Ouerproduction of Wild-typeand Mutant ACC Synthuses in E. coli-A higher level of expression of ACC synthaseand 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 grownon agar plates made with M9LB medium, pH 7.2, in the presence of 100 pg/mlampicillin 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 coenzyme 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 released 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 ofM9LB 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 theextraction buffer (Mehta et al., 1988)containing 100 m EPPS, pH 7.5,4 m D m , 10 p m PLP, 10 m EDTA, 1m PMSF, 10 pg/ml aprotinin, 10 pg/ml pepstatin, and 10 p g / d leupeptin. The cells were lysedby 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 thenused 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 a f b r sonication of 5 liters of cell cultures transformed with wild-type ACC synthase were mixedwith 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.Fractions (12 ml) eluted with the binding buffer (10m EPPS, pH 8.3,l II~M DIT, 1 m EDTA, 20 nm NaC1, 1 pg/ml leupeptin, 1pg/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 effectedwith NaCl and
~~

6910

COOH Terminus Role in ACC Synthase Function


Barn HI

0' /.'
pCRACS-61 4.5 Kb
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yN:
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4.2 Kb

+ IPTG

- IPTG

1) Nru I / Barn Hldigestlon

Gel purtficatlono f 4.2 Kb &A'\

L .

c1 c2

' . . . . . . .
. " " " .

c5 .-. C6 c7
1) Nco I l Barn HIdouble dlgestlon

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2) Gelpuriltcation of DNA fragments encoding wlld type (ACS) and mutant (del) ACC synthases. Ncol del-1 del-2 deb3 del-4 del-5 del-6 del-7 ACS

'
1

Nco I

barn^/

-.

del-1 del-2 del-3 del-4 del-5 del-6 del-7 FIG.2. Quantification of extracellularACC in E. coli harboring the wild-type (wt) or m u t a t e d constructs (del-l&l-7). The open and dashed bars indicate IPTG-induced and uninduced cultures, respectively. pET represents thecontrol without the insert.

wt

" .

lmmunoblot
PET
Llgatlon

Stain
WT

del-1 del-2

IPTG
kDa 97.4 67 -

-+-+"+-+

" "" "

-+-+-+-+

PET

WT

del-1 del-2

FIG.1. The s t r a t e g y e m p l o y e d for the preparation of various

43 -

ACC synthase constructs for expression in E. coli. pCRACS-B1,

" " " previously called pTACC-Bl (Li et al., 1992b), is the wound-inducible tomato fruitACC 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 29 and Lac2 represent the repressor for Lac operon and P-galactosidase gene, respectively. p E T l l d isthe overexpression vector (Novagen, Madison, WI). Dashed and dotted horizontal bars labeled C 1 4 7 repreFIG.3. SDSPAGE and immunoblot anaiysis o f the wild-type sent the double-stranded oligonucleotides. The horizontal bars on the (WT), del-1 ( d e l - 1), and del-2 (del-2)ACC synthases expressed in left indicate the wild-type (ACS) and mutant ( d e l - 1 4 1 - 7 )ACC syn- E. coli. Lane 1, standard molecular weight markers; lanes 2.3, and 10, thases. 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 14, 15. proteins from del-1 mutant;lanes 8,9, and 16, 17, proteins from pH gradients of 0.02 M to 0.3 (or 0.5) M NaCl and pH 8.3-7.3. Each del-2 mutant. Even numbered lunes represent the uninduced cultures, fraction (3 ml) wascollected a t a flow rate of 1.5 ml/min. Aliquots (5-20 whereas the odd numbered lanes from lane 3 onward represent the pl) were assayed for ACC synthase activity (Fig. 4). The peak activity IPTG-induced cultures. Cell-free extracts (20 pg of protein equivalentl fractions were collected and refractionatedon the MonoQ column under lane) of E. coli cultures harboring the indicated ACC synthase conthe same conditions. T h e fractions containing higher ACC synthase structs were fractionated by SDS-PAGE, the gels were then either activity were pooled and loaded onto two 5-ml hydroxylapatite columns stained with Coomassie Blue (Stain) or immunoblotted and reacted in series (Econo-Pac HTP Cartridges, Bio-Rad). Approximately 25 mg of with the anti-ACC synthase antibody (Immunoblot). 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 V,,.J(l+ KJs + s/Ki)(Haldane, 1965) using the curve-fitting program activity (Fig. 5). The fractions containing peak ACC synthase activity of Slidewriter software. The protein concentration used in each assay were pooled, dialyzed against gel-filtration buffer, and finally concen- was predetermined by the Rose Bengal method (Elliott, 1978) or by trated to 0.6-0.8 ml. The concentrate was loaded (0.2 rnUrun) onto two fractionating the proteins on 10% SDS-polyacrylamide gels together consecutively linked Superose-12 columnsto determine the native size with bovine serum albumin (50500 ng) followed by staining withCooof the active wild-type and mutant ACC synthases. The purification of massie Blue (Marderet al., 1986). the wild-type, del-1 anddel-2 mutant ACC synthases is summarized in Molecular Weight Determination by Size Exclusion Chromarespectively. Tables 11, 111, and N, 10/30) columns linked by a plastic tube tography--Two Superose-12 (HR Determination of Kinetic Parameters for ACC Synthase-The puri- were used to determine the native size of the wild-type and mutant ACC fied ACC synthase proteins were assayed for enzyme activity in 4-10-pl synthases expressed in E. coli. The totalbed volume (V,) ofthe columns aliquots containing8-220 ng of protein a s described previously (Mehta was 48 ml, and the flow rate was0.3 mumin with a backpressure of 2.2 et al., 1988). The reaction assay contained 50 m M EPPS, pH 8.2, 10p~ MPa (22 bar, 319 psi). Each sample containing 0.015-2.4 mg of protein PLP, 2 m M D'IT and different concentrationsof SAM. Four to 12 repli- in 200 p1 was loaded per run using a buffer containing 50 m M K2HP04, cates were performed a t each S A M concentration. The kinetic param- pH 7.0, 150 m M NaCI, 2 m~ D m , 5 p~ PLP, 0.25 m M EDTA, 1 pg/ml and K,,were determined from substrate saturation eters, K,, V,,, leupeptin, 1pg/ml aprotinin, and 0.1m~ PMSF. Forty-five fractions of kinetics data using a combination of Sigmaplot curve-fitting software 400 pl each were collected for each run. The column was calibrated (Jandel Scientific, Corte Madera,CA) and Slidewriter curve-fitting soft- using blue dextran (2,000,000 kDa), aldolase (158 kDa), bovine serum ware (Advanced GraphicSoftware, Inc., Qunnyvale, CA). The plots albumin (67 m a ) , ovalbumin (43 kDa), carbonic anhydrase (30 kDa), were drawn according to Haldane's substrate inhibition equation u = and cytochrome c (12.4 kDa). A molecular weight standardfor curve the

-"""

COOH Terminus Role in ACC Synthase Function

6911
0

1 L

10 -

0.42
I I

WT
8.00 0 0 0

0
0

0.34

E 6.00.26 4.00.16

! z
, n
Y

2 .

0 '
t

z B
P
W

2.0-

0.0 -

0.10 0.42

25

10

30

1 .o
0.8

l i 30 E

e
F 4
W

50

0.34 40

I
I

E.
0.26

2.
20

6
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a
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F

10

0.16

o.6

0.0

0.1 0

0.4

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E 0.9

0.0

10

15

20

25

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4

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0.6-

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.
0.0

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p 0.3

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w

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0.10 20
30

0.0 10

40

50

Fnctlon Number

0.6

'E 2
~

0.4

4
1 5

FIG.5. Hydroxylapatite column chromatographyof wild-type (WT) and del-1 and del-2 ACC synthases. Peak ACC synthase fractions from the MonoQcolumn containing 1.2-7mgof protein were loaded ontoa hydroxylapatite column at a flow rate of 0.8 ml/min, and elution waseffected using a phosphate gradient (indicated by the dashed line). A total of 50 fractions (1 ml each) werecollected and analyzed for ACC synthase activity (solid line). thase molecular weight than the semilog method whichtends to either over- or underestimate the value. Gel Electrophoresis and Immunoblotting-Proteinswere fractionated by SDS-PAGE on 10% polyacrylamidegels according to Laemmli (1970). Half of each gel was stained with silver (Wray, 1981) and the other half electrophoreticallytransferred to nitrocellulose paper. Immunodetection 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

0.2

0.0

10 15 20 25 30 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 ontoa 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 collectedand 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 1h at 30 "C.
determination ofACC synthase native size was established using a 4th degree polynomial curve fitting equation, M W , = exp[exp (exp(2.005exp(-5.902*K.,)))1, with an estimated standard deviation of 3.4kDa (Fig. M). In contrast, the estimated standard deviation of these molecular standards on the semilog curvewas 26 kDa (Fig. 6 B ) . Therefore, the exponential equation established here between Kav and molecular weight gives a more precise estimation of the native ACC syn-

Deletion Analysis of ACC Synthase-Optimum alignment of deduced amino acid sequencesof various ACC synthases shows high conservation of roughly the 430 NH2-terminal amino acids but a distinct, hypervariableregion of 18-85 amino acids in the COOH-terminal domain(Park et al., 1992;O'Neill et al., 1993). Because of this high variabilityof 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


TABLE I1

Purification of wild-type ACC synthase expressed in E. coli The abbreviations used are: PPPE,,polyethyleneimine precipitated protein;G-25, Sephadex G-25; HTP, Hydroxylapatite column.
Purification
SkP

Total volume
rnl

Total protein mg

Total activity unitD

Specific activity

Purification -fold

Yield
%

unit lmg"

725.580

PPPE3316
G-25 17.8 MonoQ

120 950 10.7 0.48 845.400 180.640

40 HTP 33 15.778 168.828 12 890 Superose 12195.000 93.600 a unit = 1 pmol of ACC formedh at 30 "C.

0.219 0.890 10.148

1 4 46 72

100 116.5 24.9 23.3

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
rnl

Total protein rng

Total activity

Specific activity

Purification

Yield
%

unit"

unitlmg"

-fold

478.25 1175.48 385.00

(NH4)2SO,
G-150

MonoQ HTP 6.60 12 Superose 2.00 a 1 unit = 1 m o l ofACC formedh at 30 "C.

270 575 35 1436.05 10.5 3.6

822.25 28.00 12.17

11796.30 7222.00 6.42

8.78

7.98 61.22 51.28

1 1.10

100 9.96

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
rnl

Total protein rng

Total activity
unit"

Specific activity

Purification

Yield
%

unit I m g '

-fold

(NH4)2SO, 150 191.25 Batch HTP 300 120.00 2.394 MonoQ 18 HTP 10.4 8 3.6 Superose 12 a 1 unit = 1 m o l ofACC formed/h at 30 "C.

994.500 462.000 62.75 0.064

1.37 18.00 39.58 1228.05

0.19 0.26 7.52 162.50 233.33

100 9.41 5.43 0.73

855.26

constructed a mutant clone (pTACC-B1A)in which 57 deduced amino acids from the COOH terminus were deleted and exfind that theprotein pressed it in E. coli, we were surprised to et al., 1993). Reconproduced is enzymatically inactive (Mattoo 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 beand Ser439to delineate the region(s) at the COOH tween Ala428 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 proteindetected on the immunoblots (Fig. 7B). The specific activity ofACC 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 Ser439and 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 andwild-type ACC synthases. When VGVEKS in del-1 mutant wasreplaced entirely with positively charged LysArg 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 arebeneficial for maintaining a higher activity of the expressed enzyme. Likewise, replacement of the COOH terminus segmentRIRRF in del-2 mutant with AIAAF' (constituting thedel-6 mutant) reduced the specific activity by approximately 45-fold (Fig. 7A, compare del-2 with del-6). Together, the results with del-1 versus del-7, and del-2 versus del-6, suggest thatpositively charged residues imparta higher specific activity to ACC synthase. Additional deletions toward the NH2 terminus caused a dramatic reduction in the enzymatic activity of the protein expressedin E. coli, maximum loss and were deleted (del-3 to del-4). occurring when Ile430 When was deleted, the protein expressed was non-functional (Fig. 7A, del-5). Immunoblot analysis of the lysatesfrom these different culto ascertain the expression level and size tures was carried out of the expressed ACC synthase protein. The unaltered, wildtype ACC synthase wasexpressed as a major, full-size product of M, 54,000 (Fig. 7B, wt)with two additional, but minor proM, 52,000 and 48,000. All the truncatedforms tein bands with 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 1 1 more amino acids at the COOH terminus. Exthey have 6 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 ACC Synthase Function


tm

6913

140

120

20
01
0.20

1
0.25
0.30 0.35 0.40 0.45

t E

.
0.20

0.25

0.30

0.35

0.40

0.45

Kav FIG.6. Standard curves used for the molecular weight deter two Superose 12 colmination of ACC synthases gel-filtered on u m n s in series. A, the indicated equation was formulated to fit the exponential curve relating molecular weightto Kav.The asterisk in the equation represents the multiplication factor used in the Slidewriter software program (AdvancedGraphic 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 theproteolytic processingat the COOH terminus ismost 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 MutantsBecause the del-1 and del-2 mutant ACC synthases hadhigher specific activity than the wild-type enzyme, we surmised that the respective deletions might result in changed kinetic properties 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-2ACC 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 A M concentraenzyme exhibited high substrate inhibition at S tions higher than 0.13 m M . The best fit of the data points

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 S A M and aV,, 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, surprisingly, vastly different, with a K , >12-foldhigher and a V,, >4-foldhigher than the wild-type enzyme (Fig. 9B and Table VI. However, additional deletion of 6 residues, VGVEKS, giving resulted in restoration of sensitivity to rise to the del-2 enzyme, 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 internal sequence VGVEKS might influence enzyme catalysis. The close correlation between higher substrate affinity and greater substrate inhibition, and vice versa, suggest that thetwo may be interlinked. Alternatively, the unusual catalytic characteristics 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 theCOOH terminus might be involved in influencing the native structure ofACC synthase. Therefore, the purified wild-typeand 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 to native molecular weight of the wild-type enzyme was found be 97,600 2 4,000while onSDS-PAGE an apparent M, of 53,350 was obtained (Figs. 8 and lo), suggesting that the fulllength 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. Onthe 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 bedue to a conformational change resulting from the presence of VGVEKS at the COOH terminus. Thus, it would seem that the COOH terminus ofACC 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 of52 amino acid residues results in a dramatic increase in the specific activity of the trun-

del-1 -ALARIRRNGVEKS del-2 -ALARIRRF del4 -ALARIR del4 -ALAR

307.6 785.8 97.7 3.8 0.0 17.6 627.7

wt
FIG. 7.

del-1 del-2 del-3

del4 del-5 del-6 del-7

COOH-terminal deletions of ACC synthase and their expression i n E. coli. A, deletion mutants (del-141-5) and site-directed

mutants (del-6 and del-7) of ACC synthase were constructed via recombinationof a series of double-stranded oligonucleotides(see'Experimental Procedures") with pETlld expression vector. The first NH,-terminal Met of ACC synthase deduced fromits open reading frame was used as the translation initiation site. No fusion protein was found amongthese mutants. The del-1, -2, -3, -4, and -5 mutants represent respective deletions 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 arrow on the upper side of wild-type ( w t ) ACC synthase indicates the last Phe4s3which is highly conservedacross all ACC synthases sequenced 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 a t 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 Deletion encompassing the proteins expressed in E. coli. highly conserved completely abolished the enzyme acis important for the sustetivity; thus conservation of nance of some enzyme activity. When one comparesthe catalytic efficiency, denoted by the ratio V,,,JK,,,, of the wild-type and truncated forms ofACC synthase, del-2 enzyme (created by the deletion of the 52 amino acidresidues 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 was illustrated when the sequence example of this role 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, whenthe 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 thesubstrate affinity of the full-length, wild-type ACC synthase is comparable to the del-2 enzyme suggests that at least two domainswithin 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 natureof the COOH terminus of ACC synthase makes it prone to cleavage. The 48kDa 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 ~ sequence. ~~-Ly Using a similar estimation, the 52-kDa fragment may arise by

A r e 2 9

Arez9

cleavage of the parent protein within or around the sequence Gl~~~-Sefi~~ ACC -Va synthase l ~ ~ ~ . fromtomato(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 associatedwith 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 cellsare homogenized. This phenomenonmay 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 encompassed 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 affectedthe 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, underscores 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 isevidence for both monomeric and dimeric forms ofACC 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 activethan the dimeric, fullslength ~ ~ enzyme. Both ACC synthase and aspartate (and tyrosine) amino-

COOH Terminus Role in ACC Synthase Function

6915

wt
stain blot
200 -

del-I del-2
stain blot stain blot

97.4

66.2

0.00

0.25

0.00

0.50 0 75 [SAM]. mM

1.00

40 240

200 80 160

120

l/[SAM], mM-'

45

31

1 2

5 6
020 10

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 areindicated.
FIG.8. SDS-PAGEandimmunoblot

30

40

50

60

l/[SAM], mM-'

transferases share sequence similarity at the binding site for del-2 PLP (Nakajima et al., 1990; Van der Straeten et al., 1990; 0 . 0 10 Huang et al., 1991).Based on this, an attempt has been made to relate the two proteins in structural and evolutionary as0.008 pects. In this regard, we note that alanime amino acid trans- / / z l o ' o ferase is active only as a dimeric form because dissociation of the dimer into monomer inactivates the enzyme activity (Herold, 1990). However, ACC synthase can exist (for instance as is more active than the del-2 enzyme) in monomeric form which the full-length dimeric ACC synthase. Our data suggest that the conformation ofACC synthase as related to function may be 0.00 0.25 0.50 0.75 1.00 quite different from that of alanime amino acid transferase. [SAM]. mM The primary sequence homology found between alanime amino 0.000 0 40 80 120 160 200 240 acid transferase and ACC synthase may have, therefore, arisen as a product of convergent evolution. I/[SAM], mM-l It is possible that limited proteolysis ofACC synthase ocFIG. 9. Determination of kinetic constants of wild-type, del-1, curs under natural situations. Signals generatedby wounding of plant tissues (Pearce et al., 1991;Pena-Cortes et al., 1989) and del-2 ACC synthases. ACC synthase activity was measured a t different S A M concentrations as indicated in a reaction mixture conor by elicitors (Chalutz et al., 1984)result ina burst of ethyl- taining 50 m~ EPPS, pH 8.2,lO p~ PLP and2 m~ D m . The amount of ene biosynthesis. The target enzyme in ethylene biosynthesis protein used for each reaction was 21, 220, and 8 ng, respectively, for A), del-1 ( B ) , and del-2 (C). Each point represents an for such a trigger has generally been the ACC synthase. The wild-type (wt, mechanism of this activation of ACC synthase is a matter of average value of four replicates. Both substrate saturationcurves (insets) and the Lineweaver-Burk plots are shown. The curves were drawn conjecture. However, wounding is known to cause activation of using Slidewritersoftware based on Haldane's high substrate inhibition proteases (Mehta, 1993).Also, PMSF and soybean trypsin in- equation (see "Experimental Procedures"). hibitor, which are inhibitors of protease action, have been shown to inhibit the development of ACC synthase during gested that specific proteolytic activity in vivo may be associwound induction (Mattoo and Anderson, 1984) and following ated with the ethylene induction processes (Mattoo and elicitor treatment (Anderson et al., 1982).These results sug- Anderson, 1984).In view of our findings, it is now tempting to

6916

COOH Terminus Role in ACC Synthase Function


TABLE VI The native and subunit molecular weights of wild-type, del-1, and del-2 ACC synthases determined respectively by Superose 12gel filtration and SDS-PAGE
Enzyme Mass on Superose 12 Mass on SDS-PAGE Enzyme conformation

TABLE V Biochemicalparameters of purified wild-type (wt), del-I, and del-2 ACC synthases expressed in E. coli
V,, Enzyme
wt

K,,,
m M
pmol h-I
mg-I

Ki
m M

V d L
pmol h " mg" mK'

del-1 del-2

0.022 0.280 1.533 0.042

96 420 880

0.690 0.620

4,364 1,500 20,952

kDa
wt

del-1 del-2

96 f 5.7 60 e 2.3 47 52 e 1.8

54 46

Dimer Monomer Monomer

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

del-1

speculate that one of the mechanisms by which certain endogenous or exogenous stimuli cause activation of ACC synthase may involve specific processing at the COOH terminus, forming monomeric ACC synthase having 4-fold or higher specific enzyme activity thanthe 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 constructive critiques on the original version of the manuscript, Michael Reinsel for preparation and purification of oligonucleotides and for technical assistance, and DingboZhoufor assistance with the preparation of Fig. 9 . REFERENCES

del4

20,""
15
7

h
IC

wt

del-1

Anderson. J. D., Mattoo, A. K., and Lieberman, M. (1982) Biochem. Eiophys. Res. Commun. 107.588594 Bailey, B. A., Ami, A,, Li, N., Mattoo, A. IC, and Anderson, J. D. (1992) Plant Physiol. 100, 1615-1616 Bleecker, A. B. (1987) Cur,: Ibp. Plant Biochem. Physiol. 6.15-24 r t e c a , R. N., and Phillips. A. Bottela. J. R.. Arteca, J. M., Schlagenhaufer, C. D., A T .(1992) Plant Mol. Biol. 18, 793-797 . P., Nelson, N., Edelman, M., and Mattoo, A. K. (1989) Callahan, F. E., Wergin, W Plant Physiol. 91, 629-635 0 Chalutz, E., Mattoo, A. K, Solornos, T., and Anderson, J. D. (1984) Plant Physiol. 74,99-103 e Dong, J. G., Kim, W .T . , Yip, W . K, Thompson, G. A., Li, L., Bennett, A.B., and a b c d I\ Yang, S. F. (1991) Planta 185.38-45 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, Cambridge Herold, M., and Kasper, K (1990) Biochemistry 29,1907-1913 . H., and Theologis, A. (1991) P r o c .Natl. Huanp, P. L.. Parks, J. E., Fbttmann, W Ac&. Sci. U.S.A. 88,7021-7025 Laemmli, U. K. (1970) Nature 227,680-685 Li, N., Parsons, B.L., Liu, D., and Mattoo, A. K (1992a) Plant Mol. Biol. 18, 477487 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. Natl. Acad. Sci. U.S.A. 89, 11046-11050 0.00-1. ,. ,. , . , ., . . ,. . , . , . , . , . , - , . , . ,. , . , . , . , . 2 4 6 8 101214161820222426283032343638404244 Liu. D.. Li. N.. Dube. S.. Kalinski. . A,. . Herman. E., and Mattoo, A. K (1993)Plant Cell'PhysioZ. 34, hi-1157 Fraction number Marder. J. B., Mattoo, A. K., and Edelman, M. (1986) Methods Enzymol. 118, 384-396 FIG. 10. Determination of the size o f native wild-type, del-1. and del-2 ACC synthasesby gel filtration. The peak ACC synthase Mattoo, A. K, and Anderson, J. D. (1984) in Ethylene: Biochemical, Physwl@cal and Applied Aspects (Fuchs, Y., and Chalutz, E., eds) pp. 139-147, Martinus activity fractions from the hydroxylapatite column were pooled and Nijhoff Publishers, The Netherlands passed through Superosel2 column. Wild-type(in three batches at 0.2, Mattoo, A. K, and White, B. W. (1991) in The Plant Hormone Ethylene (Mattoo, A. 0.24, and 2.4 mg of protein), and del-1 and del-2, both in four batches a t K, and Suttle, J. C., eds) pp. 21-42, CRC Press, B o a Raton, FL protein concentrations from 0.1 to 0.45 mg, ACC synthases were ana- 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. lyzed. Each run was followed with one or two calibrations with standard 223-231, Kluwer Academic Publishers, Dordecht protein markers. The shift in the absorbance peak a t 280 nm did not exceed 0.1fraction volume (40pl) among the separations. Aliquots (100 Mehta, R. (1993)Molecular Aspects ofRipening and Stress-related Protein Metabolism inHigher Plants, Ph. D. dissertation, University of Maryland a t Baltimore pl) from each fraction were mixedwith 50 p l of loading buffer, and 45 pl County from this solution were fractionated on SDS-PAGE and analyzed by Mehta, r o c .Natl. A. M., Jordan, R. L., Anderson, J. D., and Mattoo, A. K (1988)P immunoblots. A, immunoblot analysis of wild-type (wt), del-1, and del-2 Acad. Sei. U.S.A. 86,8810-8814 ACC synthases fractionated on Superose 12. Fractions from B which Nakajima, N., and Imaseki, H. (1986) Plant Cell Physwl. 27,969-980 were immunoblotted are listed on the top in A. B , elution profiles of Nakajima, N., Nakagawa, N., and Imaseki, H.(1988)Plant Cell Physiol. 29,989998 wild-type (closed circles), del-1 (closed squares), and del-2 (closed triangles) ACC synthase activity from Superose 12 column. Each data Nakajima, N., Mori, H., Yamazaki,K, and Imaseki. H. (1990) Plant Cell Physwl. 31,1021-1029 point represents an average of three separate assays from each indiS.D., Nadeau, J.A,, Zhang, X.S . , Bui,A. Q., and Halevy,A. H.(1993)Plant cated fraction indicated a t the bottom of C. C, a representative elution O'Neill, Cell 5,419432 profile for standard protein markers on the Superose 12 column. The Park, K. Y., Drory, A., and Woodson, W.R. (1992) Plant Mol. Bwl. 18,377486 molecular mass markers used were: aldolase, 158 kDa (a); bovine se- Pearce, G., Strydom, D., Johnson, S., and Ryan, C. A. (1991) Science 253,895-898 rum albumin, 67 kDa (b);ovalbumin, 43 kDa ( c ) ; carbonic anhydrase, Pena-Cortes, H., Sanchez-Serrano, J. J., Mertens, R., Willmitzer, L., and Prat. S. 29 kDa (d); cytochrome c, 12.4 kDa (e). (1989) Proc. Natl. Acad. Sei. U.S. A. 86,9851-9855

. .

COOH Terminus Role in ACC Synthase Function


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 Enzymol. 166,60-89 Theologis, A. (1992) Cell 70, 181-184 Van der Straeten, D. Van Wiemeersh, L., Goodman, H. M., and Van Montagu, M.

6917

(1990) Proc. Natl. Acad. Sci. U.S. A 87,48594663 Van der Straeten, D., Rodrigues-Pousada,R. A,, Wllarroel, R.Hanley, S., Goodman, H. M., and Van Montagu, M. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 99699973 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