Bacterial Expression of the Catalytic Domain of ... - Wiley Online Library

Eur. J. Biochem. 2.33, 506-513 (1995) FEBS 1995

Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-CoAreductase (isoform HMGR1) from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase Susan DALE', Montserrat ARRO', Beatriz BECERRA', Nick G. MORRICE', Albert BORONAT", D. Grahame HARDIE' and Albert FERRER'

' Biochemistry Department, Thc University, Dundee, UK Unitat de Bioquirnica, Facultat de Farmhcia, Universitat de Barcelona, Spain

' MRC Protein Phosphorylation Unit, The University, Dundee, U K

' Departament de Bioquirnica i Fisiologia, Facultat de Quiinica, Universitat de Barcelona, Spain (Received 3 April 1995)

~

EJB 95 0529/1

The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoforin 1 (HMGRI cd) from Aruhidopsis thulium has been expressed in Escherichiu coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source. HMGRlcd activity was completely blocked by the HMGR inhibitor mevinolin (IC5,, = 12.5 nM). No significant differences were observed between the K,,, values of HMGRlcd for NADPH (71 2 7 pM) and (S)-.?-hydroxy-3-methylglutaryl-CoA (8.3 -t 1..5 pM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGRl cd was reversibly inactivated by phosphorylation at a single site by Bmssica olrmcru HMGR kinase A, which is functionally related to the mammalian AMP-activated protein kinase. The site of phosphorylation is Ser.577 i n the complete sequence of A. tlzulinntr HMGRI. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least i n a cell-free system. Our results also reinforce the view that the AMPactivated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system. Keyvords: isoprenoid biosynthe higher plants ; h ydroxymethylglutaryl-CoA reductase ; hydroxymethylglutaryl-C'oA reductase kinase ; protein phosphorylation.

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the NADPH-dependent double-reduction of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) to mevalonate. This reaction is generally considered as a key controlling step in plant isoprenoid biosynthesis [I -41. However, although HMGR is one of the most studied enzymes of isoprenoid biosynthesis i n plants, its rolc in the overall control of this metabolic pathway remains to be unequivocally established. The characterization of HMGR has been hampered by the fact that it is a membrane-bound enzyme which is difficult to purify from plant sources 12, 51. Consequently, many of the properties of plant HMGR did not emerge until the genes encoding the enzyme had been cloned. In contrast to animal systems, where only one form of HMGR has been reported in each species, it has become clear ~ ~ ~ ~ - ~ . e . s ~ 7 10 ~ ~A. fi~ Ferrer, / e ~ i [ Unitat ,~, de Bioquirnica, Facultat de Farmicia. Avda. Diagonal 643, E-08028-Barcelona,Spain Fux: + 34 3 402 1X 96. Ahhreriations. HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A : HMGR. HMG-CoA reductase; HRK-A and HRK-B, HMGR kinases A and B ; HMGRlcd, expressed catalytic domain of HMGRl. Enzynws. 3-Hydi-oxy-3-niethyIglutaryl-CoA reductase (EC 1 . I . I .34): 3~hydroxy-3-niethylglutaryl-CoAreductase kinase (EC 2.7.1.109): protein phosphatasc (EC 3.1.3.16).

that plants contain multiple genes encoding several HMGR isoforms. The exact number of genes ranges from two in Amhidopsis tlzalinnu [ 6, 71 to at least nine reported to occur in potato 141. On the basis of amino acid sequences predicted from the DNA sequences, a structural model for plant HMGRs has been proposed 181. The HMGRs of plants consists of an N-terminal region (highly divergent), a conserved membrane anchor domain (containing two hydrophobic sequences), a short linker region (also highly divergent) and a highly conserved C-terminal catalytic domain. Several reports indicate that plant HMGR activity responds to a variety of developmental and environmental stimuli, such as light, phytohormones, pathogen attack, wounding, isoprenoid derivatives and endogenous protein factors 12, 4, 91. It is now clear that plants regulate HMGR activity at the level of mRNA by differential expression of the HMGR gene family members. There is also emerging evidence of the post-translational control of HMGR activity [4]. Some previous studies had suggested that plant HMGR activity could be regulated by reversible phosphorylation [lo, 111, as in animals. Mammalian HMGRs are inactivated by phosphorylation at a serine residue close to the C-terminus, by the AMPactivated protein kinase [ 12- 141. AMP-activated protein kinase is dramatically activated by elevation of the AMP/ATP ratio, via

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a dual mechanism involving direct allosteric activation [IS - 171 and phosphorylation by an upstream protein kinase [18, 191. Elevation of the AMP/ATP ratio occurs in mammalian cells when they are experiencing a stress (e.g. heat shock, hypoxia, chemical stress) which causes ATP depletion. Phosphorylation and inactivation of HMGR and other biosynthetic enzymes under these conditions may conserve ATP until the stress situation is resolved [20]. Recent amino acid sequencing and cDNA cloning [21, 221 has suggested that AMP-activated protein kinase is the mammalian homologue of the product of the SNFl gene from the yeast Saccharornyces cerevisiae, also known as CAT1 or CCR3 123-2.51. Studies with snfl mutants show that the wildtype protein k.inase is required for the response to starvation for glucose [23, 261, which for yeast cells growing in logarithmic phase represents an acute stress, since they do not maintain reserves of glycogen or any other carbohydrate. DNAs encoding protein kinases related to SNFl (e.g. RKINI from rye) have also been cloned from several higher plant species [27-301. They appear to represent true homologues of S N F l since they restore the ability of snfl mutant yeast to grow on non-fermentable carbon sources [27, 301. Activities with biochemical similarities to AMP-activated protein kinase have also been characterized from several plant species and purified from Brussica oleracea (cauliflower) inflorescences [31]. These probably correspond to the products of the SNFI-related genes, although this remains to be conclusively established. They are not directly activated by AMP, but phosphorylate mammalian HMGR at the same serine residue as mammalian AMP-activated protein kinase, and also inactivate HMGR in potato microsomes, an effect reversed by adding protein phosphatase [31]. We therefore refer to them as HMGR kinases (HRKs). Recently we have separated two forms of HRK from B. oleracea. The major form (HRK-A) has a native molecular mass of around 200 kDa and a catalytic subunit of 58 kDa, corresponding to the subunit mass predicted for the RKINl family. The minor form (HRK-B) has a molecular mass of 45 kDa and appears to be a monomer [32]. In the present study we report the expression in Escherichia coli of the C-terminal catalytic domain of the HMGRl isoform from A. thaliana. The catalytic domain of HMGRl has been purified and characterized. The pure enzyme is fully active, but is inactivated by phosphorylation at a single site by B. oleracea HRK-A. The site of phosphorylation is Ser577 which exactly corresponds to the single regulatory phosphorylation site on mammalian HMGR.

MATERIALS AND METHODS Materials. Restriction enzymes and T4 DNA ligase were obtained from Promega. Affi-Gel Blue (100-200 mesh) was from Bio-Rad Laboratories. Radiochemichals were from Amersham International. All other chemicals were of the highest commercial grade available. The bacterial expression vector pT7.7 was kindly provided by S. Tabor (Harvard Medical School, Boston MA). HRK-A was purified from B. olemcea and assayed as described previously (321. Construction of the expression vector pHMGRlcd. The overall scheme for the construction of plasmid pHMGRlcd is summarized in Fig. 1. The oligonucleotides 5'-TATGGAATC3' and 5'-GATTCCA-3' were annealed to generate an adapter sequence which was designed to have a 5' NdeI-cohesive end and a 3' blunt end. In addition, it contains the nucleotide sequence corresponding to nucleotides +496 to +SO0 of A. thaliana HMGRl cDNA. Plasmid pLB12, bearing the full-length cDNA of A. thaliana HMGRI, [6] was digested with XrnnI and

pLBl2 5.3 Kb

BamHl

Xmnl

I

\

w I

BamHl

I

Ligation

r:d 4.2Kb

BamHl

CATATGGAATCGC-CCTGAGG AAGAC Met Glu Ser Leu Pro GIu GIU ASP

Fig. 1. Construction of plasmid pHMGR1cd. Plasmid pLB12 contains the full-length cDNA coding for A. thaliana HMGRI. The regions of the HMGRl cDNA coding for the N-terminal region and the membrane anchor domain (hatched), the linker region (stippled), and the C-terminal catalytic domain (black solid) are shown. Open areas correspond to the 5' and 3' non-coding regions. The complete sequence of the adapter is provided. The predicted nucleotide and deduced amino acid sequence of the N-terminal end of HMGRlcd is also shown. The last six amino acids of the HMGRl linker region are underlined. Position and orientation are shown for the T7 RNA polymerase promoter (410) and for the p-lactamase gene (bla). The plasmid ribosome binding site (rbs) is also indicated.

BarnHI. The resulting 1735-bp fragment, which contains the sequence encoding amino acids 166-592 plus the 3' non-coding region, together with the adapter sequence, were ligated into expression vector pT7.7 1331 at NdeIIEarnHI sites by a three-piece ligation. The clones of interest were initially selected in E. coli HMS174 and further introduced into E. coli BL21(DE3) for expression ctudies. Plasmid pHMGRlcd, as confirmed by sequencing, contained the cDNA sequence in the correct reading frame. Expression in E. coli. BL21(DE3) cells harbouring the expression plasmid pHMGRl cd were grown with aeration at 37 "C in Luria-Bertani medium [34] supplemented with ampicillin (100 pg/ml) to an A,,,,, of 0.6, and then induced by the addition of 0.4 mM isopropyl 8-D-thiogalactoside. After incubation for 6 h at 22"C, cells were harvested by centrifugation at 7000Xg for 5 min at 4°C. The collected cells were resuspended in 4 ml buffer A/g wet mass (buffer A = 100 mM sucrose, 40 mM sodium phosphate pH 7.5, 30 mM EDTA, 50 mM sodium chloride, 10 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.25% by vol. Triton X-100) and disrupted (0.5 min/ml suspension) in an ultrasonic disintegrator (MSE, 60 W) while being

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chilled in a -10°C bath. Cell debris was removed by centrifugation (lSOOOXg, 20 min, 4°C) and the supernatant was used as a starting material for enzyme purification. For exclusive labelling of the expressed protein, cells were grown until an A,,,, of 0.6 and then induced by the addition of isopropyl ,!h-thiogalactoside. Rifampicin (200 pg/ml) was added 30 min after induction and cells were pulse-labelled with 6 pCi/ml of ["SImethionine for 6 h. Cells from 2 ml of culture were resuspended in 300 p1 buffer A, and the soluble fraction prepared by sonication as described. Aliquots of 5 p1 were subjected to SDSI PAGE. Purification of A. thaliana HMGRlcd. All procedures for enzyme purification were performed at room temperature unless otherwise stated. Cell-free extracts from 1 1 isopropyl P-D-thiogalactoside-induced cultures of E. coli cells transformed with pHMGRlcd were concentrated by the addition of a saturated ammonium sulfate solution (pH 7.2) to give a 50% saturated solution. After stirring for 30 min, the precipitate was collected by centrifugation at 25000Xg for 15 min and redissolved in 7 ml buffer B (100 mM sucrose, 40 mM sodium phosphate pH 7.5, 30 mM EDTA, 1 M NaCl, 33 % by vol. glycerol, 10 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The concentrated extract was heated at 65°C for 10 min, rapidly cooled using an ice-water bath and centrifuged at 1OOOOOXg for 45 min. The cleared sample was diluted fivefold with buffer C (buffer A lacking both NaCl and Triton X-100) and applied to a 25-ml Affi-Gel Blue column ( 8 x 2 cm; 1 ml . min-') equilibrated in buffer C. The column was washed extensively with the same buffer and then eluted with a 700-ml linear gradient of 0.2-2.5 M NaCl in buffer C. HMGR activity eluted as a single peak at 1.2 M NaCl. Active fractions were pooled and concentrated to a volume of 10 ml using an Amicon ultrafiltration cell with a PMlO filter; 40 ml buffer C was added and the sample concentrated again to 10 ml. This step was repeated once more and glycerol added to give a final concentration of 50% (by vol.). The enzyme was further concentrated to 0.5 mg/ml and stored frozen at -70°C. This preparation was used for all kinetic studies. To obtain HMGRl cd for the phosphorylation experiments, the enzyme eluting from the column was concentrated using the same protocol but with 100 mM Tris/HCl pH 7.5, 1 mM EDTA, 10 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, rather than buffer C. Enzyme assays. HMGR activity was dctermined by the radiometric assay described by Bach et al. [35], with the enzyme being diluted using buffer A supplemented with BSA (0.5 mg/ ml). For the experiments shown in Figs 4 and 5, we used a spectrophotometric assay at 37°C (total volume 500 pl) containing HMG-CoA (0.3 mM), NADPH (0.2 mM) and dithiothreitol (4 mM) in 100 mM potassium phosphate pH 7.0. The decrease in absorbance at 340 nm was monitored before and after addition of HMG-CoA, which was used to start the reaction. For both assays, one unit of HMGR activity is defined as the amount of enzyme which converts 1 pmol HMG-CoA into mevalonate (equivalent to 2 pmol NADPH oxidized/min at 37°C. Production of antibodies against HMGR1. Antipeptide antibodies were raised against the sequence CRDISGATTTTTT corresponding to amino acids 579-590 of the A . thaliana HMGRl with an additional cysteine residue at the N-terminus (underlined). The peptide was conjugated to keyhole limpet haemocyanin through the Cys residue by the MBS procedure 1361 and the conjugate (0.5 mg) emulsified in Freund's complete adjuvant (1 :1) before being injected subcutaneously into New Zealand white rabbits. Two additional 0.5-mg booster injections were given at 3-week intervals. Sera was collected 7 days after the last injection. An ELISA [37] was used to titer for antipeptide antibodies using the peptide antigen. The antibodies were

concentrated by precipitation with ammonium sulfate and dialysis against phosphate-buffered saline (pH 7.5). Phosphorylation of HMGRl by HRK-A. HMGRlcd (0.15-0.2 mg/ml) was phosphorylated at 30°C in incubations which contained 20 mM TrisMCl, 30 mM Na/Hepes pH 7.0, 12 mM NaCl, 40 pM EDTA, 2.6 mM dithiothreitol, 20% (by vol.) glycerol, 5 mM MgC12, 0.2 mM [yJ2P]ATP (150200 cpmhmol by Cerenkov counting) and HRK-A ( 5 U/ml). Phosphorylation was estimated as radioactivity precipitable by trichloroacetic acid [38]. HMGR activity was estimated by diluting aliquots into 4 vol. 20 mM EDTA (to chelate MgZ+and stop phosphorylation), followed by the spectrophotometric assay as described above. For analysis of the phosphorylation site, reactions were terminated in SDS/PAGE sample buffer and the mixture separated on 10% SDS/PAGE gels. After detection by Coomassie blue staining, the '2P-labelled 50-kDa HMGRlcd polypeptide was excised and subject to CNBr digestion. CNBr digestion, isolation and structural analysis of the phosphorylated peptide. The excised gel pieces were ground in 400 p1 70% (by vol.) formic acid in a I-ml ground-glass homogenizer. CNBr (= 10 mg) was added and, after standing at 4°C for 16 h, the supernatant was removed and dried in a centrifugal vacuum concentrator to 100 pl. The sample was diluted to 1 ml with water, taken to dryness, and reconstituted in 400 p1 0.1 % (by vol.) trifluoroacetic acid. The sample was chromatographed on a Vydac microbore C18 column (150X2.1 mm) attached to an Applied Biosystems 140B HPLC system, and eluted at 0.2 ml/min with a 0.1 % trifluoroacetic acid gradient from water to acetonitrile (increasing by 0.5 %/min). Peptides were detected at 214 nm and radioactive peptides by Cerenkov counting. Amino acid sequencing and mass spectrometry. An aliquot (30 pmol) of the isolated phosphopeptide was sequenced by pulsed liquid Edman chemistry on an Applied Biosystems 476A sequenator. Another aliquot was covalently coupled to a Sequelon AA (Milligen) filter according to the manufacturer's protocol, and sequenced on an Applied Biosystems 470/120A gas-phase sequencer using a protocol modified to determine the cycle containing ["P]phosphate [391. The peptide, dissolved in 0.1% (by vol.) formic acid, 50% (by vol.) acetonitrile was also analysed by positive-mode electrospray mass spectrometry on a Fisons VG Quattro instrument. SDSPAGE and detection of proteins in gels. Denaturing polyacrylamide gel electrophoresis was performed by the procedure of Laemmli [40]. After electrophoresis, proteins were detected by Coomassie blue staining, fluorography [41], autoradiography in the case of "P-labelled protein, or Western blot [42] using antipeptide HMGRl antibodies and '"I-protein A. Determination of protein. Protein concentration was determined by the method of Bradford [43] using bovine serum albumin as a standard.

RESULTS Expression of HMGRlcd in E. coli. Plasmid pHMGRlcd contains a fragment of the A. thalianu HMGRl cDNA, coding for amino acids 1666592 of the HMGRl isoform, directly attached to the translation initiation codon of the expression vector pT7.7 (Fig. 1).The encoded protein represents a truncated form of the enzyme comprising the last six amino acids of the linker region and the entire catalytic domain. We therefore refer to it as HMGRlcd. After 6 h induction with isopropyl P-D-thiogalactoside at 22 "C in the presence of [ "S]methioninc and rifampicin,

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B 1

2

Table 2. Kinetic parameters of A. thaliunu HMGRlcd expressed in E. coli and HMGR purified from different plant species. n.d., parameter not determined.

C 3

4

KDa

97

-

K,,, for

Sample source

50 -+66

NADPH

(S)-HMG-CoA

71 5 7 n.d. 27 25 107

8.3 ? 1.5 13 1.5 6.4 10

45

29 -

Fig. 2. Fluorography, SDWAGE and Western blot analysis of the A . thuZiuna HMGRlcd. (A) Analysis by fluorography of the cell-free extract (2 pg protein) from E. coli cells harbouring pHMGR1cd induced in the presence of ['5S]methionine and rifarnpicin. (B) Coomassie blue

A. thulium (this study) Hevea brasiliensis [44] Radish [35] Potato [45] Maize [S]

staining of fractions from each purification step resolved on SDS/PAGE. Lane 1 , cell-free extract (50 pg protein) ; lane 2, ammonium sulfate fraction (60 pg); lane 3, supernatant fraction after 65°C treatment (14 pg); lane 4, Affi-Gel blue fraction (2 pg). (C) Western blot analysis of the Affi-Gel blue fraction (50 ng) using antibodies raised against a C-terminal peptide. The migration of HMGRlcd is shown by the arrow. Numbers on the left indicate the molecular masses of standards and estimated molecular mas?, of the A. thaliana HMGRlcd.

210kDa 1 1 6 kDa 98 kDa 68 kDa

-

45 kDa

-

29 kDa

-

Top of gel

Table 1. Purification of A. thaliana HMGRlcd expressed in E. coli. Results are from a 1-1 culture of E. coli cells transformed with the expression vector pHMGRlcd. Sample source

Protein

Activity

SpePurifi- Yield cific cation activity

Dye front

mg Crude extract Ammonium sullfate precipitate Heat treatment, 65 "C Affi-Gel blue

U

355.8 234 334.2 216 63 330.1 5.5 93.3

U/mg

-fold

%

1.52 1.55 5.24 16.96

1 1.1 3.5 11.2

100 94 93 26

cells harbouring pHMGRlcd synthesized a single labelled polypeptide migrating with an apparent molecular mass of 50 kDa (Fig. 2A) which is in reasonable agreement with the molecular mass predicted from the cDNA sequence (45289 Da). The labelled polypeptide comigrated with a prominent protein band displaying the same apparent molecular mass. (Fig. 2B, lane 1). In addition, very high levels of HMGR activity (1.52 U/mg) were measured in this fraction. E. coli cells containing the expression vector pT7.7 without the HMGRl cDNA fragment showed neither synthesis of the 50-kDa polypeptide nor HMGR activity (data not shown). Based on these criteria, the 50-kDa protein was deduced to correspond to the catalytic domain of HMGRl.

Purification of HMGRlcd. The procedure used for the purification of HMGRcd was based on that reported for the purification of the catalytic domain of rat liver HMGR [46], and is described in detail in Materials and Methods. Samples from each purification step were analyzed by SDS/PAGE (Fig. 2B). As shown in Fig. 2 B (lane 4) the purified HMGRlcd (2 pg protein) migrated as a single band, although minor contaminating bands were observed when gels were overloaded. Table 1 summarizes the results of a typical purification procedure from 1 1 E. coli culture. To further confirm the identity of the purified protein, we performed Western blot analysis of the Affi-Gel blue fraction using antibodies raised against a synthetic peptide representing

Fig. 3. Coomassie-blue-stained gel and autoradiogram of purified HMGRlcd after phosphorylation to >0.9 moVmol 50-kDa subunit using [y-'*P]ATP and HRK-A.

a short amino acid sequence (residues 579-590) of the A. thaliaim HMGRl C-terminus. The antipeptide serum (Fig. 2C), but not pre-immune serum (not shown) recognized the 50-kDa polypeptide in purified HMGRIcd.

Catalytic properties of purified HMGRlcd. The activity of HMGRlcd was analyzed at pH values ranging over 5.0-10.0. Maximal HMGR activity was obtained at pH 7.5. Activity was completely blocked by the HMGR inhibitor mevinolin, with the apparent IC,,, value being 12.5 nM. The effect of varying NADPH and (S)-HMG-CoA concentrations on the rate of the reaction catalyzed by HMGRlcd was also analyzed. The apparent K,, value for NADPH was 71 ? 7 pM and that for (S)-HMGCoA was 8.3 2 1.5 pM. Table 2 shows the apparent K,,, values of the HMGRlcd for both substrates compared with those obtained in previous studies performed either with purified HMGR from radi5h [35], potato [45] and Hevea brasiliensis [44] or partially purified HMGR from maize [ S ] . Phosphorylation and inactivation of HMGRlcd by HRK-A from Brassica oleracea. Fig. 3 shows that HRK-A purified from B. oleracea phosphorylates HMGRl cd. Phosphorylation was rapid and stoichiotnetric, reaching >0.9 mol/mol subunit (Fig. 4). Fig. 4 also shows that there was an excellent inverse correlation between phosphorylation and HMGR activity. The inactivation was caused by the phosphorylation, because it was completely reversed by addition of homogeneous catalytic subunit of bovine protein phosphatase-2A (Fig. 5).

510

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Dale et al. ( E M J . Riochem. 233)

1.o

,- 100

8-

0.8

:- 80

6-

0.6

_ - - _--

4-

0.4

2o

t/

I

20

-

I

40

60

80

-60

- 40

- 20

0.2

V

/

10

20

30

40

50

60

70

Time (min)

Fig. 6. Reverse-phase HPLC analysis of radioactive CNBr peptides derived from "*P-labelledHMGRlcd. The labelled SO-kDa polypepFig.4. HMGR activity (C, 0 ) and phosphorylation (W, 0)during incubation of HMGRlcd with MgATP in the absence (0,O) or pres- tide was excised from an SDS/PAGE gel and the gel slice digested with CNBr. The resulting peptides (monitored by radioactivity) were analysed ence (0, W) of 5U/ml HRK-A. Phosphorylation in the absence of on a C18 column (2SXO.45 cm) at 1 mllmin in 0.1 96 (by vol.) trifluoroA HRK-A was negligible: only the YO-min time point is shown (0). acetic acid and eluted using the indicated gradient from water to acetoniinolecular inass of SO kDa was used for the calculation of stoichiometry of phosphorylation of HMGRlcd. HMGR activity is expressed as the trile. Radioactivity was monitored by Cerenkov counting using a Reeve Analytical on-line monitor. perccntage activity of the control at time zero, which was 0.6.5 U/ml.

E

400 300

200 100

I

I

20

40

60

I

80

Time (min)

Fig. 5. Inactivation of HMGRlcd by HRK-A is reversible. HMGRlcd and presence ( 0 )of 5 U/ was incubated with MgATP in the abscnce (0) ml HRK-A. At the point shown by thc arrow. EDTA (to prevent further phosphorylation) was added to one portion of the incubation, together with homogeneous catalytic subunit of bovine protein phosphatase-2A (W). Aliquots were removed at various times to measure HMGR activity, which is exprewed a\ percentage of the activity in the control at time Lero (100% activity was 0.65 U/inI).

Phosphorylation to > 0.9 mol/mol suggested that, as for the phosphorylation of mammalian HMGR by the AMP-activated protein kinase [ 13, 471, phosphorylation of A . thaliana HMGR by MRK-A occurred at a single site. This was strengthened by results showing that cyanogen bromide digestion of the "P-labellcd 50-kDa polypeptide excised from the SDSPAGE gel resulted in a single radioactive peptide resolvable by reverse-phase HPLC (Fig. 6). This peptide was purified on a microbore reverse-phase HPLC column and subjected to 14 cycles of Edman degradation using both the convcntional pulse liquid protocol, and a solid-phase protocol to determine the location of the "P label. The sequence (Fig. 7) corresponded to residues 573 -587 of the predicted sequence of A. thaliana HMGRI, with the radioactivity all emerging at cycle 5, corresponding to Ser577 in the complete sequence. The pulsed liquid sequencer run gave a blank at this position, with only a very small peak of the Ser derivative. This is the expected result because phosphoserine is broken down to p-alanine and other products during the cleavage step. The phosphopeptide was also analyzed by electrospray mass spectrometry. This gave a series of rnlz peaks consistent with a single molecular species of 2242 Da, corresponding exactly to the predicted mass of the expected C-terminal CNBr peptide of HMGRI (KYNRSSRDISGATTTTTTTT) plus one

CycleNo: Aminoacid:

1 2 3 4 5 6 7 8 9 1011121314

K Y N RS S R D I SG A T T

Fig. 7. Recovery of phenylthiohydantoin derivatives of amino acids (0) and 32Pradioactivity).( during repetitive Edman degradation of CNBr peptide derived from HMGRlcd. The amino acid identified is indicated by its single letter code at the bottom, and the recovery is plotted using a logarithmic scale (left axis). Only trace amounts of the Ser derivative were recovered in cycle 6, and the assignment at this position is based on the recovery of radioactivity in this cycle (phosphoserine breaks down due to p-elimination during sequencing) and the prediction of Ser at this position by the DNA sequence. Radioactivity is plotted using a linear scale (right axis). The amino acid identification and recovery of radioactivity were determined in separate runs, the former using a conventional pulsed liquid sequencer protocol and the latter using a solid-phase protocol (see Materials and Methods).

phosphate group. This result confirms that the expressed protein has the expected C-terminus, and that the CNBr peptide contains a single phosphate group.

DISCUSSION Plant HMGR is known to be regulated by a number of developmental and environmental stimuli [2, 4, 91. Although it has been reported that plant HMGR is regulated both at transcriptional and post-transcriptional levels 141,the molecular mechanisms controlling the enzyme activity are largely unknown. For many years the kinetic and molecular characterization of plant HMGR has been hampered by difficulties in obtaining large amounts of pure enzyme from plant sources [2, 51. Moreover. the occurrence of multiple HMGR isoforms in plants [4] hinders the isolation of individual isoforms by using conventional protein purification techniques starting from plant tissues. This fact

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Dale et al. (Eur J. Biochenz. 2331

represents a further complexity for the identification of the role of each of the plant HMGR isoforms in the overall control of plant isoprenoid biosynthesis. An approach to overcome these difficulties is the production of large amounts of the individual HMGR isoforms through the expression of the corresponding cDNA clones in a heterologous system. In a previous paper [48] we reported that the expression in E. coli of a radish HMGR isoform, comprising both the membrane and the catalytic domains, produced an insoluble and catalytically inactive enzyme. This behaviour was attributed to the presence of the two highly hydrophobic sequences within the membrane domain. However, the expression of a truncated form of the radish enzyme, containing only the catalytic domain, resulted in the production of high levels of soluble and active enzyme (0.093 U/mg). Since our objective was to obtain large quantities of pure and fully active A. thulium HMGR to enable its further characterization, we elected to express only the catalytic domain of the enzyme in order to overcome the solubility problem. The expressed polypeptide, named HMGRlcd, contained six amino acid residues of the linker region followed by the entire catalytic domain. As expected, very high levels of HMGR activity (1 .52 U/mg) were produced in the bacterial system after 6 h induction with isopropyl p-D-thiogalactoside. For the purification of the A. thalianu HMGRl cd we adapted the procedure previously described for the purification of mammalian HMGR 1461. The high efficiency of the bacterial expression system, together with the simplicity of the purification procedure, resulted in the attainment of large quantities of pure enzyme (about S mg/l culture), with a specific activity of = 17 U/ mg, this is higher than any other report we can find for HMGR purified from a plant source [35, 44, 451. As yet, all eukaryotic and prokaryotic HMGR enzymes are inhibited by mevinolin, a highly specific competitive inhibitor of HMGR [49]. The activity of the purified HMGRlcd is also completely blocked by mevinolin, with an IC,,, of 12.5 nM. In addition, the purified polypeptide is recognized by antibodies raised against a synthetic peptide reproducing a sequence (residues 579-590) close to the C-terminus of A. thulium HMGRI (Fig. 2C). Taken together, these results unequivocally demonstrate that the purified 50-kDa polypeptide (Fig. 2B, lane 4) corresponds to the fully active catalytic domain of A. thuliana HMGRl. As shown in Table 2, no significant differences are observed between the K,,, values for NADPH and (S)-HMG-CoA of bacterially expressed A. thaliunu HMGRlcd, and those of pure HMGR preparations obtained from a number of different plant sources. Thus, our results show that the bacterial expression system and the purification procedure adopted in this study are suitable for the isolation of very large amounts of catalytically active A. tkaliana HMGRlcd, thereby allowing the functional and structural analysis of the enzyme. This expression system should prove useful for the purification and characterization of the catalytic domain of other plant HMGR isoforms. Our results confirm that a higher plant HMGR is regulated by direct phos;phorylation, at least in a cell-free system. The HMG-CoA reductase kinases from B. oleracea (HRK-A and HRK-B) were originally isolated using as substrate a synthetic peptide derived from mammalian acetyl-CoA carboxylase [31 , 321. We had previously shown that HRK-A would also phosphorylate and inactivate mammalian HMGR at Ser871, the residue phosphorylated by mammalian AMP-activated protein kinase [31]. In addition, it inactivated a crude preparation of potato HMGR, an effect that was time-dependent, ATP-dependent and was reversed by adding purified protein phosphatase [31]. We therefore adopted the name HMG-CoA reductase kinase, since it seemed likely that HMGR would be a direct substrate, and no other substrates were known. Nevertheless it remained formally

Consensus motifs: Chinese hamster A. thaliana HMG1 A. thaliana HMG2 Tomato 1 Tomato 2 Tomato 3 Pea 1 Pea 2 Pea 3 Hevea brasiliensis 7 Hevea brasiliensis 2 Hevea brasiliensis 3 Radish Nicotiana sytvestris Catharanthus roseus Potato Rice Camptotheca acuminata

i

t

@XpxxsXXX@ @rnSXXX@

m S K I N L Q D L HEMyNRSSR D I SGA WYNRSSRDISGP WYNRSIKDISQV WYNRSTKDWKA WYNRSSKDVTK WYNRSSRDITKI WYNRSSKDVTKI WYNRCKDVSKV

Wm&WDHSKA HHIiyNRSSKDVSKA mYNRSAKDVSK1 H13yyNRsSRDI SGA WYNRSTKDWKA IIMLiyNRSSKDITNI WYNRSIKDISK HNMyNRSsKDvm

-SNKDVTKA

Fig. 8. Alignment of two consensus recognition motifs for HRK-A [51] and relevant regions of sequences of HMGR from one mammalian source (Chinese hamster) and 17 higher plant isoforms. In the consensus motifs, @ represents a residue with a bulky hydrophobic side chain (M, V, L, 1 or F) and hydrophobic residues in HMGRs which align with these are shown in bold type. represents a basic residue (R, K or H) and basic residues in HMGRs which align with these are underlined. The phosphorylated serine is in bold and underlined, and marked with an arrow. All HMGR sequences may be obtained from the EMBL, GENBANK or SWISSPROT databases.

possible that the inactivation of potato HMGR was due to phosphorylation of some other protein whose phosphorylation state indirectly affected the activity of HMGR. The results in this paper conclusively demonstrate that the inactivation is due to direct phosphorylation. Fig. 8 compares the sequence around the site phosphorylated on HMGRI with the consensus recognition motif for HRK-A and HRK-B, defined using synthetic peptide substrates [32, SO]. Also compared are the equivalent sequences around the phosphorylation site on a mammalian HMGR (Syrian hamster) and 16 other plant HMGRs, including HMGR2 from A. thaliana. Like its mammalian homologue AMP-activated protein kinase, HRK-A requires a hydrophobic residue (@) at P-5 and P + 4 (i.e. five residues N-terminal and four residues C-terminal to the phosphorylated Ser), plus at least one basic residue (Arg, Lys or His: represented by /l in Fig. 8) between the phosphorylated hydrophobic residue and the Ser. Recently we have refined this analysis with a new set of peptides [SI] and shown that HRKA requires the Arg to be at P-4 or P-3, although the latter is preferred. The sequence around Ser577 in A. thaliana HMGRl fits this consensus perfectly, with Met at P-5, Lys at P-4 and Ile at P+4. With the exception of rice (Oryia sativa, where the sequence has not yet been confirmed at the amino acid level), all other higher plant HMGRs also contain this motif, with Met at P-5, Lys at P-4, and Ile, Val or Met at P+4. One would therefore expect all of these HMGR isoforms in all of these species, both mono- and di-cotyledonous, to be regulated by HRK-A. Due to the lack of sufficient material, we were unable to do the same analysis with other combinations of HMGR and HRK isoforms, i.e. HMGR2 and HRK-B 1311. However the bacterially expressed catalytic domain of A. thaliarza HMGR2 was phosphorylated by HRK-A (data not shown) and the recognition mo-

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tif for the protein kinase is perfectly conserved in the predicted sequence of HMGR2 (Fig. 8). In addition, HRK-A and HRK-B recognize very similar motifs [32], so that HRK-B would be expected to phosphorylate both H M G R I and HMGR2. All animal HMGRs sequenced to date (from humans to Drosophila melumguster) also contain a form of this motif near the C-terminus, although interestingly they have His at P-3 rather than Lys at P-4. This is consistent with the finding that the recognition motifs for animal AMP-activated protein kinase and plant HRK-A are very similar [50, 511. Phosphorylation site sequences have been determined previously for a small number of plant proteins, including phosphoenolpyruvate carboxylase and sucrose phosphate synthase 152, 531. However the latter enzymes are not found in animals, and HMGR may represent the first documented case where a regulatory phosphorylation site motif has been conserved over the billion years since the divergence of animal and plants. This reinforces our view that the AMP-activated protein kinase/SNFl family is an ancient and highly conserved protein kinase system 1541. The histidine at P-6 (His571 in A. thulium H M G R I ) is conserved in all HMGRs sequenced to date, even in the yeast Sacchuromyces cerevisiae and eubacteria and archaebacteria, where the phosphorylation site is not conserved. This residue (corresponding to His865 in the Syrian hamster enzyme) is essential for activity in bacterial and mammalian H M G R ; Rodwell and coworkers have proposed that it acts as an acid-base catalyst which protonates the Co-A anion released during the hydrolysis of the mevaldyl-CoA intermediate 155, 561. They further propose that phosphorylation of the Ser side chain six residues C-terminal to this His (Ser871 in the Syrian hamster enzyme) inhibits the enzyme by the formation of an electrostatic interaction between the phosphate group and the His side chain, preventing the latter from acting as a proton donor [571. Since the present work shows that the six-residue spacing between this essential His and the phosphorylated Ser is conserved in higher plants, this mechanism would also explain the inactivation of HMGRs in plants. These studies were supported by an Agricultural & Food Research Council project grant (to DGH), a Medical Research Council research studentship (to SD) and grants PB90-0492 and PB93-0753 from the Direccirin General de lnvextigacidn Cientqica y Te'cnicu (to AB). We gratefully acknowledge Joaquim Ros for assistance in the preparation of antibodies, David Andreu for peptide synthesis, Dirk Zahn for the construction of the expresion plasmid pHMGRlcd and Angela Mehlert for the mash 5pectrometric analysis.

REFERENCES 1. Gray. J. C. (1987) Arlv. Rotn. Res. 7. 25-91. 2. Bach, T. J., Wettstein, A., Boronat, A,, Ferrer, A,. Enjuto, M.. Gruissem, W. & Narita. J. 0. (1991) in Physiology and hiochemistq @plant sterols (Patterson, G. W. & Nes, W. D., eds) pp. 29-49, American Oils Chemical Society, Champain IL. 3. Bach. T. J. (1995) Lipids, in the press. 4. Stermer, B. A,, Bianchini. G. & Korth, K. L. (1994) J . Lipid Rex 35. 1133-1140. 5 . Bach, T. J., Motel, A. & Weber, T. (1990) in Biochemistry of the nzevalonic acid pat1ipv.c.n):to terpenoids (Towers, G. H. N. & Stafford, H. A,, eds) vol. 24, pp. 1-82, Plenum Press, New York. 6. Caelles. C., Ferrer, A,, Balcells, L., Hegardt. F. G. & Boronat, A. (1989) Plant Mol. Biol. 13, 627-638. 7. Enjuto, M., Balcells, L.. Campos, N., Caelles, C., Arr6, M. & Boronat. A. (1994) Proc. Natl Acurl. Sci. USA 91, 927-931. 8. Monfar, M., Caelles, C.. Balcells, L., Ferrer, A,, Hegardt, F. G. & Boronat, A. (1 990) in Biochemisfp of the nievalotiic acid pathwwy fo tr/pnoid.s (Towers, G. H. N. & Stafford. H. A., eds) vol. 24. pp. X1-97. Plenum Press, New York.

9. Bach, T. J., Boronat, A,, Caelles, C., Ferrer, A,, Weber. T. & Wettstein, A. (1991) Lipids 26, 637-648. 10. Sipat, A. B. (1982) Phyfochemistry 21, 2613-2618. 11. Russell, D. W., Knight, J. S. & Wilson, T. M. (1985) in Current topics in plant biochemistry and physiology (Randall, D. D., Blevins, D. G. & Larson, R. L., eds) vol. 4, pp. 191-206, University of Missouri-Columbia. 12. Clarke, P. R. & Hardie, D. G. (1990) FEBS Lett. 269, 213-217. 13. Clarke, P. R. & Hardie, D. G. (1990) EMBO J. 9, 2439-2446. 14. Sato, R., Goldstein, J. L. & Brown, M. S. (1993) Proc. Natl Acrtd. Sci. USA 90, 9261-9265. 15. Ferrer, A., Caelles, C., Massot. N. & Hegardt, F. G. (1985) Biochem. Biophy.s. Res. Commun. 132. 497-504. 16. Ferrer, A,, Caelles, C., Massot, N. & Hegardt, F. G. (1987) J. Biol. Chem. 262, 13507-13512. 17. Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur: J. Biochem. 186, 129-136. 18. Moore, F., Weekes, J. & Hardie, D. G. (1991) Eul: J. Biochem. 199, 691 -697. 19. Weekes, J., Hawley, S. A., Corton. J., Shugar, D. & Hardie, D. G. (1994) Eur: .I. Biochem. 219, 751-757. 20. Corton, J. M., Gillespie, J. G. & Hardie, D. G. (1994) Curr: Biol. 4, 315 -324. 21. Carling, D., Aguam, K., Woods, A,, Verhoeven, A. J. M., Beri, R. K, Brennan, C. H. Sidebottom. C., Davison, M. D. & Scott, J. (1994) J. Biol. Chem. 269, 11442- 11 448. 22. Mitchelhill, K. I., Stapleton, D., Gao. G., House, C., Mitchell, B., Katsis, E, Witters, L. A. & Kemp. B. E. (1994) J. B i d . Chem. 269, 2361 -2364. 23. Celenza, J. L. & Carlson, M. (1986) Science 233, 1175-1180. 24. Ciriacy, M. (1077) Mol. Gen. Genet. 154. 213-220. 25. Zimmermann, F. K., Kaufmann, I., Rasenberger, A. & Haussman, P. (1977) Mol. Gen. Genet. 151, 95-103. 26. Thompson-Jaeger, S., Francois, J.. Gaughran, J. P. & Tatchell. K. (1991) Genetics 12Y, 697-706. 27. Muranaka, T., Banno, H. & Machida, Y. (1994) Mol. Cell. B i d . 14, 2958-2965. 28. LeGuen, L., Thomas, M., Bianchi, M., Halford. N. G. & Kreis, M. (1992) Gene 120, 249-254. 29. Halford, N. G., Vicente-Carbajosa, J., Sabelli, P. A,, Shewry, P. R., Hannapel, LJ. & Kreis, M. (1992) Plant J. 2, 791 -797. 30. Alderson, A., Sabelli, P. A,, Dickinson, J . R., Cole, D., Richardson, M., Kreis, M., Shewry, P. R. & Halford, N. G . (1991) Pro