Methionine synthase is localized to the nucleus in Pichia pastoris and Candida albicans and to the cytoplasm in Saccharomyces cerevisiae

Methionine synthase (MS) catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of S-adenosylmethionine. Here, we report that MS is localized to the nucleus of Pichia pastoris and Candida albicans but is cytoplasmic in Saccharomyces cerevisiae. The P. pastoris strain carrying a deletion of the MET6 gene encoding MS (Ppmet6) exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of P. pastoris MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation. In silico analysis of the PpMS structure indicated that PpMS may exist in a dimer-like configuration in which Arg-742 of a monomer forms a salt bridge with Asp-113 of another monomer. Biochemical studies indicate that R742A as well as D113R mutations abrogate nuclear localization of PpMS and its ability to reverse methionine auxotrophy of Ppmet6. Thus, association of two PpMS monomers through the interaction of Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization. When PpMS is targeted to the cytoplasm employing a heterologous nuclear export signal, it is expressed at very low levels and is unable to reverse methionine and adenine auxotrophy of Ppmet6. Thus, nuclear localization is essential for the stability and function of MS in P. pastoris. We conclude that nuclear localization of MS is a unique feature of respiratory yeasts such as P. pastoris and C. albicans, and it may have novel moonlighting functions in the nucleus.

methionine as well as adenine auxotrophy due to homocysteine accumulation and defective purine biosynthesis (12). Thus, in addition to its role in methionine biosynthesis, MS may have other unknown functions in certain yeast species.
The genome of P. pastoris, a methylotrophic yeast, encodes a cobalamin-independent MS (PpMS), which shares 77 and 79% amino acid sequence identity with the MS of S. cerevisiae (ScMS) and CaMS, respectively (13). Here, we report that deletion of MET6 results in methionine as well as adenine auxotrophy in P. pastoris but only methionine auxotrophy in S. cerevisiae. MS is uniquely localized to the nucleus of P. pastoris and C. albicans, although it localizes to the cytosol in S. cerevisiae. Arg-742 and Asp-113 located in the C-and N-terminal regions of PpMS interact with each other and facilitate association between two monomers in a dimer-like configuration. Interaction between Arg-742 and Asp-113 is essential for the stability, catalytic activity, and nuclear localization of MS. Taken together, our results suggest that MS is present in the nucleus of respiratory yeasts such as P. pastoris and C. albicans, and it possesses unique biochemical properties that have not been reported in any other species.

Deletion of P. pastoris MET6 results in methionine as well as adenine auxotrophy
Our laboratory has been studying key transcription factors involved in the regulation of carbon metabolism in the respiratory yeast, P. pastoris (14 -21). While studying methionine metabolism, a methionine auxotroph (Ppmet6) was generated by replacing the coding region of MET6 encoding MS with a Zeocin expression cassette in the P. pastoris GS115 strain (Fig.  1, A and B). MS deficiency resulted in methionine auxotrophy, and Ppmet6 was unable to grow in a methionine-deficient medium, as expected (Fig. 1C). However, methionine supplementation did not restore the growth of Ppmet6 (Fig. 1D) under conditions in which it readily restored the growth of S. cerevisiae met6 strain (Scmet6) (Fig. 1, E and F). Of the various components tested, adenine when added to YNBD ϩ methionine medium restored growth of Ppmet6 (Fig. 1, G and H) indicating that MS deficiency results in methionine as well as adenine auxotrophy in P. pastoris.

MS localizes to the nucleus of P. pastoris and C. albicans
To further characterize P. pastoris MS (PpMS),we first examined its subcellular localization. PpMS was expressed as a histidine-tagged protein in E. coli, purified, and injected into rabbits to generate anti-PpMS antibodies that reacted with a protein of ϳ86 kDa in the cell lysates of GS115 but not Ppmet6 (Fig. 2, A and B). Immunofluorescence studies revealed that PpMS is present in the nucleus of P. pastoris cells cultured in YPD, YPG, or YPM media (Fig. 2C). To further validate these results, P. pastoris strain expressing chromosomally tagged PpMS-GFP (Pp-PpMSGFP) was constructed, and the localization of GFP fusion protein was visualized by direct examination of GFP fluorescence as well as immunofluorescence of DAPI-stained cells using anti-GFP and anti-PpMS antibodies. The results indicate that PpMSGFP localizes to the nucleus (Fig. 2, D and E). To further confirm nuclear localization of PpMS, P. pastoris GS115 strain was transformed with pGAP-PpMS Myc plasmid expressing c-Myc epitope-tagged PpMS (PpMS Myc ) from the GAPDH promoter, and immunofluorescence studies were carried out with anti-c-Myc antibodies. The results indicate that PpMS Myc localizes to the nucleus (Fig. 2F). PpMS Myc expression was confirmed by SDS-PAGE followed by Western blotting of whole-cell extracts using anti-c-Myc as well as anti-PpMS antibodies (Fig. 2G).
Because nuclear localization of MS has not been reported in any species thus far and to study the generality of this phenomenon, MS localization was examined in C. albicans, another

Nuclear localization of yeast methionine synthase
respiratory yeast, which also exhibits methionine as well as adenine auxotrophy when MET6 is deleted (3). We confirmed that CaMS is immunoreactive to anti-PpMS antibodies by Western blotting (Fig. 2H). Immunofluorescence studies indicate that CaMS localizes to the nucleus in C. albicans as well (Fig. 2I). Because the S. cerevisiae met6 strain does not exhibit adenine auxotrophy, we examined MS localization in this yeast species. ScMS was expressed as a GFP fusion protein (ScMSGFP) in S. cerevisiae met6 strain to generate Sc-ScMSGFP strain. Direct visualization of GFP fluorescence (Fig. 3A) as well as immunofluorescence using anti-GFP antibodies (Fig. 3B) indicates that ScMSGFP is localized to the cytoplasm. Interestingly, when ScMSGFP was expressed in the Ppmet6 strain (Pp-ScMSGFP), it localized to the nucleus as evident from the direct visualization of GFP fluorescence (Fig. 3C) and by immunofluorescence using anti-GFP antibodies (Fig. 3D). When PpMSGFP was expressed in S. cerevisiae (Sc-PpMSGFP), it localized to the cytoplasm as evident from the direct visualization of GFP fluorescence ( Fig. 3E) and by immunofluorescence using anti-GFP antibodies (Fig. 3F). Differential subcellular localization of MS in P. pastoris and S. cerevisiae was further confirmed by confocal microscopy (Fig. 3G). Western blot analysis revealed that PpMS is present in the cytoplasm as well as nuclear fractions of P. pastoris, although it is present only in the cytoplasm of S. cerevisiae (Fig. 3H). Taken together, these results indicate that ScMS is cytosolic in S. cerevisiae but nuclear in P. pastoris, and PpMS is nuclear in P. pastoris but cytoplasmic in S. cerevisiae.

Identification of nuclear localization signal of PpMS
To understand the mechanism of nuclear localization of PpMS, we examined whether catalytic activity is required for its nuclear localization. In CaMS, the Asp-614 residue in the active site is essential for binding to homocysteine, and consequently, CaMS D612A mutant enzyme possesses only 2% of the native enzyme activity (22). Because this aspartate residue is conserved in PpMS (Fig. 4A), we introduced the D612A mutation into PpMSGFP. The Ppmet6 strain expressing PpMS D612A GFP was catalytically inactive as evident from its inability to grow in YNBD medium (Fig. 4B). Direct fluorescence as well as immunofluorescence using anti-GFP antibodies revealed that PpMS D612A GFP localizes to the nucleus (Fig. 4, C-E) indicating that catalytic activity is not required for nuclear localization of PpMS.
Analysis of PpMS amino acid sequence using PSort II, Pre-dictNLS, and NetNTS databases indicated that PpMS does not Nuclear localization of yeast methionine synthase possess a canonical nuclear localization signal (NLS). To examine whether PpMS possesses a non-canonical NLS, we generated pGAPZA vectors encoding PpMS⌬99GFP in which 99 N-terminal amino acids of PpMS are deleted. PpMS⌬99GFP was localized to the nucleus (Fig. 4, C-E), indicating that the 99 N-terminal amino acids are not required for nuclear localization. To examine the role of C-terminal amino acids, we generated pGAPZA vector encoding the PpMS⌬661-768GFP mutant carrying a deletion of 107 C-terminal amino acids, and when expressed in the P. pastoris GS115 strain, it remained in the cytosol (Fig. 4, C-E). Thus, the C-terminal region is essential for nuclear localization of PpMS.
Methionine is converted to S-adenosylmethionine by methionine adenosyltransferase (MAT) also known as S-adenosylmethionine synthetase. In mammalian cells, MATI/III was reported to be present in the nucleus, and basic amino acid residues in the C-terminal region of MATI/III are important for nuclear localization (23). The C-terminal region of PpMS also contains basic amino acid residues, many of which are conserved in ScMS as well as CaMS (Fig. 4F). To examine their role in nuclear localization, we substituted specific arginine/lysine residues present in the C-terminal region of PpMS by alanine. P. pastoris strains expressing PpMS K740A GFP, PpMS R742A GFP, PpMS K759A GFP, or PpMS R762A GFP were generated, and their expression was confirmed by Western blotting with anti-GFP antibodies (Fig. 4G). Analysis of MS-GFP localization by fluorescence microscopy indicates that all the mutants localize to the nucleus except PpMS R742A GFP, which remained in the cytosol (Fig. 4H). Expression of PpMS R742A GFP in Ppmet6 did not result in the restoration of the growth of cells cultured in methionine-and adenine-deficient medium (Fig. 5, A and B).
To understand the effect of R742A mutation on enzyme function, the same mutation was introduced into ScMS to generate ScMS R742A GFP. ScMS R742A GFP as well as PpMS R742A GFP were expressed in the Scmet6 strain, and their expression was confirmed by Western blotting using anti-GFP antibodies (Fig.  5C). ScMS R742A GFP and PpMS R742A GFP failed to reverse the methionine auxotrophy of Scmet6 (Fig. 5, D and E) indicating that the R742A mutant is catalytically inactive.
To examine the effect of extranuclear localization on enzyme stability and function, we generated Ppmet6 strains expressing PpMSGFP-NES and PpMSGFP-MAS in which a nuclear export signal (NES) or a membrane anchor signal was fused to the C terminus of PpMS, respectively (Fig. 6A). We also generated PpMSGFP-NLS, in which a nuclear localization signal was fused to the C terminus of PpMSGFP (Fig. 6A). As expected, PpMSGFP-NES and PpMSGFP-NLS were targeted to the cytoplasm and nucleus, respectively (Fig. 6B). Although PpMSGFP-MAS was targeted to the plasma membrane, nuclear localization was not completely abrogated (Fig. 6, B and C). Although expression of PpMSGFP-NLS and PpMSGFP-MAS was observed in almost all cells, expression of PpMSGFP-NES was restricted to a small subset of cells (Fig. 6B). Western blot analysis indicated that expression of PpMSGFP-NES was the lowest among the three proteins (Fig. 6D), and as a result, PpMSGFP-NES expression does not result in the reversal of methionine and adenine auxotrophy of Ppmet6. Pp-PpMSGFP-NES strain exhibited severe growth retardation even in medium containing methionine and adenine (Fig. 6, E and F).

Interaction between Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization
To understand the mechanism by which the R742A mutation affects enzyme function, structural modeling exercise was carried out to examine the role and significance of Arg-742 at the C-terminal end on the enzyme activity. BLAST search H, Western blot analysis of cytoplasm and nuclear extract of Sc-ScMSGFP strain and P. pastoris GS115 strain cultured in YNBD and YPD, respectively, using anti-GFP, anti-PpMS, antiphosphoglycerate kinase (PGK), and anti-histone H3 antibodies. Anti-phosphoglycerate kinase and histone H3 served as cytoplasmic and nuclear markers, respectively. Protein molecular mass markers (kDa) are indicated.

Nuclear localization of yeast methionine synthase
revealed that PpMS shares the highest similarity with CaMS (PDB codes 3PPC, 3PPF, and 3PPH) with a query coverage of 99%, e-value of ϳ0, and amino acid sequence identity of 78% (Fig. 7A). 3PPC was used as the base template, and the short stretches of 8 and 6 residues that were missing in the N and C termini, respectively, were modeled based on corresponding regions in 3PPH (Fig. 7A). Superposition of all the structural templates revealed that the substrate-binding site for homocysteine, zinc ion, and the methyl donor have distinct pockets and are seen in between the N-and C-terminal barrel domains (6 -8). In our structural model, Arg-742 is located at a distance of 8 Å from the nearest atom in the substrate-binding pocket and clearly far away from the substrate-binding pocket. Hence, it cannot have any direct effect on the binding of any of the substrates, nor can it be critically involved in stabilizing the pocket. Therefore, we focused our attention on the quaternary structure of cobalamin-independent MS of C. albicans (3PPC) and T. maritima (PDB code 1XPG). The biological assemblies obtained from the PDB database for these two proteins consisted of two subunits of the same polypeptide chain that form a "dimer-like" assembly. With this information, a corresponding dimer-like association was modeled for the PpMS as well, by superposing the whole-length polypeptide chain of our protein with the A and B chains of the structural templates as shown in Fig. 7B. A subsequent energy minimization of the interface of the dimer-like association was also carried out. It was observed that Arg-742 of one subunit makes extensive interactions, mainly hydrogen bonding and ionic in nature, with the residues of the other subunit. Of the many interactions, ionic interaction of Arg-742 with Asp-113 was identified, whose side chains were positioned appropriately for forming a salt bridge (Fig. 6, C and D, and supplemental Fig. 1). Apart from the Arg-742-Asp-113 salt bridge, hydrogen-bonding interactions of Thr-106 -Ser-527, Gln-449 -Lys-85, Ser-711-Lys-681, Arg-103-Glu-23, Glu-745-Lys-179, Glu-745-Arg-82, and Arg-748 -Asp-180 could also be deciphered from the model. The full-length model of the protein in the PDB format is given as supplemental material. The extensive nature of interactions between the two subunits was suggestive of a biological association between the two subunits rather than as an artifact of crystallization. Thus, structural modeling indicated that Arg-742 could play an important role in the stabilization of a dimer-like assembly through an ionic interaction with Asp-113 of a neighboring subunit.
To validate these in silico studies, detailed biochemical investigations were carried out. Because R742A mutation results in an inactive enzyme, we examined the effect of the D113R/ D113A mutation on enzyme function. PpMS D113A GFP and PpMS D113R GFP were expressed in Scmet6 as well as Ppmet6 strains. Western blot analysis of multiple clones of each mutant indicated that PpMS D113A GFP and PpMS D113R GFP are expressed at lower levels than PpMSGFP (Fig. 8, A-C) suggesting that Asp-113 is essential for the stability of the protein. D113A mutation abrogates enzyme function as evident from the inability of PpMS D113A GFP/PpMS D113R GFP to restore the growth of Scmet6 and Ppmet6 in methionine-deficient media (Fig.  8, D and E). Subcellular localization studies indicate that PpMS D113R GFP is localized to the cytoplasm of P. pastoris cells (Fig. 8, F and G). Thus, mutation of either Arg-742 or Asp-113 results in catalytically inactive, cytosolic PpMS.

Discussion
Recruitment of metabolic enzymes to the nuclear compartment for alternative functions is well-documented. For example, enzymes of carbon metabolism such as phosphoglycerate kinase, aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase, fumarase etc., known conventionally as cytoplasmic enzymes with housekeeping functions, localize to the nucleus and perform moonlighting functions in gene transcription, DNA replication, DNA repair, DNA methylation, nuclear RNA export, etc. (24 -28). Similarly, the mitochondrial pyruvate dehydrogenase complex translocates from the mitochondria to the nucleus to provide acetyl-CoA necessary for histone acetylation (29). Among the enzymes of methionine metabolism, MATII localizes to the nucleus of mammalian cells and serves as a transcriptional corepressor of Maf oncoprotein (30). Thus far, there is no report on nuclear localization of MS in any species. In this study, we demonstrate that MS is present in the nucleus as well as the cytoplasm of P. pastoris and C. albicans but only in the cytosol of S. cerevisiae. As a first step toward understanding the function of PpMS in the nucleus, we first focused our attention on the mechanism of nuclear translocation of PpMS. Analysis of various mutants indicated that deletion of 107 C-terminal amino acids or more specifically, mutation of a C-terminal amino acid (R762A) abrogates nuclear localization of PpMS. When expressed in Ppmet6 and Scmet6 strains, the cytosolic PpMS R742A mutant was unable to reverse methionine auxotrophy. Thus, R742A mutation abrogates catalytic activity as well as nuclear localization of PpMS. On the contrary, D612A mutation within the active site abrogates catalytic activity but not nuclear localization of PpMS. Although cobalamin-independent MS has been studied in a number of bacterial and yeast species, the importance of Arg-742 has not been examined thus far, and therefore a detailed investigation was undertaken. PpMS shares a high degree of structural similarity with the CaMS and T. maritima MS, and an in silico analysis indicated that these enzymes may exist in a dimer-like configuration in which Arg-742 of a monomer can

Nuclear localization of yeast methionine synthase
form a salt bridge with Asp-113 of another monomer. To confirm this, biochemical studies were carried out, and the results indicate that the D113R/D113A mutation abrogates catalytic activity as well as nuclear localization of PpMS. Furthermore, PpMS D113R /PpMS D113A are expressed at much lower levels than PpMS in S. cerevisiae and P. pastoris indicating that this mutation may affect the stability of the protein as well. In the case of PpMS R742A GFP and PpMS D113R GFP, the inability to associate in a dimer-like configuration may lead to their cytoplasmic retention, poor stability, and loss of function. Attempts to demonstrate the existence of a PpMS dimer by native PAGE and gel filtration chromatography of recombinant PpMS or chemical cross-linking and coimmunoprecipitation of epitope-tagged PpMS expressed in P. pastoris cell extracts were not successful. Thus, we were unable to provide direct evidence for dimer formation. It is possible that the association between PpMS monomers is not sufficiently strong or is transient thus obscuring the detection through conventional biochemical techniques (6).
It is interesting to note that cytosolic localization of PpMS-GFP in Scmet6 results in reversal of methionine auxotrophy. However, PpMSGFP-NES is unable to reverse methionine and adenine auxotrophy of Ppmet6. This may be due to very low levels of the protein. It is possible that forceful targeting of the protein to cytoplasm renders it unstable and susceptible for proteolytic degradation. Thus, nuclear association is essential for the stability and function PpMS in P. pastoris but not S. cerevisiae. The factor(s) that contributes to the stability of PpMS in P. pastoris remains to be investigated. It is also pertinent to note that methionine and adenine fail to completely rescue the growth of Ppmet6. Thus, in addition to its role in methionine and adenine metabolism, PpMS may have other moonlighting functions in the nucleus in P. pastoris.
Homocysteine is a substrate not only for MS but also for cystathionine ␤-synthase. The latter catalyzes the first step of transsulfuration pathway involving the synthesis of cystathionine from serine and homocysteine (Fig. 9). In S. pombe, which lacks the enzymes of transsulfuration pathway, MS deficiency leads to accumulation of homocysteine resulting in defective purine biosynthesis and adenine auxotrophy (12). Expression of enzymes of the transsulfuration pathway in the MS-deficient strain of S. pombe, restores growth in the absence of adenine (12). The genome of P. pastoris encodes cystathionine ␤-synthase (PAS_chr2-2_0137) as well as cystathionine ␥-lyase (PAS_ chr1-4_0489), and thus, adenine auxotrophy of Ppmet6 is unlikely to be due to homocysteine toxicity. Methionine biosynthesis is intimately linked to purine biosynthesis as THF generated by MS-catalyzed homocysteine remethylation reaction is essential for de novo purine biosynthesis. It is possible that a decrease in the intracellular pool of THF in Ppmet6 may lead to defective purine biosynthesis and impaired growth. Because the accumulation of purine biosynthetic intermediates

Nuclear localization of yeast methionine synthase
such as 5Ј-phosphoribosyl-5-aminoimidazole-4 carboxamide (AICAR) interferes with methionine biosynthesis (31), it is possible that accumulation of 5-methyl-THF in Ppmet6 results in THF deficiency leading to defective purine biosynthesis. These aspects are schematically represented in Fig. 9.
Respiratory yeasts such as P. pastoris, Hansenula polymorpha, and C. albicans favor respiratory growth, which is characterized by low glucose uptake and high biomass yield. S. cerevisiae follows a respiro-fermentative growth characterized by high glucose uptake and low biomass yield. Key differences

Nuclear localization of yeast methionine synthase
have been reported between the respiro-fermentative and respiratory yeasts. For example, RNA interference has not been demonstrated in S. cerevisiae but is effective in C. albicans (32).
Although respiratory yeasts such as C. albicans possess a mitochondrial dihyroorotate dehydrogenase, S. cerevisiae possesses a cytosolic enzyme whose activity is independent of ubiquinone

Nuclear localization of yeast methionine synthase
and the presence of oxygen, enabling the latter to grow under anaerobic conditions (33)(34). Cyanide-insensitive alternative oxidase involved in the oxidation of ubiquinone by molecular oxygen is present in aerobic yeasts but not in S. cerevisiae (35). The fact that MS, a key enzyme required for multiple metabolic pathways, is cytosolic in S. cerevisiae but nuclear in P. pastoris and C. albicans suggests that differential localization of MS is another key feature that distinguishes respiratory yeasts from respiro-fermentative yeasts. We speculate that nuclear localization of MS may be a common feature of many respiratory yeasts, and MS may have novel moonlighting functions in the nucleus of these yeasts. Furthermore, methylotrophic yeasts may harbor novel sulfur metabolic pathways as was shown recently in H. polymorpha in which methionine is synthesized only through cysteine and cystathionine, and the sulfur metabolism is centered on cysteine rather than methionine (36). Thus, understanding sulfur metabolism of methylotrophic yeasts may lead to better exploitation of these yeasts for the production of high-value sulfur-containing amino acids and metabolites.

Measurement of growth rate of yeast cells
A single colony of the indicated yeast strain was inoculated into 5 ml of YPD and grown overnight at 30°C in a shaker incubator. Absorbance was measured at 600 nm, and cells corresponding to 0.1 A 600 units were inoculated into flasks containing 25 ml of YNBD medium containing (Met ϩ ) or lacking (Met Ϫ ) methionine and cultured at 30°C. Aliquots were taken at 2-6-h intervals, and A 600 was recorded. Growth rate experiments were performed in triplicate. In some experiments, adenine was added at a final concentration of 20 mg/liter. For spot tests, cells were pre-grown on YPD medium overnight, spun down, washed twice in sterile distilled water, resuspended to a density of 2.7 ϫ 10 7 cells/ml (A 600 1.0), and serially diluted (1:10 dilutions). Five l of each dilution was spotted onto YNBD ϩ agar plates and incubated at 30°C for 72 h.

Nuclear localization of yeast methionine synthase
(GenBank TM accession number AY601648) and used in a PCR to amplify the MET6 gene from P. pastoris genomic DNA. The XhoI and HindIII restriction sites are underlined. cDNA was digested with XhoI and HindIII and cloned into the E. coli pRSETA vector (Invitrogen) in-frame with the vector-encoded histidine tag, and PpMS was expressed as histidine-tagged protein in E. coli (BL21) cells (39). The E. coli cell lysate expressing PpMS was subjected to SDS-PAGE, and the band corresponding to PpMS was visualized by CuCl 2 staining (40) and excised from the gel. The protein was electroeluted, and its identity was confirmed by Western blotting with anti-His tag antibodies and used for immunization of rabbits. PpMS (ϳ300 g) was mixed thoroughly with an equal volume of Freund's complete adjuvant and injected into rabbit subcutaneously at multiple sites. Three weeks later, 100 g of PpMS mixed with Freund's incomplete adjuvant was injected three times at 10-day intervals. The animal was bled; serum was collected and stored at Ϫ20°C in aliquots.

Preparation of yeast nuclear extract and cytosol
Nuclear extract was isolated as described (41) with modifications. Briefly, yeast cells were cultured in YPD at 30°C to A 600 between 4 and 5. A 2-liter culture yielded ϳ10 g of cells (wet weight). The cell pellet was resuspended in 1 volume (10 ml) of zymolyase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1.2 M sorbitol, 30 mM DTT) and incubated at room temperature for 15 min. Cells were pelleted at 4000 rpm (Beckman JA-20) for 5 min at 4°C, and the pellet, resuspended in 3 volumes (30 ml) of Zymolyase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1.2 M sorbitol, 1 mM DTT) containing 1 ml of long life zymolyase (1.5 units/l from G-Biosciences) and incubated at 37°C for 3-5 h with gentle stirring until a 10-fold decrease in A 600 (in presence of 1% SDS), was observed. Spheroplasts were pelleted by centrifugation at 4000 rpm for 5 min and at 4°C. The pellet was resuspended in 0.1 ml of 18% Ficoll 400 (which was dissolved in 20 mM KH 2 PO 4 at pH 7.5 and 0.5 mM CaCl 2 ) per g of cells using a glass rod. The cells were then diluted with 3 ml of 18% Ficoll and homogenized in a Dounce homogenizer with a Teflon pestle (ϳ10 strokes). The mixture was then diluted with equal volume of 0% Ficoll (1 M sorbitol and 0.5 mM CaCl 2 ), and 10 ml of it was carefully loaded on preformed Percoll gradient. A 32.5% Percoll gradient was formed by mixing 0 and 100% (1 M sorbitol and 0.5 mM CaCl 2 at pH 7.5) of Percoll in an appropriate ratio (Percoll was purchased from Sigma). Percoll mixture (34 ml) was centrifuged at 27,000 ϫ g for 50 min in an S28 rotor in an ultracentrifuge (Beckmann Coulter). Nuclei thus obtained were loaded onto the Percoll gradient and centrifuged at 9500 ϫ g for 4 h in a swinging bucket rotor. Among the three bands formed, the top band containing nuclei was isolated, mixed with 2 volumes of 0% Percoll, and then centrifuged to obtain a nuclear pellet. This pellet was resuspended in an equal volume of nuclear lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgSO 4 , 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 1ϫ protease inhibitor mix), and the purity was examined in a fluorescence microscope after staining with DAPI. After careful examination under a microscope and DAPI staining, we found that the top layer had the least contamination of unbroken cells and cellular debris. Purity of the nuclear fraction was ascertained by Western blotting using appropriate nuclear markers.
For the preparation of cytosol, yeast cells were cultured overnight in YPD, and 5 ml of cells were pelleted and resuspended in 2 volumes of zymolyase buffer containing 20 l of long life zymolyase (1.5 units/l from G-Biosciences) and incubated at 37°C for 30 -60 min. Spheroplasts were washed twice with zymolyase buffer and resuspended in 2 volumes of yeast lysis buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM MgCl 2 , 10 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 7 mM ␤-mercaptoethanol, 1ϫ protease inhibitor mix). Cells were lysed by pipetting up and down using a pipetman and incubated on ice for 20 min. Following centrifugation at 10,000 ϫ g for 30 min at 4°C, the supernatant (cytosol) was removed, stored at Ϫ80°C in aliquots, and subjected to SDS-PAGE and Western blot analysis.

Nuclear localization of yeast methionine synthase Fluorescence microscopy
Late log phase cultures of P. pastoris were treated with 3.7% formaldehyde for 1 h, and spheroplasts were prepared by treating the cells with Zymolyase (1.5 units/l) in a Zymolyase buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT, and 1 M sorbitol for 1 h at 30°C. Cells were centrifuged at 1600 rpm for 10 min at room temperature in a microcentrifuge (Eppendorf) and resuspended in PBST (phosphatebuffered saline containing 0.05% Tween 20). The cells were spread onto glass coverslips. Following treatment with chilled methanol and acetone for 7 min and 30 s, respectively, the coverslips were air-dried. They were incubated sequentially with blocking buffer (PBS containing 20 mg/ml BSA), blocking buffer containing appropriate primary and secondary antibodies. Finally, the smears were treated with 1 g/ml 4Ј,6-diamidino-2-phenylindole (DAPI, Sigma) for 5 min for staining the DNA. The coverslips were washed in PBS and air-dried, and cells were visualized by either a fluorescent microscope (Leica) or a confocal microscope (Confocal Zeiss LSM880-Airyscan). MS-GFP fusion proteins were also localized by direct fluorescence of yeast cells in the fluorescent microscope using a FITC filter. Rabbit anti-histone H3 antibodies (ab1791) were obtained from Abcam. Mouse anti-c-Myc antibodies (sc-9996) were obtained from Santa Cruz Biotechnology. Mouse anti-GFP antibodies (OP10) were obtained from Millipore.

Construction of P. pastoris strain expressing chromosomally GFP-tagged PpMS (Pp-PpMSGFP)
The chromosomal MET6 gene of P. pastoris was tagged inframe with the DNA sequence encoding green fluorescent protein (GFP) so that the PpMET6-GFP gene is expressed under the control of the MET6 promoter. The PpMET6-GFP expression cassette consisting of 1000 bp of 3Ј-flanking region of PpMET6 followed by GFP ORF and Zeocin expression cassette was generated by four different PCRs. First, 1000 bp of MET6 gene encompassing ϩ1305 to ϩ2304 bp were amplified from P. pastoris genomic DNA by PCR using the primer pair 5Ј-CTTGCCTTTGTTCCCAACCACCACC-3Ј (1ЈF, nucleotides from position ϩ1305 to ϩ1330 bp of MET6) and 5Ј-CTCCT-TTACTAGTCAGATCTACCATattctgagcgtacttttcacggtag-3Ј (1ЈR, ϩ25 to ϩ1 bp of GFP (uppercase) and ϩ2305 to ϩ2281 bp of MET6 (lowercase)). In another PCR, a 714-bp region from the pREP41GFP vector (42) was amplified using the primer pair 5Ј-ctaccgtgaaaagtacgctcagaatATGGTAGATCTGACTAGTA-AAGGAG-3Ј (2ЈF, position from ϩ2279 to ϩ2304 bp of MET6 (lowercase), position ϩ1 to ϩ25 bp of GFP (uppercase)) and 5Ј-AGCTATGGTGTGTGGGGGATCCGCATTAgtggtggtggctagctttgtatag-3Ј (2ЈR, 962-990 bp of pGAPZA vector (uppercase) and ϩ741 to ϩ713 bp of GFP (lowercase)). In the third PCR, the Zeocin expression cassette was amplified using the primer pair 5Ј-ctatacaaagctagccaccaccacTAATGCGGATCC-CCCACACACCATAGCT-3Ј (3ЈF, ϩ717 to ϩ714 bp of GFP (lowercase) and 962 to 989 bp of pGAPZA vector (lowercase)) and 5Ј-TGCTCACATGTTGGTCTCCAGCTTG-3Ј (3ЈR, 2161 to 2136 bp of pGAPZA vector). Final PCR was carried out using all the above three PCR products (50 ng each) together with 1ЈF and 3ЈR primers to generate the PpMET6-GFP expression cassette (2941 bp), which was transformed into P. pastoris by electroporation. Zeocin-resistant colonies in which the PpMET6-GFP expression cassette was integrated into the chromosomal PpMET6 locus by homologous recombination were selected by plating the transformants on YPD plates containing Zeocin (100 g/ml). P. pastoris strain expressing chromosomally GFPtagged MS (PpMSGFP) was designated as Pp-PpMSGFP.

Analysis of PpMS structure
The query PpMS sequence (Uniprot accession number C4QZU2, 768 amino acids long) was searched against the Protein Data Bank (43) using protein BLAST (44) (basic local alignment tool) and BLOSUM62 substitution matrix to find the structural templates that were homologous to PpMS. A homology model was obtained using Modeler (version 9) (45), and the structural model was verified to have acceptable geometrical and stereo-chemical parameters using PROCHECK (46). A loop refinement process using an inbuilt modeler script was also carried out to correct the loop regions. A structural similarity search was carried out using Dali (47), and the identified homologs were aligned using Mustang (48). Energy minimization of the modeled protein was carried out using Chiron (49). Calculation of the interface contacts was carried out using Protein Interactions Calculator server (50). PyMOL (57) was used for structure visualization and generation of images.
Because MET15 was already deleted in BY4741, in order to study methionine auxotrophy of Scmet6met15 caused by the deletion of MET6 alone, it became necessary to express MET15 in Scmet6met15. MET15 was amplified from S. cerevisiae genomic DNA using the primer pair 5Ј-CGCGGATCCATGC-CATCTCATTTCGATACTGTTCAACTAC-3Ј and 5Ј-CCG-CTCGAGCTACAGATCCTCTTCTGAGATGAGTTTTTGT-TCTGGTTTTTGGCCAGCGAAAAC-3Ј. The BamHI and XhoI restriction sites in the primers are underlined. DNA encoding Myc tag in the reverse primer is shown in italics. The PCR product was cloned into pRS416TEF vector (51) and transformed into Scmet6met15 strain, and expression of MET15 gene product (O-acetylhomoserine-O-acetylserine sulfhydrylase) was confirmed by Western blotting using anti-Myc antibodies (details will be provided on request). This strain in which ScMET6 is deleted but ScMET15 is functional is designated as Scmet6.
The gene encoding ScMSGFP was amplified by PCR using the primer pair 5Ј-AAAACTGCAGATGGTTCAATCTGCT-GTCTTAGG-3Ј and 5Ј-CCGCTCGAGCTAGTGGTGGTG-GCTAGCTTTG-3Ј from pIB3-ScMSGFP vector. PstI and XhoI sites are underlined. The PCR product was digested with PstI and XhoI and cloned into pRS415TEF vector (52) to generate the pRS415-ScMSGFP plasmid that was transformed into the electrocompetent Scmet6 strain. Transformants were selected on LEU Ϫ plates. S. cerevisiae expressing ScMSGFP was designated as Sc-ScMSGFP.

Construction of P. pastoris strain expressing c-Myc epitopetagged PpMS (Pp-PpMS Myc )
To express PpMS as c-Myc epitope-tagged protein in P. pastoris, the cDNA encoding PpMS was amplified by PCR from pRSETA-PpMS plasmid using the primer pair 5Ј-CCGCTCG-AGGGAATGGTTCAATCATCTGTCTTAGGT-3Ј and 5Ј-ATACATACGCGGCCGCAATTCTGAGCGTACTTTTCAC-3Ј. The XhoI and NotI sites are underlined. Following digestion with XhoI and NotI, the PCR product was cloned into pGAPZA vector, in-frame with vector-encoded c-Myc epitope to generate pGAPZA-PpMS vector, which was linearized with AvrII and transformed into electrocompetent P. pastoris GS115 strain to generate the Pp-PpMS Myc strain expressing c-Myc-tagged PpMS (PpMS Myc ).

Nuclear localization of yeast methionine synthase
To generate pGAPZA expressing PpMS⌬661-768GFP, we carried out three different PCRs. First, PpMET6 gene (1983 bp) was amplified by PCR using the primer pair 5Ј-CCGCTCGAGatggttcaatcatctgtc-3Ј (PpMSGFP-FP, XhoI site is underlined, and ϩ1 to ϩ18 bp of MET6 gene are shown in lowercase), and 5Ј-cctttactagtcagatctaccatCAAATCAGAGTAACAGAAGT-GAG-3Ј (MSGFP-RP, ϩ1 to ϩ23 bp of GFP gene are shown in lowercase, and ϩ1960 to ϩ1983 bp of PpMET6 are shown in uppercase). In the second PCR, the gene encoding GFP was amplified using the primer pair 5Ј-ctcacttctgttactctgatttgATG-GTAGATCTGACTAGTAAAGG-3Ј (MSGFP-FP, ϩ1960 to ϩ1983 bp of PpMET6 gene are shown in lowercase, and ϩ1 to ϩ23 bp of GFP gene are shown in uppercase) and 5Ј-ATAAG-AATGCGGCCGCctagggtggtggctagctttg-3Ј (GFP-RP, NotI site is underlined, and ϩ744 to ϩ723 bp of GFP gene are shown in lowercase). In the third and final PCR, the products of the first two PCRs were used as templates, and PCR was carried out with PpMSGFP-FP and GFP-RP primers. The final PCR products were restricted with XhoI and NotI and cloned into pGAPZA vector (Invitrogen). All the recombinant pGAPZA plasmids were linearized with AvrII and transformed into P. pastoris GS115 strain. Zeocin-resistant colonies were screened for GFP expression.
All the PCR products were cloned into pIB3 vector, and the recombinant plasmids were linearized with SalI and transformed into Ppmet6.

Generation of S. cerevisiae strains expressing PpMS R742A GFP, PpMS D612A GFP, and ScMS D612A GFP
The PpMS R742A GFP and PpMS D612A GFP were amplified from pIB3-PpMS R742A GFP and pIB3-PpMS R742A GFP plasmids, respectively, by PCR and cloned into pRS415TEF vector. The recombinant plasmids were transformed into Scmet6 strain, and transformants were selected on Leu Ϫ plates. pScMS D612A plasmid was generated by introducing D612A mutation into pScMSGFP vector by site-directed mutagenesis using the QuickChange method (Stratagene, CA). Following confirmation of the mutation by DNA sequencing, recombinant pScMS D612A GFP was transformed into Scmet6 strain by electroporation. Transformants were plated on YNBD Leu Ϫ agar and screened for GFP expression.
Author contributions-U. S. and P. N. R. planned the experiments and organized the data; U. S., H. R. V. K. and S. S. K. performed the experiments; R. B. and N. C. carried out the structural analysis. U. S. and P. N. R. take the responsibility for integrity of the data and accuracy of data analysis. P. N. R. wrote the paper.