Rubisco Small and Large Subunit N-Methyltransferases BI- AND MONO-FUNCTIONAL METHYLTRANSFERASES THAT METHYLATE THE SMALL AND LARGE SUBUNITS OF RUBISCO*

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the a-amino group of the Nterminal methionine of the processed form of the small subunit (SS), and at the e-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both Nmethylation of the SS and N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the a-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the ␣-amino group of the Nterminal methionine of the processed form of the small subunit (SS), and at the ⑀-amino group of lysine-14 of the large subunit (LS) in some species.The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco.We determine here that both pea and tobacco Rubisco LSMT also exhibit ␣ N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both ␣ Nmethylation of the SS and ⑀ N-methylation of the LS.A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco.Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the ␣-amino group of the N-terminal methionine of the SS of Rubisco.Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) experiences several types of post-translational modifications during the expression, import, and assembly of the protein.The large subunit (LS) 1 is encoded by a plastid gene and is trans-lated and assembled into Rubisco holoenzyme within the stroma of the chloroplast.Mass spectral and amino acid sequence analysis of peptides prepared from Rubisco has demonstrated that the LS is processed to the mature form by removal of the N-terminal Met-1 and Ser-2 residues and acetylation of Pro-3 (1,2).In addition the LS from many species, but not all, contains a trimethyllysyl residue at Lys-14 (2,3,4).The small subunit (SS) of Rubisco is also post-translationally modified.The polypeptide is post-translationally imported into chloroplasts and processed by a stromal processing peptidase that removes the targeting presequence.The resultant N-terminal methionine residue of the processed SS is subjected to N-methylation (5) prior to assembly with the LS into the holoenzyme.
The observation that LS polypeptides from some species are methylated at Lys-14, whereas methylation is not detected in species such as spinach is a curious observation.Preliminary studies indicated that spinach contained LSMT-like DNA sequences.In this study we report a homologue of Rubisco LSMT from spinach that encodes an enzyme that methylates the SS of Rubisco (Rubisco SSMT).In addition, pea and tobacco Rubisco LSMT are demonstrated to catalyze ␣-methylation of the processed form of Rubisco SS.Thus, pea Rubisco LSMT is a bifunctional enzyme that catalyzes methylation of both the ␣-amino group of the SS and the ⑀-amino group of Lys-14 of the LS.This is the first reported eukaryotic ␣ N-methyltransferase and of a bifunctional methyltransferase that methylates both ␣and ⑀-amino groups.

EXPERIMENTAL PROCEDURES
Plant Material-Spinach (Spinacea oleracea L. cv.Melody) plants were cultured in ProMix TM soil medium in a greenhouse at approximately 20 °C with a natural light photoperiod during the winter season (Lexington, Kentucky).
Cloning and Sequencing of rbcMT-S cDNAs-rbcMT-S cDNAs were obtained by reverse transcription-polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE).Total RNA was isolated from spinach leaves using Trizol (Life Technologies, Inc.).Reverse transcription-PCR was performed by reverse-transcription of 5 g of total spinach RNA with an oligo d(T)17 primer.The first-strand cDNA product was amplified by PCR with Taq polymerase (Life Technologies, Inc.) using degenerate oligonucleotide primers to conserved peptide sequences between pea (6) and tobacco (7) Rubisco LSMTs.The forward primer (SF-8, 5Ј-CGA TGG GCA TTT GGA ATT CTC AGA TCA AGG GC) included an EcoRI site and corresponded to the amino acid sequence WAFGILRSRA.The reverse primer (SR-2, 5Ј-GGC CAA GGC CAA GAT CTT TAA GCC TCC TTT C) included a BglII site and corresponded to peptide sequence ERRLKDLGLA.Conditions for PCR were 35 cycles of: 94 °C 1 min, 50 °C 1 min, and 72 °C 1.5 min and a final extension of 72 °C 10 min.The PCR product was digested with EcoRI and BglII, cloned into Bluescript II KS(ϩ) vector (Stratagene), and designated pS25.
RACE was performed to obtain 5Ј cDNA sequences as described above except an rbcMT-S-specific primer (SR-3, 5Ј-ACT GTA ACC AGA AAG AGC AGA TGT ACA) primed cDNA synthesis in the mid-coding region and was followed by poly(dC) tailing as described previously (9).The dC-tailed products were amplified using a nested primer (SR-5, 5Ј-G TTC TCT AGA GAA TAG ACC AGC TCC), which included a XbaI restriction site and a poly(dG/dI)-containing oligonucleotide.The PCR products were digested with SacI and XbaI, gel purified, and cloned into Bluescript II KS(ϩ) vector for sequencing.Two different spinach cDNAs were identified: as a long form (pS40) with a 12-bp insertion and as a short form (pS38).
For 3Ј RACE, 5 g of total RNA from spinach leaves was reversetranscribed with an adapter-primer (AP-1, 5Ј-GGC CAC GCG TCG ACT AGT ACT (T)16).Amplification was performed by PCR as described above with oligonucleotide AP-1 and a spinach-specific primer, (SF-9, 5Ј-CAA TAC GAT TTG AAC AAG AGC AAT GC).The PCR product was cloned into pCR-Scrip Direct SK(ϩ) vector (Stratagene) for sequencing and designated pS2.
Two to five independent clones were chosen for sequencing from each of the above constructs.Both strands of each clone were sequenced by the dideoxy chain termination method (10) using Sequenase (U.S. Biochemical Corp.) and 35 S-dATP (NEN Life Science Products) with M13 reverse and Ϫ40 primers.In addition, 18 -27-mer oligonucleotides were synthesized from DNA sequence obtained and used as primers for additional sequencing.
Both full-length S-38 and S-40 cDNAs (accession numbers AF071544 and AF071545, respectively) were obtained by ligation of clones pS2 and pS25 into pS38 or pS40 based on restriction sites within the overlapped regions.N-terminally truncated forms of S-38 and S-40 were produced by deletion of the putative chloroplast transit peptide through PCR with primers designed to remove the first 43 amino acids and replace Cys-44 with a Met.Both full-length and N-terminally truncated forms of S-38 and S-40 were cloned into pET-23d for expression in pLysS cells.
Isolation and DNA Gel Blot Analysis of the rbcMT-S-The rbcMT-S gene (accession number AF071543) was cloned by PCR.Spinach nuclear DNA was isolated using Floraclean (Bio101, Inc.).Approximately 100 ng of the nuclear DNA was amplified by PCR with Taq polymerase (Life Technologies, Inc.) using a forward primer (SF-1, 5Ј-AGA AGC TTC ACC ATG GCA ACT TTA TTC ACT CTC ATC) and a reverse primer (SR-1, 5Ј-AGT TTG CAT TTT GAT TGT AGT TTC CAC).The PCR product was cloned into pCR-Script SK(ϩ) for sequencing.
RNase Protection-Clone pS40XR0.4 was transcribed with T3 RNA polymerase in the presence of [ 32 P]ATP (800 Ci/mmol) to produce a radiolabeled antisense transcript.The radioactive probe (ϳ100,000 cpm) was precipitated with 5 g of tRNA and 0, 2.5, 10, or 20 g of total RNA isolated from mature spinach leaves.A heterologous RNA (10 g of maize mitochondrial RNA) was included as a control.The RNAs were dissolved in 10 l of hybridization buffer (80% formamide, 40 mM PIPES-NaOH, pH 6.4, 400 mM NaCl, 1 mM EDTA), denatured, and allowed to hybridize at 45 °C overnight.The duplexes were digested by addition of 0.3 ml of RNase digestion buffer (10 mM Tris-HCl, pH 7.6, 300 mM NaCl, 5 mM EDTA) with 200 units of RNase T1 (Roche Molecular Biochemicals) and 250 ng RNase A (Sigma).The duplexes were digested for 1 h at 30 °C, and the reaction terminated by addition of 100 g of proteinase K and SDS to a final concentration of 0.6% and incubation for 30 min at 37 °C.The protected fragments were electrophoresed on 6% polyacrylamide gels with 8 M urea.Gels were dried and analyzed by autoradiography.
Rubisco SSMT Activity Assays, Immunoblot, and Purification Procedures-Full-length and N-terminally truncated forms of S-40 and S-38 were cloned and expressed with the pET-23d vector in pLysS host cells.Bacterial cells were cultured at 37 °C for 3.5 h in 5 ml of LB broth with 50 g/ml carbenicillin and 35 g/ml chloramphenicol.After induction with 0.5 mM IPTG, cultures were incubated for 2.5 h at 25 °C or 37 °C, and cells were collected by centrifugation at 5000 ϫ g for 5 min at 4 °C.The cells were washed twice with deionized water and resuspended in 100 l of buffer A (50 mM Tris-HCl, pH 8.2, 5 mM MgCl 2 , 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml leupeptin) and fro-zen at Ϫ80 °C.The activity of Rubisco LSMT was determined in the thawed cell lysates as described previously (11).Rubisco SSMT activity was determined by incubation of pLysS cell lysates, or purified S-38 and S-40, with lysates from spinach chloroplasts (12).Reactions contained 2.5-5 Ci of [ 3 H-methyl]AdoMet (70 -80 Ci/mmol), 10 -20 g of chloroplast lysate protein, and either 10 g of protein from pLysS cell lysates or 1 g of purified S-38 or S-40 protein.After incubation at 30 °C for 30 min, reactions were terminated by addition of SDS-PAGE sample preparation buffer, and samples were electrophoresed on 15% acrylamide gels.After transfer of proteins to a PVDF membrane, radioactivity was visualized using a 3 H PhosphorImager screen (Molecular Dynamics model 425, Sunnyvale, CA), and polypeptides were visualized by staining with Coomassie Brilliant Blue R-250.Alternatively, the PVDFblotted proteins were subjected to Immunoblot analysis using antibodies prepared against pea Rubisco LSMT or S-38 protein.
Full-length and N-truncated forms of S-38 and S-40 were purified from pLysS cell lysates.pLysS cells were cultured at 37 °C, and inclusion bodies were harvested after cell lysis and washed twice with buffer A before dissolving in 6 M urea.Cell debris was removed by centrifugation (5 min, 12,000 ϫ g), and the remaining supernatant was dialyzed against buffer A for 4 h.The dialysate contained 1-2 mg/ml purified protein and was used directly in activity assays and to elicit antibodies (Charles River PharmServices, Southbridge, MA).

RESULTS
Isolation of rbcMT-S cDNAs-Degenerate oligonucleotide primers were designed to highly conserved sequences of pea and tobacco Rubisco LSMT and were used to amplify a 786-bp cDNA fragment from total spinach leaf RNA.DNA sequence analysis of the 786-bp fragment indicated that the spinach cDNA had extensive sequence identity with pea and tobacco LSMT.A complete cDNA sequence of the spinach cDNA was obtained by 5Ј and 3Ј RACE of spinach leaf RNA (13).
Amplification of 5Ј and 3Ј coding sequences was performed by RACE.PCR amplification resulted in the identification of two 5Ј products (836-and 848-bp fragments).The 848-bp product had a 12-bp insertion relative to the 836-bp fragment.The 5Ј RACE products and the initial PCR product amplified with LSMT-specific primers had complete sequence identity in the regions that overlapped, except for the 12-bp insertion.Amplification of 3Ј coding sequence was performed by RACE, and a single 761-bp PCR product was obtained.Complete cDNA sequences have been assembled from these overlapping clones of the rbcMT-S and have been submitted to GenBank TM as accession numbers AF071544 and AF071545.
The spinach rbcMT-S cDNAs encode polypeptides of 495 (S-40) and 491 amino acids (S-38) with predicted molecular masses of approximately 55 kDa, similar to the masses of pea (55.0 kDa) and tobacco (56.0 kDa) Rubisco LSMT (Fig. 1).In addition, the deduced amino acid sequence of the spinach cDNAs included five imperfect copies of a leucine-rich repeat (14), similar to pea and tobacco LSMT (7).The deduced amino acid sequence from the spinach rbcMT-S cDNAs shows 60 and 62% identity with the amino acid sequences of pea and tobacco Rubisco LSMT, respectively (Fig. 1).
Transcription and Expression of rbcMT-S-A 3.1-kilobase genomic clone that included the entire coding region of the spinach rbcMT-S gene was obtained and sequenced (Gen-Bank TM accession number AF071543).The spinach rbcMT-S gene included six exons and five introns and has similar genomic organization with the tobacco Rubisco LSMT gene (rbcMT-T).DNA gel blot analysis indicated that the rbcMT-S is a single copy gene in spinach (data not shown).
The 12-bp insertion in the rbcMT-S S-40 cDNA corresponds to the 3Ј 12 nucleotides of intron III (Fig. 2).RNase protection studies were performed to analyze the relative expression of the S-38 and S-40 mRNA in spinach leaves.A 390-nucleotide subclone of the S-40 cDNA was prepared that spanned exons 3 and 4 (Fig. 3A).The S-40 mRNA (long form) would protect the entire 390-nucleotide region, whereas S-38 mRNA (short form) would protect 306-and 72-nucleotide fragments.A 460-nucleotide antisense probe was produced by transcription with T3 RNA polymerase (Fig. 3B, lane 1) and hybridized to total spinach leaf RNA.Digestion with RNase A and T1 revealed that the S-40-specific fragment (390 nucleotides) and the S-38-specific fragments (306 and 72 nucleotides) were present in approximately equal abundance (lanes 3-5).Control reactions included omission of spinach leaf RNA (lane 2) or substitution of heterologous RNA (maize mitochondrial RNA; lane 6), and these reactions failed to protect any fragments.Both S-40 and S-38 mRNAs were not detectable in total RNA from spinach roots, stems, or flowers (data not shown).Thus, alternative 3Ј splice site selection produces two transcripts for the rbcMT-S gene that are expressed at similar abundance in spinach leaves.
Examination of the N-terminal sequence of S-38 and S-40 bodies were prepared against the purified N-terminally truncated S-38 and used to detect spinach SSMT polypeptides.The SSMT antibody recognized a single polypeptide from spinach chloroplast lysates at a molecular mass expected for the processed form of S-38 (ϳ50.6 kDa; Fig. 4).It was not possible to distinguish between S-38 and S-40 protein forms on SDS-PAGE because the expected molecular mass shift is only 560 daltons.Duplicate protein blots were probed with antibody raised against Rubisco, which demonstrates that the single immunoreactive band observed with S-38 antibodies is not due to cross-reactivity to the LS of Rubisco (Fig. 4).
Spinach rbcMT-S Catalyzes Methylation of the ␣-Amino Group of the Processed Form of the SS of Rubisco-The presence of chloroplast-localized translation products for S-38 and S-40 with homology to both pea and tobacco Rubisco LSMT suggested that these proteins may have a different but related type of methyltransferase activity.Pea Rubisco LSMT catalyzes incorporation of tritiated methyl groups from [ 3 H-methyl] AdoMet into Rubisco holoenzyme isolated from unmethylated species such as spinach (11).Truncated S-38 and S-40 proteins exhibited no methyltransferase activity toward purified spinach Rubisco or toward purified Rubisco from other plant species.A recent report of ␣-methylation of the N terminus of the processed form of the SS of Rubisco ( 5) lead us to examine the possibility that the S-38 and S-40 cDNAs coded for an ␣ Nmethyltransferase for the SS of spinach Rubisco.N-terminally truncated S-38 and S-40 recombinant proteins were assayed for methyltransferase activity with spinach chloroplast lysates as a proteinaceous substrate (Fig. 5).Tritium radiolabel incorporation from [ 3 H-methyl]AdoMet into spinach chloroplast polypeptides was analyzed by SDS-PAGE, electrophoretic transfer to PVDF membranes, and PhosphorImager analysis (Fig. 5A).Incorporation of radiolabel was detected into a region corresponding to the SS of Rubisco (Fig. 5A) that was dependent on the addition of the chloroplast lysate.A small amount of label incorporation was observed in the region of the SS in the absence of IPTG but was dramatically increased with the addition of IPTG and is a consequence of leaky expression of the pET-23d construct.No radiolabel was observed in pLysS cell lysates or chloroplast lysates alone.Both S-38 and S-40 were capable of catalyzing methylation of the SS (Fig. 5A).Highly purified S-38 was obtained from inclusion bodies and refolded as a soluble protein and was shown to exclusively catalyze transfer of radiolabel into the region corresponding to the SS of Rubisco (Fig. 5B).Similar results were obtained with purified S-40 (data not shown).These results suggest that a small pool of free SSs exists in the stromal fraction from spinach chloroplasts that function as a substrate for methylation.A pool of free SSs has been previously demonstrated in chloroplasts from Chlamydomonas (17) and peas (18).
Unequivocal demonstration that purified recombinant S-38 catalyzed SS methylation at the N-terminal Met of processed SS was documented by Edman degradation of the SS with simultaneous determination of radioactivity.During the first cycle of Edman degradative sequencing, 92% of the radiolabel incorporated into the small subunit region of the PVDF blot was released (Fig. 6).The amino acid residue in this cycle did not correspond to any of the standard amino acids but migrated as a unknown with a retention time just slightly greater than N-diphenylurea, similar to that reported for methylated Met in other work (5).The next 11 amino acid residues corresponded exactly to the known N-terminal residues of the SS of spinach Rubisco (19).Given that a thorough chemical and physical structural analysis of the N-terminal residue of the SS of spinach Rubisco has already identified this residue as N-methyl-methionine, these results demonstrate that the S-38 and S-40 cDNAs for the rbcMT-S gene encode enzymes that can methylate the N-terminal Met residue of the small subunit of

FIG. 5. S-38 and S-40 Expression-dependent incorporation of 3 H-radiolabel from [ 3 H-methyl]AdoMet into the small subunit of
Rubisco in spinach chloroplast lysates.A, lysates (ϳ15 g of total protein) from induced (ϩIPTG) and non-induced (ϪIPTG) pLysS cells harboring S-38 or S-40 cDNAs cloned into pET23d expression vector were incubated in the presence and absence of spinach chloroplast lysates (ϳ12 g of total protein) and [ 3 H-methyl]AdoMet (1.8 M, 70 -80 Ci/mmol).After incubation at 30 °C for 30 min, samples were electrophoresed on 15% SDS-PAGE gels and electrophoretically transferred to PVDF membranes.The PVDF membranes were briefly stained with Coomassie Brilliant Blue R-250 and then destained with 100% methanol, washed with distilled water, and imaged for radioactivity.LS and SS refer to the locations of the large and small subunits of Rubisco, respectively.B, purified S-38 (ϳ2 g) was incubated in the presence and absence of spinach chloroplast lysates (ϳ18 g of total protein) and [ 3 H-methyl]AdoMet (1.8 M, 70 -80 Ci/mmol) for 30 min at 30 °C.After electrophoresis and transfer, the PVDF membrane was stained and imaged as described above.FIG. 6. Radiosequencing of the spinach rubisco SS.A spinach chloroplast lysate containing 100 g of total protein was incubated with purified S-38 protein (10 g) and [ 3 H-methyl]AdoMet (1.8 M, 70 -80 Ci/mmol) for 30 min at 30 °C.The sample was electrophoresed on a 15% SDS-PAGE gel, and proteins were electrophoretically transferred to a PVDF membrane.The PVDF membrane was briefly stained with Coomassie Brilliant Blue R-250 and then imaged for radioactivity, and the protein band at the position of the SS of Rubisco was subjected to Edman degradative sequencing.During each cycle of sequencing a portion of the phenylthiohydantoin-derivatized amino acid pool was diverted prior to separation by high pressure liquid chromatography for determination of radioactivity.Amino acid residues identified are indicated, and all other residues were Ͻ5 pmol.LS and SS refer to the locations of the large and small subunits of Rubisco, respectively.The sequence obtained, identified at the bottom of the figure, is identical with the sequence reported for the SS of spinach Rubisco beginning with the second lysyl residue (19).The first residue released during sequencing, identified as a modified Met residue (indicated by an asterisk in the sequence bar graph) migrates slightly behind N-diphenylurea and is consistent with the sequence and recently reported mass spectroscopy identification of methyl-N-methionine as the N-terminal residue of the SS of spinach Rubisco (5).
Rubisco.We propose that these enzymes be referred to as Rubisco small subunit ␣ N-methyltransferase or Rubisco SSMT.
Pea and Tobacco Rubisco LSMT Exhibit SSMT Activity-The high degree of sequence identity between spinach SSMT and pea and tobacco Rubisco LSMT as well as the evidence for SS methylation in these species (Ref.5 and this work) suggest that the previously characterized pea and tobacco Rubisco LSMT may also possess SSMT activity.Purified pea chloroplast Rubisco LSMT and pea and tobacco recombinant LSMT bacteria were tested for N-methyltransferase activity toward the SS of spinach Rubisco.Pea LSMT efficiently methylated both SS and LS of Rubisco from spinach chloroplast lysates when assayed with high specific activity [ 3 H-methyl]AdoMet (Fig. 7).Control reactions included cell lysates from noninduced (without IPTG) conditions and spinach chloroplast lysates alone and did not show radiolabel incorporation into the SS of spinach Rubisco.Earlier methylation experiments (3) were performed with purified Rubisco; however, assembled Rubisco is apparently a poor substrate for ␣-methyltransferase reactions.Tobacco Rubisco LSMT had not been previously demonstrated to catalyze LS methylation and in these assays only catalyzed radiolabel incorporation into the SS of spinach Rubisco.Thus, the pea, spinach, and tobacco enzymes catalyze ␣-amino group methylation of the SS, whereas the pea enzyme is bifunctional and also methylates Lys-14 in the LS.

DISCUSSION
Rubisco is subjected to several forms of post-translational modifications, and these reactions include removal of two Nterminal amino acid residues and acetylation of Pro-3 of the LS (1, 2); ⑀-methylation of Lys-14 of the LS (2); proteolytic cleavage of the SS by the stromal processing protease (20); and ␣-methylation of the Met-1 of the processed form of the SS (5).This paper demonstrates that the ␣-methylation reaction of the SS is catalyzed by enzymes in spinach and tobacco that are closely related to the previously characterized LS methyltransferase from pea.In addition, the pea enzyme has been shown to be bifunctional and possesses the capacity to methylate both the ␣amino group of Met-1 of the SS and the ⑀-amino group of Lys-14 of the LS.
The rbcMT-S gene is present as a single copy in the spinach genome, and two splice forms of the mRNA are detected by PCR and by RNase protection.The two forms apparently result from alternative 3Ј splice site selection since: 1) DNA gel blot analysis indicated that the cDNA probe sequence is a single copy in the spinach genome; 2) sequence analysis of S-40 and S-38 cDNA clones revealed identical DNA sequence; and 3) RNase protection analysis detected both splice variants in similar abundance.S-40 transcripts include a 12-bp insertion that would result in an in-frame insertion of WVQQ into the polypeptide.The functions of the two forms of the spinach SSMT are currently unknown.Both long and short forms efficiently catalyzed SS methylation, but the previously characterized pea and tobacco methyltransferases lack the WVQQ insertion.It is possible that these proteins have different specificities and participate differently in various methylation reactions.
Both 3Ј splice sites of the spinach SSMT gene are typical 3Ј splice sites.The downstream (S-40) splice site has a typical 3Ј splice boundary (CAG*GC) and compares well with the dicot consensus (YAG*GU) (21,22).In addition, an upstream polypyrimidine stretch (UAUUUAUUU) is present, although the stretch extends 23 nucleotides upstream of the splice site and is perhaps somewhat further upstream than typical in plants.Possible branch sites exist at the A residues, which are 21 and 37 nucleotides upstream of the splice site.The short form (S-38) splice boundary (AAG*UG) includes the highly conserved AG preceding the splice site, a polypyrimidine stretch that extends upstream 10 nucleotides, and a possible branch site at 24 nucleotides upstream of the splice site.
A single immunoreactive polypeptide localized in the chloroplast was detected with polyclonal antibodies to S-38, but the small mass difference between the protein forms of S-38 and S-40 precludes determination of whether or not both of these proteins are present in vivo.Recombinant S-38 and S-40 proteins have SS methyltransferase activity.It is possible that other chloroplast proteins may be substrates for either S-38 or S-40; however, these may not be detected by methylation studies utilizing chloroplast lysates, because detection under these conditions depends on hypo-methylation of these polypeptides.In the case of the SS of Rubisco, proteolytic processing by the stromal processing peptidase is a prerequisite for methylation of the N-terminal Met residue.The presence of unassembled SS and the requirement for proteolytic processing may be responsible for a small but easily detectable pool of methylatable SS in spinach chloroplast lysates.
Methylation of the SS of Rubisco is apparently widespread because N-methyl-Met was detected as the N-terminal residue in the SS of Rubisco from pea, corn, and barley (5).We have also detected N-methyl-Met as the N-terminal residue in the SS of tobacco Rubisco (data not shown).In addition, an enzymatic assay demonstrated that chloroplast lysates catalyzed methylation of bacterially expressed SS using AdoMet as a methyl donor (5).
The expression of a spinach homologue to Rubisco LSMT from pea and tobacco was initially anomalous, given that Lys-14 in the LS of spinach Rubisco is not methylated.Structural analyses of the methylation status of Lys-14 in the LS of spinach Rubisco have unequivocally established this residue as unmodified (1,4), and spinach Rubisco is an excellent in vitro FIG. 7. SSMT Activity of pea and tobacco Rubisco LSMT.Native pea Rubisco LSMT affinity purified from chloroplasts (11) and cell lysates from bacterially expressed recombinant forms of both pea and tobacco Rubisco LSMT (in pET-23d expression vectors with pLysS E. coli cells as hosts) were incubated in the presence of spinach chloroplast lysates and [ 3 H-methyl]AdoMet (1.8 M, 70 -80 Ci/mmol or 28 M, 36 Ci/mmol as indicated) for 30 min at 30 °C.After SDS-PAGE and electrophoretic transfer to a PVDF membrane, proteins were visualized by staining with Coomassie Brilliant Blue R-250, and tritium radioactivity was located by PhosphorImager analysis.LS and SS identify the positions of the large and small subunits of Rubisco, respectively.The presence of ␤-lactoglobulin B in the lanes identified as containing purified native pea Rubisco LSMT is present as a carrier protein added during purification (11).The control lane represents results obtained with uninduced pLysS cells incubated under identical conditions in the presence of a spinach chloroplast lysate.
substrate for pea Rubisco LSMT (3,11).Additional studies in our laboratory have demonstrated that the absence of methylation at Lys-14 in the LS of spinach Rubisco is consistent throughout the development of spinach plants and is not influenced by several environmental stresses. 2In additional, the gene and coding sequences reported for spinach, pea, and tobacco Rubisco methyltransferases do not contain substantial sequence identity with any reported nucleotide or amino acid sequences, including those of other protein, DNA, or small metabolite methyltransferases.Thus, methylation of Rubisco SS in spinach is catalyzed by a homologue of a bifunctional methyltransferase in pea that methylates both LS and SS.
The bifunctionality of pea Rubisco LSMT is the first report of both an ␣ N-and an ⑀ N-protein methylation activity in a single enzyme, and in addition this is the first report of a eukaryotic gene for a ␣ N-methyltransferase. Methylation of E. coli ribosomal L11 protein involves both ␣ N-and ⑀ N-methylation (23).Mutations in the prmA gene encoding the ribosomal protein L11 methyltransferase result in an under-methylated form of the L11 protein, which may be an indication of a similar prokaryotic bi-functional protein methyltransferase, although this has not been directly demonstrated.N-terminal methylation has been described for various amino acid residues in several proteins for both prokaryotic and eukaryotic organisms (24).The functional significance of this post-translational modification is obscure and has been speculated to be involved in processes as diverse as protection against proteolytic degradation (25,26,27), proper subcellular localization of proteins, and/or assembly of macromolecular structures (28).Protection against proteolytic degradation is a possible function because the stroma of pea chloroplasts contains at least three aminopeptidases, one of which has specificity for Met residues (29).
An intriguing analogy is provided by functional comparison with the well described bacterial system of pilin processing, which involves a prokaryotic ␣ N-methyltransferase.In bacteria, membrane associated pili are involved in processes such as conjugative transfer of DNA, adherence to host tissues, and extracellular protein secretion (30,31,32).The type-IV pilins are synthesized as precursors with an N-terminal leader peptide and are processed by an endopeptidase.A second posttranslational modification occurs by methylation of the newly formed N-terminal amino acid residue (33).Surprisingly, the leader peptidase responsible for prepilin (PilD) processing is a bifunctional enzyme that also catalyzes N-methylation of the N-terminal Phe of pilins (28).These observations are interest-ing because recent studies (34) as well as earlier reports (35,36,37) have described tubular connections between chloroplasts in vivo capable of allowing the exchange of molecules between chloroplasts, similar in function and form to bacterial pili.

usingFIG. 1 .FIG. 2 .
FIG. 1.Comparison of the deduced amino acid sequences of spinach SSMT with tobacco and pea Rubisco LSMTs.Identical residues are indicated by vertical lines, and conservative substitutions are indicated by dots.Gaps introduced to maximize alignment are indicated by dashes.Leucine-rich repeat-like motifs are underlined.The four amino acid sequence (WVQQ) that is produced by an alternative splice site is shown in bold italic letters.The conserved peptide sequences which were utilized to design degenerate primers are indicated by arrows.

FIG. 3 .FIG. 4 .
FIG. 3. Two forms of rbcMT-S mRNAs are produced as a result of alternative splice site selection.A, pS40XR0.4 was produced by cloning the 390 nucleotide EcoRI/XbaI fragment of the S-40 cDNA into pBluescript SKϩ.The clone spans exon III, the alternatively spliced region, and exon IV.A 460-nucleotide antisense probe was produced by transcription from the T3 promoter to the XhoI site.The long form of the mRNA protects a 390-nucleotide fragment of the probe, and the short form of the mRNA protects a 306-fragment and a 72-nucleotide fragment.B, RNase protection was performed to demonstrate the relative abundance of the long and short forms of rbcMT-S.The 460nucleotide antisense probe (lane 1) was annealed to 0, 2.5, 10, or 20 g of total RNA from mature spinach leaves (lanes 2-5, respectively) and digested with RNase A and RNase T1.The intensities of the 390-and 306-nucleotide fragments are similar, indicating that the long and short forms are approximately equivalent in abundance.Omission of spinach RNA (lane 2) or the substitution of a heterologous RNA (maize mitochondrial RNA; lane 6) resulted in complete sensitivity of the probe to RNase.The migration of a radiolabeled DNA size ladder (250-bp ladder, Life Technologies, Inc.) is shown on the right margin.