Isolation and properties of an S-adenosyl-L-methionine binding protein from the green alga, Chlamydomonas reinhardi

• JOURNAL OF PLANT PHYSIOLOGY

J. Plant Physiol. 157.707-711 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

I Short Communication

I

Isolation and properties of an S-adenosyl-t-methionine binding protein from the green alga, Chlamydomonas reinhardi Yuka Nakano, Nozumu Koizumi, Tomonobu Kusano, Hiroshi Sano* Laboratory of Plant Molecular Breeding, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0101, Japan

Received July 6, 2000 . Accepted August 22, 2000

Summary A protein that binds S-adenosyl-L-methionine (AdoMet) was isolated by successive chromatography from vegetative cells of Chlamydomonas reinhardi. The relative molecular masses of native and denatured proteins were calculated to be 15kDa and 16kDa, respectively. The reaction was specific to AdoMet and reversible as shown by competition experiments . The optimal pH for binding ranged between 7.5 and 9.0, and the apparent Ka for AdoMet was 1.0Ilmol/L. Results suggested that the protein may function as the carrier to stabilize AdoMet, which is otherwise labile in the cellular environment.

Key words: S-adenosyl-L-methionine - Chlamydomonas reinhardi

Abbreviations: Ado Met S-adenosyl-L-methionine. - MET methyltransferase

Introduction S-adenosyl-L-methionine (AdoMet) is indispensable for biological and biochemical events in both prokaryotes and eukaryotes. In animal cells, the AdoMet concentration ranges from 50 to 500llmol/L, of which 85 % is used for creatine synthesis, 10 % for methylation, and 5 % for polyamine synthesis (Robert Hoffman, personal communication). In plant cells, AdoMet plays at least four important roles. First, it serves as the methyl donor for various methyltransferases (METs). Sec• E-mail correspondingauthor:[email protected]

ond, it provides a-aminopropionyl groups for spermidine synthesis. Third, it allosterically regulates the synthesis of amino acids, including methionine and the aspartate family. And fourth , it is a direct substrate for ethylene synthesis (Giovanelli et al. 1980). AdoMet is synthesized by AdoMet synthetase using methionine and ATP as substrates. Studies with genes encoding AdoMet synthetase from rice and C. roseus suggested several isoforms specifically associated with enzymes which use AdoMet (Ravanel et al. 1998). However, regulation of their activity in response to various stimuli is currently not clear. The average half-life of AdoMet in cells has been estimated to be approximately 10 min, and since it is 0176-1617/00/157/06-707 $ 15.00/0

708

Yuka Nakano et al.

Materials and Methods

constitutively needed for biological reactions, its storage at an adequate level is critical for maintenance of cellular homeostasis. Despite this importance, almost nothing is known about the fate of AdoMet after its synthesis and before its physiological consumption. In this paper, we report isolation from C. reinhardi of a protein that specifically binds AdoMet to form a stable complex.

A

3

80

Ec..

§.

(')

-

60 2

~

>-

(.)

c::

-... 0

:;::

10

.;;

....

--E

0,

(.)

0 ,....

Vegetative cells of C. reinhardi, strain me 1 (mating type+) were cultured at 25 'C in liquid TAP medium (Tris-acetate, pH 7.2, 1 mmol/L phosphate, 5 % 2 x Beijerlinck's solution, and 1 % trace element solution), under continous light, and bubbled with 5 % CO 2 to a density of 3x 10 6 cells per mL (Sano et al. 1984). Cells were harvested by cen-

c::

40

c::

0

:c

0

~

0.2

(.)

c::

10

a:

0.3

Q) (.)

10

]

20

0

10

...0 0-

C'2....

0

10

20

40

30

0.1 ~

0

0

60

50

~

(3

Fraction

B

E c.. (.)

(')

100

0.8

80

0.6

0 ,....

-

~

>-

';;

...

60

:c

40

:E'

20

••

10

a:

C'2.....

c::

0

10

(.)

0

§. :;::

:;::

10

--a,E

10

20

.. .. .. ..

0.4 •• ••

2

(.)

c::

0

(.)

0.2

60

50

30

cQ)::

~

c:: j

(3

...0 0-

10

~

0

0

Fraction

C

40

0.04

30

0.03 E c::

Ec..

(.) (')

0 ,....

-

~

.;;

:;:: (.)

20

10

0

:c

""

10

a:

:E'

0

IX) ('II

>-

10

""

"

""

" ""

"

0.02

10 Q) (.)

0.01

c:: 10 .c ... 0

UI

.c

«

C'2.....

0

0

5

10

15

Fraction

20

25

30

0

0.4 0.3 ~ 0.2 (3 10

0.1 ~

0

Figure 1. Purification of AdoMet binding protein. (A) DEAE-Toyopearl column chromatography. Fraction I containing 400-450mg protein was applied to an 80mL bed volume of DEAE-Toyopearl, and proteins (indicated by open circles) were eluted with a linear gradient of NaCI from O.02mo1/L to 0.3mo1/L (dotted line). The activity (indicated by closed circles) was measured, and major active fractions (indicated by shade) eluted from 0.11 mol/L NaCI were pooled (Fraction II). (B) Affi-Gel Blue column chromatography. Fraction II containing 25 mg protein was applied to a 2.5-mL bed volume of Affi-Gel Blue, and proteins (indicated by open circles) were eluted with a linear gradient of NaCI from 002mol/L to 2.0mol/L (dotted line). The activity (indicated by closed circles) was measured, and major active fractions (indicated by shade) eluted from 0.95 mol/L NaCI were pooled (Fraction III). (C) FPLC-Mono-Q column chromatography. Fraction III containing 0.72 mg protein was applied to a 1-mL bed volume of Mono-Q, and proteins (indicated by line) were eluted with a linear gradient of NaCI from 0.02mol/L to 0.4mo1/L (dotted line). The activity (indicated by closed circles) was measured, and major active fractions (indicated by shade) eluted from 0.06mol/L NaCI were pooled (Fraction IV).

S-adenosylmethionine-binding protein of Chlamydomonas trifugation, resuspended in buffer I (50mmol/L Tris-CI, pH 7.6, 0.5mol/ L KCI, 1mmol/L EDTA, 1mmol/L ~-mercaptoethanol, 5 % glycerol) and homogenized with a French press at 1,500 psi. Cell debris was removed by centrifugation at 1,000xg, and the resulting supernatant was further centrifuged at 12,000xg for 2h. After dialysis against buffer I without KCI for 16 h, the sample was further purified by repeated chromatography on DEAE-Toyopearl, Affi-Gel Blue, and Mono-Q, as detailed in the figure legend. AdoMet binding activity was assayed by incorporation of S-adenosyl[3H]methionine into protein fractions. A 50-J,lL reaction mixture containing 50 mmol/L Tris-acetate, pH 7.5, 20 mmol/L EOTA, 3 mmol/L on, 1 J,lg RNaseA, 3 J,lmol/L S-adenosyleH]methionine (specific activity, 200 GBq/mmol), and 15 J,lL of enzyme solution, was incubated at 37"C for 1 h. After addition of cold AdoMet to a final concentration of 7 J,lmol/L, the reaction mixture was spotted on a OEAE-filter paper (Whatman DE52), washed with 0.5 mol/L Na-phosphate three times, for 15 min each time, dried and subjected to radioactivity counting. One unit of binding activity was defined as incorporation of 100cpm of 3H-radioactivity. Direct AdoMet binding was analyzed by UV-cross linking method (Som and Friedman 1990). Briefly, a 36-J,lL reaction mixture containing 50 mmol/L TrisCI, pH 7.2, 10 mmol/L EOTA, 20 mmol/L NaCI, 1 mmol/L On. 1mmol/L ~-mercaptoethanol, 4 J,lmol/L S-adenosyl[3H]methionine (specific activity, 2.03-3.15 TBq/mmol), and 18 mmol/L NaOH (to neutralize AdoMet suspended in sulfuric acid) was mixed with 24 J,lL protein fraction and irradiated with 2,700 mJ/cm 2 with a UV-crosslinker (RPN 2505, Amersham). After SOS-PAGE, proteins were transferred to a PVOF membrane filter, sprayed with autoradiography enhancer (ENHANCE Spray Surface, NEN), and placed in contact with an x-ray film for several days.

709

to affinity chromatography (Affi-Gel Blue Gel 100-200 mesh, Bio-Rad), and eluted with a gradient of 0.02-2.0 mol/l NaCI solution. Total protein absorbed was 13 mg, most of which was eluted at less than 0.5mol/l NaCI. AdoMet binding activity eluted at 0.95 mol/l NaCI was pooled and designated as Fraction III, which had 0.7mg proteins with 4,350 units of activity (6,050 units/mg protein) (Fig. 1 B). After further fractionation (0.7 mg) by Mono-Q FPlC (Pharmacia), active fractions were eluted with 0.06 mol/l NaCI and designated as Fraction IV, which contained 0.1 mg protein and 590 units of activity (5,370 units/mg protein). The purification procedure is summarized in Table 1. The sudden decline in Fraction IV may be attributable to a decrease in stability as the purification proceeded, stability being evident up to Fraction III, but binding activity rapidly lost in Fraction IV, with a half-life of 12 h. In order to identify the protein that binds AdoMe!, we performed an AdoMet-binding assay, whereby, proteins were incubated with [3H]AdoMet and then cross-linked by UV light irradiation before SOS-PAGE analysis. Proteins exhibiting considerable heterogeneity were visualized by silver staining (Fig. 2A). However, when a duplicate was transferred to a nylon membrane and subjected to autoradiography, only a single band was observed at the position of 16 kOa with Fractions II through IV (Fig. 2 B). To determine the native form of this 16 kOa protein, Fraction III was assayed by gel filtration, which showed the protein to exist as the monomeric form of 15 kOa (Fig. 2 C). The AdoMet binding properties of this protein were then examined. The specificity and reversibility

Results and Discussion

were examined by competition experiments, which were performed by adding effectors to the reaction mixture 10 min af-

Actively proliferating cells were harvested from 9 l culture,

ter incubation (Fig. 3A). It was clearly shown that cold AdoMet

homogenized, and subjected to ultra-centrifugation. The

caused a decrease of radioactivity, indicating the protein to

crude supernatant designated as Fraction I contained ap-

exchange [3H]AdoMet with cold one. The structurally related

proximately 400-450 mg of proteins, and had a total of 6,700

S-adenosyl-l-homocysteine (AdoHcy) affected the binding

units of AdoMet binding activity. The calculated specific ac-

less efficiently than AdoMet, while NAOH, which also shares

tivity was 16.8 units/mg protein. Fraction I (400 mg protein)

a common structure with AdoMet, was ineffective. Efficient

was loaded onto a OEAE-cellulose column (OEAE-Toyopearl

binding was apparent in the pH range between 7.5 and 9.0,

650 mol/l, Tosoh), washed, and eluted with a gradient of 0.02-0.3 mol/l NaCI solution. Total protein absorbed was 250 mg, and AdoMet binding activity was eluted at 0.14 mol/l NaCI (Fig. 1 A). The active fractions were pooled and desig-

but a sharp decline occurred at higher or lower values. The apparent dissociation constant (Ka) estimated from kinetic analysis was 1.0 jJ.mol/l (Fig. 3 B), comparable with those re-

nated as Fraction II, which had 26 mg of proteins with 12,470 units of activity (480 units/mg protein), applied (25 mg)

ported for some METs, as examplified by EcoRI and Chlamydomonas METs with values close to 0.25 jJ.mol/l (Rubin and Modrich 1977, Sano and Sager 1980). The high dissociation

Table 1. AdoMet-bindung protein purification stages.

Fraction

I. II. III. IV.

Crude supernatant OEAE-Toyopearl chromatography Affi-Gel Blue chromatography Mono-Q-chromatography

* 1 unit= 100 cpm

Protein (mg)

Total activity (unit*)

Specific activity (unit/mg protein)

purification (-fold)

415 26 0.72 0.11

6660 12500 6816 591

16 476 6048 5374

30 376 334

Recovery (%)

100 188 102 8.8

710

Yuka Nakano et al.

A

A 200kDa

97

66

31

'i'

14

5

(W)

L......O

OL-----~----~----~~

o

B

40

20

60

Time (min)

200kDa

97

66

B

......

E Q. 10 U

(W)

31

0 ,..

-

~

Ka-l.0uM

8

>-

.:;

;:;

16

U

0

14

;:;

~

a:

~

E a.

(,) C')

0 ,..

-

40

15k

>-

C

"67k

~ as 0

'5as 10

CI)

"0 E

-32k

a:

£'

'"2... 0

20

00

25

30

\12.4k 10 35

g! "j Gi

a:

Retention Time (min) Figure 2. Identification of AdoMet binding protein. (A) SDS-PAGE of Fractions I through IV. Samples were fractionated on a 12.5 % SDS-

2 4

6

8

10

2

4

6

8

[AdoMet]( IJ. M)

til til

E ".!! 2 10 :::J (,)

0

11[AdoMe1)

as

·142k

20

-2

<2....

~

30 ·290k

~

.s:

103 'i?

0

~ 0.4

It!

C

.

0.8

It!

Figure3. AdoMet binding properties of 16kDa protein. (A) Competition experiments. After the standard reaction mixture containing Fraction III protein was incubated at 37'C for 10 min, each effector with the final concentration of 20 Ilmol/L was added. The mixture was further incubated for indicated period and assayed for bound [3H]AdoMet. Samples were the control without effector (open circles), AdoMet (closed circles), AdoHcy (open triangles) and NADH (closed triangles). (8) Effect of AdoMet concentration. The standard reaction mixture containing indicated concentrations of [3H]AdoMet was incubated at 37'C for 30 min. Double-reciprocal plots are shown for estimation of apparent Ka.

polyacrylamide gel and stained with a silver staining kit (Daiichi Chemicals, Tokyo). Protein size is indicated on the right. (8) AdoMet binding assay. After reaction with eH]AdoMet, and being subjected to UV-cross linking, samples were fractionated on a 12.5 % SDS-polyacrylamide gel and transferred to PVDF membrane, which was sprayed with enhancer solution and exposed to x-ray film at room temperature for 20 days. The binding activity was detected at the position of 16kDa. Protein size is indicated on the right. (C) Gel filtration of Fraction III. The sample was fractionated on Superdex 200, together with glutamate dehydrogenase (290kDa), lactate dehydrogenase (142kDa), enolase (67kDa), adenylate kinase (32 kDa), and cytochrome C (12.4 kDa) as marker proteins. The binding activity was eluted at the position of 15 kDa.

constant suggests the interaction between 16 kDa protein and AdoMet to be reversible, and thus the binding to be physiologically significant. There are approximately 120 types of AdoMet-dependent MET with E.C. numbers (Cheng and Blumenthal 1999). They catalyze methylation of a variety of substances, including DNA, RNA, proteins, lipids, polysaccharides, and chemicals with small molecular masses (Cheng and Blumenthal 1999). Their molecular size is variable, ranging from 190 kDa for

5-adenosylmethionine-binding protein of Chlamydomonas DNA-MET (Bester and Ingram 1985) to the 25 kDa of catechol-O-MET (Vidgren et al. 1994) and L-isoaspartyl-MET (Mudgett and Clarke 1996), which are the smallest METs reported to date. Amino acid sequence analyses, however, have revealed that AdoMet is bound by a common region, spanning over 150 amino acids (Cheng and Blumenthal 1999). Since the presently-reported protein is 15-16 kDa, it presumably only constitutes the AdoMet binding region, and therefore may lack any enzymatic activity. Considering the small size and high affinity, it is conceivable that it functions as a carrier or stabilizer of AdoMe!. Further analyses are now needed to determine its physiological function by isolating the encoding gene. Acknowledgements. This work was partly supported by a grant from CREST, Japan Science and Technology Corporation.

711

References Bester TH, Ingram VM (1985) Proc Nail Acad Sci USA 82: 2674-2678 Cheng X, Blumenthal RM (1999) S-Adenosylmethionine-Dependent Methyltransferases: Structure and Functions. World Scientific, Singapore Giovanelli J, Mudd SH, Datko AH (1980) In: Miflin BJ (ed) The Biochemistry of Plants. Vol 5 Amino Acids and Derivatives. Acadmic Press, New York, pp 453-505 Mudgett MB, Clarke S (1996) Plant Mol Bioi 30 723-737 Ravanel S, Gakiere B, Job D, Douce R (1998) Proc Natl Acd Sci USA

95 7805-7812 Rubin RA, Modrich P (1977) J Bioi Chem 252: 7265-7272 Sano H, Grabowy CT, Sager R (1984) Mol Cell Bioi 4: 2103-2108 Sano H, Sager R (1980) Eur J Biochem 105: 471-480 Som S, Friedman S (1990) J Bioi Chem 265 4278-4283 Vidgren L, Svensson LA, Liljas A (1994) Nature 268: 354-358