Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris*

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1. We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris.

eral transcription factors that regulate carbon metabolism have been identified in P. pastoris. These include the following: Mxr1p, Rop1p, Trm1p, Mit1p, and Nrg1p (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The role of these transcription factors in the regulation of amino acid metabolism has not been examined. Here, we demonstrate that P. pastoris can utilize amino acids as the sole source of carbon, and Mxr1p but not Trm1p or Rop1p is essential for this process. Mxr1p regulates the synthesis of several key enzymes involved in amino acid metabolism such as GDH2, AAT1, AAT2, MDH1, MDH2, and GLN1 at the transcriptional or post-transcriptional level.

Enzymes Essential for the Utilization of Amino Acids as the Sole Source of Carbon by P. pastoris and Regulation of Their
Biosynthesis by Mxr1p-The ability of P. pastoris to utilize amino acids as the sole source of carbon and nitrogen has not been investigated. We therefore examined the ability of P. pastoris GS115 strain to grow in media such as yeast nitrogen base (YNB) without amino acids and 0.5% ammonium sulfate supplemented with 2.0% glucose (YNBD), 1.0% glutamate (YNB ϩ Glu), 1% aspartate (YNB ϩ Asp) or YNB ϩ Glu without ammonium sulfate. The results indicate that P. pastoris GS115 strain but not ⌬mxr1 strain (Table 1) can grow in these media (Fig.  1A) indicating that Mxr1p is essential for the utilization of amino acids as the sole source of carbon and nitrogen. Rop1 and Trm1p, which regulate the expression of genes of methanol utilization pathway (8,9), have no role in the utilization of amino acids because growth of ⌬rop1 and ⌬trm1 strains is not affected when cultured in YP medium (1.0% yeast extract and 2.0% peptone) (Fig. 1A). S. cerevisiae was unable to grow in YP medium as expected (Fig. 1B) (3). Subcellular localization studies employing P. pastoris strain expressing a FLAG-tagged Mxr1p (13) indicates that Mxr1p localizes to the nucleus of cells cultured in YP as well as YPM (YP ϩ 2% methanol) but was cytosolic in cells cultured in YPD (YP ϩ 2% glucose) (Fig. 1C). Cell lysates of GS115 and ⌬mxr1 strains cultured in YP were subjected to SDS-PAGE, and proteins were visualized by Coo-massie Blue staining (Fig. 1D). Proteins a-f, which are differentially expressed in GS115 and ⌬mxr1, were selected; protein bands were excised and subjected to in-gel trypsin digestion, and the tryptic peptides were analyzed by MALDI-TOF mass spectrometry (supplemental data). Proteins a-c were identified as GDH2, alcohol oxidase, and formate dehydrogenase, respectively, and proteins d-f were identified as aconitase, malate synthase, and citrate synthase, respectively ( Fig. 1D and supplemental data). To confirm their differential expression in GS115 and ⌬mxr1, qPCR analysis was carried out with RNA isolated from cells cultured in YP medium. The results indicate that AOXI and FDH transcripts are present in higher levels in GS115 than ⌬mxr1, whereas transcript levels of ACO, MS, and CS are higher in ⌬mxr1 than GS115 cultured in YP (Fig. 1E). However, GDH2 mRNA levels were comparable in GS115 and ⌬mxr1 cultured in YP (Fig. 1E). Because GDH2 has a key role in the utilization of amino acids in S. stipitis and GDH2 protein but not mRNA levels were consistently lower in ⌬mxr1 than GS115 in several independent experiments, a detailed study was undertaken. P. pastoris GS115 and ⌬mxr1 were transformed with pIB3-GDH2 His , and expression of Gdh2p His was examined in cells cultured in different carbon sources by Western blotting. GDH2 His expression levels were higher in cells cultured in YP, YNB ϩ Glu, and YNB ϩ Asp than those cultured in YPD or YNBD (Fig. 1, F and G). Furthermore, GDH2 His is expressed at higher levels in GS115 than ⌬mxr1 cultured in YP, YNB ϩ Glu, and YNB ϩ Asp media (Fig. 1, F and G). Immunofluorescence studies indicate that GDH2 His is localized to the cytosol (Fig. 1H). Thus, Mxr1p is required for the synthesis of Gdh2p but not GDH2 mRNA suggesting that Mxr1p regulates GDH2 expression at the post-transcriptional level.
To confirm whether GDH2 is required for the utilization of amino acids as the sole source of carbon, P. pastoris ⌬gdh2 strain was generated (Fig. 2, A and B), and its ability to grow in YPD, YP, YNB ϩ Glu, and YNB ϩ Asp media was examined. Growth of ⌬gdh2 strain was normal in YPD medium but severely impaired when cultured in YP, YNB ϩ Glu, and YNB ϩ  (13,14). This trans-activation domain mediates carbon source-dependent repression and activation of Mxr1p-regulated genes (13,14). To examine the role of this N-terminal trans-activation domain in the regulation of GDH2 expression, P. pastoris ⌬mxr1 strain overexpressing Mxr1p N400 was generated and named MXR1 N400 -OE (Fig.  3A). Expression of MXR1 and MXR1 N400 mRNA in cells cultured in YP and YNB ϩ Glu media was confirmed by qPCR (Fig.  3B). Overexpression of Mxr1p N400 resulted in partial restoration of growth of cells cultured in YP but not YNB ϩ Glu (Fig.  3C). Overexpression of Mxr1p but not Mxr1p N400 results in the restoration of Gdh2p His levels in ⌬mxr1 (Fig. 3, D and E). However, deletion or overexpression of Mxr1p or Mxr1p N400 had no significant effect on GDH2 mRNA levels (Fig. 3F). These results not only confirm post-transcriptional regulation of GDH2 FIGURE 1. Growth of P. pastoris GS115 and ⌬mxr1 in media containing amino acids as the sole source of carbon and regulation of GDH2 expression by Mxr1p. A, growth of P. pastoris GS115 and ⌬mxr1 strains in media containing amino acids as the sole source of carbon and nitrogen. In YNB ϩ Glu-(NH 4 ) 2 SO 4 medium, glutamate serves as the sole source of carbon as well as nitrogen. B, comparison of growth of P. pastoris and S. cerevisiae in YP medium. C, subcellular localization of FLAG-tagged Mxr1p in cells cultured in different media as analyzed by immunofluorescence using anti-FLAG antibodies. DAPI was used to stain the nucleus. D, protein profile of lysates of cells cultured in YP medium. Proteins were resolved on SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue R. Proteins a-f, which are differentially expressed in GS115 and ⌬mxr1, are identified by mass spectrometry (see supplemental data). E, qPCR validation of genes differentially expressed in GS115 and ⌬mxr1. F, Western blotting analysis of lysates of GS115-GDH2 His and ⌬mxr1-GDH2 His strains expressing Gdh2p His using anti-His tag antibodies. Cells were cultured in different media as indicated. ZTA1 (23) served as loading control. G, quantitation of data presented in F. H, cytosolic localization of Gdh2p His in cells cultured in YP as examined by immunofluorescence using anti-His tag antibodies. *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.0005; ns, not significant. expression by Mxr1p but also demonstrate that the N-terminal 400 amino acids alone are not sufficient for this process.
Mxr1p Is a Key Regulator of Aspartate Aminotransferase-Because the growth of ⌬gdh2 can be restored by the expression of GDH2, it was of interest to examine whether overexpression of GDH2 in ⌬mxr1 can restore the growth in YP and YNB ϩ Glu media. GDH2 was expressed as a Myc-tagged protein (Gdh2p Myc ) at high levels from the GAPDH promoter or moderate levels from S. cerevisiae CUP1 promoter (15) in ⌬mxr1 to generate ⌬mxr1-GDH2-OE1 and ⌬mxr1-GDH2-OE2 strains respectively. Gdh2p Myc expression in cells cultured in YP medium was confirmed by Western blotting with anti-Myc tag antibodies (Fig. 4, A and B). Overexpression of Gdh2p Myc from either of these promoters was unable to restore growth of ⌬mxr1 in YP and YNB ϩ Glu media (Fig. 4C). The fact that expression of GDH2 results in the restoration of growth of ⌬gdh2 ( Fig. 2F) but not ⌬mxr1 strain indicates that Mxr1p may regulate the expression of other enzymes that are required for utilization of amino acids as the sole source of carbon in P. pastoris.
To examine whether mAAT and cAAT are required for the utilization of amino acids as the sole source of carbon in P. pastoris, they were expressed as Myc-tagged proteins, and their localization in mitochondria and cytoplasm, respectively, was confirmed by immunofluorescence using anti-Myc tag antibodies (Fig. 5A). P. pastoris ⌬aat1 and ⌬aat2 strains were generated, and their growth was examined in media containing different carbon sources. The results indicate that ⌬aat2 strain is unable to grow in YP, YNB ϩ Glu, and YNB ϩ Asp media indicating that cAAT is essential for the growth of P. pastoris in these media (Fig. 5B). qPCR analysis indicates that AAT1 and AAT2 are expressed at low levels in ⌬mxr1 strain (Fig. 5C). Overexpression of either Mxr1p or Mxr1p-N400 in ⌬mxr1 strain results in up-regulation of AAT1 and AAT2 expression in cells cultured in YP medium. However, only Mxr1p but not Mxr1p N400 restores the expression of these genes in cells cultured in YNB ϩ Glu (Fig. 5C).
Mxr1p Regulates the Expression of Genes Encoding mMDH, cMDH, and GLN1-In view of the crucial role of mMDH and cMDH in the transport of malate between cytoplasm and mitochondria (4), a detailed study was undertaken to examine their role during the utilization of amino acids as the sole source of carbon in P. pastoris. Immunofluorescence studies indicate that mMDH and cMDH are indeed localized in mitochondria and cytoplasm, respectively, in P. pastoris cultured in YP medium (Fig. 6A). P. pastoris ⌬mdh1 and ⌬mdh2 strains were generated, and their growth was examined in different media.
The results indicate that the growth of ⌬mdh1 is severely impaired in cells cultured in media containing glucose or amino acids as the sole source of carbon (Fig. 6B). However, the growth of ⌬mdh2 is affected only in cells cultured in YP or YNB ϩ Glu media (Fig. 6B). Thus, the generation of malate in the cytoplasm and its conversion to oxaloacetate in the mitochondria are important for the normal growth of cells cultured in YP and YNB ϩ Glu media. The expression of MDH1 as well as MDH2 is regulated by Mxr1p only in cells cultured in YNB ϩ Glu but not YP medium as evident from qPCR studies (Fig. 6C). Mxr1p also regulates the expression of GLN1 in cells cultured in YNB ϩ Glu as well as YP media (Fig. 6D). GLN1 was expressed from its own promoter as a Myc-tagged protein, and its localization in the cytoplasm was confirmed by immunofluorescence using anti-Myc tag antibodies (Fig. 6E). The function of GLN1 could not be examined further as a ⌬gln1 strain could not be generated, despite several attempts. Thus, overexpression of Mxr1p in ⌬mxr1 strain results in the restoration of synthesis of several key enzymes involved in amino acid metabolism in cells cultured in YP as well as YNB ϩ Glu media. In contrast, Mxr1p N400 restores the expression of only AAT1 and AAT2 in cells cultured in YP medium. It does not activate the transcription of any other target gene of Mxr1p in cells cultured The gene encoding Gdh2p His was expressed from its own promoter in different strains as indicated. E, quantitation of data presented in D. F, analysis of GDH2 mRNA levels by qPCR in different P. pastoris strains cultured in YP and YNB ϩ Glu media. Error bars indicate mean Ϯ S.D. *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.0005; ns, not significant. One-way analysis of variance followed by Tukey's multiple comparison test was carried out (n ϭ 3).
in YNB ϩ Glu medium. These results are summarized in Table 3.
Identification of Mxr1p Response Elements (MXREs) in the Promoters of AAT2, MDH2, and GLN1-Mxr1p regulates transcription of target genes by binding to MXREs present in their promoters. The MXREs identified so far contain a core motif whose consensus sequence is 5Ј-CYCCNY-3Ј ( Fig. 7A) (6,7,9,13). A sequence similar to this core MXRE is present between Ϫ151 and Ϫ146 bp (site A) of the promoter of AAT2, Ϫ213 and Ϫ208 bp of the promoter of MDH2, and Ϫ528 and Ϫ523 bp of FIGURE 4. Analysis of the ability of Gdh2p to restore the growth of ⌬mxr1. A, Western blotting analysis of lysates of ⌬mxr1, ⌬mxr1 overexpressing GDH2 from GAPDH promoter, or CUP1 promoter. B, quantitation of data presented in A. C, analysis of ability of Gdh2p overexpressed from GAPDH or CUP1 promoters to restore the growth of ⌬mxr1 cultured in YP and YNB ϩ Glu media. FIGURE 5. Function and regulation of mAAT and cAAT encoded by AAT1 and AAT2, respectively, in P. pastoris cells cultured in YP, YNB ؉ Glu, and YP ؉ Asp media. A, localization of Myc-tagged mAAT and cAAT in the mitochondria and cytoplasm, respectively, by immunofluorescence using anti-Myc tag antibodies. Hsp70 and DAPI were used as mitochondrial and nuclear markers, respectively. B, effect of deletion of genes encoding cAAT and mAAT on the growth of P. pastoris in YP, YNB ϩ Glu, and YP ϩ Asp media. C, quantification of AAT1 and AAT2 mRNAs by qPCR in various P. pastoris strains as indicated. Error bars indicate mean Ϯ S.D. *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.0005. One-way analysis of variance followed by Tukey's multiple comparison test was carried out (n ϭ 3). the promoter encoding GLN1 (Fig. 7, B, D, and E). In the region between Ϫ118 and Ϫ113 bp (site B) of AAT2 promoter, the sequence 5Ј-CCCCGA-3Ј is present (Fig. 7B), which is similar to the consensus sequence 5Ј-CYCCNR-3Ј. Studies with AOX1 promoter have shown that Mxr1p binds only to 5Ј-CYCCNY-3Ј but not 5Ј-CYCCNR-3Ј (6). To examine the ability of Mxr1p to bind to site A or site B of the AAT2 promoter, oligonucleotides carrying mutations in either site B (pAAT2-MXRE-M1), site A (pAAT2-MXRE-M2), or both (pAAT2-MXRE-M3) were synthesized (Fig. 7C), and their ability to bind to recombinant Histagged Mxr1p N150 (6,9) was examined by EMSA. Similarly, oligonucleotides carrying point mutations within the putative core MXREs of promoters encoding MDH2 and GLN1 were synthesized (Fig. 7, D and E), and their ability to bind to recombinant His-tagged Mxr1p N150 was examined by EMSA. The results indicate that Mxr1p 150 binds specifically to pAAT2-MXRE as well as pAAT2-MXRE-M1 but not pAAT2-MXRE-M2 and pAAT2-MXRE-M3 (Fig. 7F). Thus, Mxr1p binds specifically to site A but not site B of the AAT2 promoter. Addition of anti-His tag antibodies resulted in the supershift of the DNA-protein complexes, confirming the presence of recombinant Mxr1p N150 in these complexes (Fig. 7F). Mxr1p N150 also binds specifically to pMDH2-MXRE and pGLN1-MXRE but not to pMDH2-MXRE-M1 and pGLN1-MXRE-M1carrying point mutations within the core MXREs (Fig. 7F).
Chromatin immunoprecipitation (ChIP) studies were carried out to study the ability of Mxr1p N400 to bind to MXREs present in the promoters of AAT2 and GLN1 in cells cultured in YP medium because Mxr1p N400 activates the transcription of AAT2 but not GLN1 under these culture conditions (Table 3). Extracts were prepared from MXR1 N400 -OE strain cultured in YP, and the presence of Myc-tagged Mxr1p N400 was confirmed by Western blotting (Fig. 7G). Mxr1p N400 was cross-linked to chromatin and immunoprecipitated with anti-Myc antibodies. DNA was extracted, and PCR was carried out with primers that amplify AAT2 and GLN1 promoter regions containing MXREs (Fig. 7H). Only AAT2 but not GLN1 promoter was amplified by PCR ( Fig. 7I) indicating that Mxr1p N400 binds to the promoter of AAT2 but not GLN1 in cells cultured in YP medium. Thus, there is good correlation between promoter occupation in vivo and transcriptional activation by Mxr1p N400 (Fig. 7J).

Discussion
The integration of carbon and nitrogen metabolism with energy production is crucial for the survival of living organisms. Although yeasts such as S. cerevisiae utilize amino acids primarily for nitrogen metabolism, respiratory yeasts such as P. pastoris and P. stipitis have evolved pathways for efficient utilization of amino acids both as a source of carbon and nitro-gen. The generation of TCA cycle intermediates such as ␣-ketoglutarate and oxaloacetate from glutamate and aspartate is the first step in the utilization of amino acids as the sole source of carbon. While examining the role of Mxr1p in the regulation of various metabolic pathways, it was observed that P. pastoris GS115 but not ⌬mxr1 can grow in media containing amino acids as the sole source of carbon. This led us to study the enzymes involved in the utilization of amino acids as well as to examine the role of Mxr1p in the regulation of their biosynthesis. We demonstrate that enzymes such as GDH2, cAAT, mAAT, cMDH, and mMDH, which play a crucial role in the inter-conversion of amino acids and keto acids in the cytosolic and mitochondrial compartments as well as glutamine synthetase involved in glutamine synthesis, are important for the utilization of amino acids as the sole source of carbon and nitrogen in P. pastoris. Disruption of genes encoding cAAT and cMDH impairs the ability of P. pastoris to grow in media containing amino acids as the sole source of carbon. Although mAAT is not required for growth in YP, its disruption results in growth retardation in YNB ϩ Glu medium. Disruption of MDH1 severely impairs growth in media containing not only amino acids but also glucose as the sole source of carbon.
An important aspect of this study is the identification of Mxr1p as a regulator of synthesis of key enzymes involved in the utilization of amino acids. Biosynthesis of AAT1, AAT2, MDH1, MDH2, and GLN1 is regulated by Mxr1p at the level of transcription as evident from the significant reduction in mRNA levels of genes encoding these enzymes in ⌬mxr1 strain. Promoters of AAT2, MDH2, and GLN1 contain MXREs to which recombinant Mxr1p binds specifically in vitro. Using AAT2 and GLN1 as examples, we have demonstrated the importance of promoter occupancy by Mxr1p in vivo for transcriptional activation of these genes. Key enzymes identified in this study, whose synthesis is regulated by Mxr1p, are depicted schematically in Fig. 8A. We conclude that Mxr1p functions as a global regulator of multiple metabolic pathways in P. pastoris (Fig. 8B).
Amino acids enhance growth rate as well as recombinant protein production in several yeast species when added to media containing conventional carbon sources, primarily by serving as precursors for the synthesis of proteins as well as participating in anaplerotic reactions (16 -19). In the case of P. pastoris, mixed feeds of methanol and a multicarbon source instead of methanol as the sole carbon source have been shown to improve recombinant protein production (20). However, metabolic flux analysis during methanol metabolism in the presence of amino acids as a second source of carbon has not been examined thus far. The various P. pastoris strains described in this study can be exploited for these studies. Such FIGURE 6. Function and regulation of mMDH and cMDH in P. pastoris cells cultured in YP and YNB ؉ Glu media. A, localization of his-tagged mMDH and cMDH in the mitochondria and cytoplasm, respectively, by immunofluorescence using anti-His tag antibodies. The genes were expressed from their own promoters. Hsp70 was used as mitochondrial marker. DAPI was used as nuclear as well as mitochondrial marker. B, effect of deletion of genes encoding mMDH and cMDH on the growth of P. pastoris in YPD, YNB ϩ 2% glucose (YNBD), YP, and YNB ϩ Glu media. C and D, quantification of MDH1, MDH2, and GLN1 mRNAs by qPCR in various P. pastoris strains as indicated. Error bars indicate mean Ϯ S.D. *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.0005; ns, not significant. One-way analysis of variance followed by Tukey's multiple comparison test was carried out (n ϭ 3). E, localization of Myc-tagged glutamine synthetase (GLN) in the cytoplasm by immunofluorescence using anti-Myc tag antibodies. GLN1 was expressed from its own promoter. DAPI was used to stain nuclei. SEPTEMBER 23, 2016 • VOLUME 291 • NUMBER 39

JOURNAL OF BIOLOGICAL CHEMISTRY 20595
studies may provide new insights into the potential interactions between methanol metabolism and amino acid metabolism on heterologous protein production, leading to novel biotechnological applications.
A surprising result of this study is the regulation of GDH2 expression by Mxr1p at the translational level rather than the transcriptional level as evident from the reduction in GDH2 protein but not mRNA levels in the ⌬mxr1 strain. Furthermore,  6, 7, 13). B, nucleotide sequence of AAT2 promoter region between Ϫ105 and Ϫ162 bp. The nucleotide sequence of site A exactly matches with the pAOXI-MXRE consensus sequence (6) and that of site B differs by one nucleotide. C, nucleotide sequence of pAAT2-MXRE oligonucleotides used in EMSA. Mutated bases are indicated by arrows. D, oligonucleotides encompassing the MDH2 promoter region between Ϫ191 and Ϫ230 bp. The nucleotide sequence between Ϫ208 and Ϫ213 bp exactly matches with the MXRE consensus sequence. Mutated base is indicated by an arrow. E, oligonucleotides encompassing the GLN1 promoter region between Ϫ477 and Ϫ536 bp. The nucleotide sequence between Ϫ528 and Ϫ523 bp exactly matches with the MXRE consensus sequence. Mutated base is indicated by an arrow. F, EMSA with radiolabeled oligonucleotides and histidine-tagged recombinant Mxr1p containing 150 N-terminal amino acids encompassing the DNA binding domain (Mxr1p 150 ) as indicated. Mxr1p 150 has been described (6, 7). Supershift of DNA-Mxr1p 150 complex by the addition of anti-His antibodies is shown. G, Western blotting analysis of lysate of MXR1 N400 -OE strain cultured in YP medium using anti-Myc antibodies. H, schematic representation of location of primers used in the PCRs for the amplification of promoter regions of AAT2 and GLN1 following ChIP. ChIP was carried out using anti-Myc antibody. Black boxes indicate core MXREs (5Ј-CYCCNY-3Ј). I, analysis of enrichment of ChIP-ed DNA over the reference sample by qPCR (21,22). The data are expressed as Mxr1p N400 binding (ChIP/input) to AAT2 and GLN1 relative to binding (ChIP/input) at the TEL (telomere) region. TEL was used as a reference (14). Input refers to the amount of chromatin used in ChIP. The data represent the average of two independent experiments. J, table depicting correlation between promoter occupation by Mxr1p N400 in vivo and transcript levels of target genes (Table 3) in MXR1 N400 -OE strain cultured in YP medium. expression of Mxr1p but not Mxr1p N400 in ⌬mxr1 restores GDH2 protein levels indicating that the N-terminal region of Mxr1p alone is not sufficient for the post-transcriptional regulation of GDH2. GDH2 is the first example of a gene whose expression is regulated by Mxr1p at the post-transcriptional level (Fig. 8C). Mxr1p may not be directly involved in the regulation of translation of GDH2 mRNA because it localizes to the nucleus of cells cultured in YP medium. We hypothesize that Mxr1p activates the transcription of a gene whose product (protein X) is required for the efficient translation of GDH2 mRNA (Fig. 8C).

Experimental Procedures
Media and Culture Conditions-P. pastoris (GS115, his Ϫ ) was cultured in either minimal medium containing 0.17% yeast nitrogen base (YNB) without amino acids and 0.5% ammonium sulfate supplemented with 2.0% glucose (YNBD), 1.0% glutamate (YNB ϩ Glu), or 1% aspartate (YNB ϩ Asp) or nutrientrich YP medium (1.0% yeast extract and 2.0% peptone) alone or YP medium containing 2.0% glucose (YPD) or 2% methanol (YPM). S. cerevisiae BY4741 strain (Euroscarf) was cultured in YPD/YP medium. Yeast strains were grown at 30°C in an orbital shaker at 180 rpm. For growth assays, colonies were first cultured overnight in YPD medium, then washed with sterile water, and shifted to different media with initial A 600 of ϳ0.1. To assay growth in liquid medium, P. pastoris cells grown overnight in YPD were diluted in duplicate with fresh YPD to A 600 of 0.1, and aliquots of cells were removed at regular intervals, and A 600 was measured. Escherichia coli DH5␣ and BL21 (DE3) strains were used for the isolation of recombinant plasmids and expression of recombinant proteins, respectively. Bacterial and yeast transformations were done by CaCl 2 and electroporation method (Gene Pulser, Bio-Rad) respectively.
Antibodies and Other Reagents-Oligonucleotides and anti-FLAG antibodies were purchased from Sigma (Bangalore, India). Anti-His tag and anti-c-Myc tag antibodies were purchased from Thermo Fisher Scientific (Bangalore, India) and Merck Millipore (Bangalore, India) respectively.
Generation of P. pastoris Strain Expressing GDH2 His from Its Own Promoter-The gene encoding GDH2 along with 545 bp of its promoter was cloned into pIB3 vector (Addgene) as a histidine-tagged protein (GDH2 His ). GDH2 along with Ϫ545 bp of promoter was amplified from genomic DNA of P. pastoris by the primer pair 5Ј-CCGGAATTCCTCTCATGTTCGATATTC-CAGCGGCTTTC-3Ј and 5Ј-CCGCTCGAGCTAATGATGAT-GATGATGATGCAATCCCCGAGACTTGTAC-3Ј. EcoRI and XhoI sites are underlined. The histidine tag-encoding region is shown in italics. The PCR product was digested with EcoRI and XhoI and cloned into the pIB3 vector at the EcoRI and XhoI sites to obtain pIB3-GDH2 His . After linearization with BspHI, the plasmid was transformed to P. pastoris GS115 and ⌬mxr1 strains by electroporation to obtain GS115-GDH2 His and ⌬mxr1-GDH2 His strains, respectively.
Generation of P. pastoris ⌬mxr1 Strain Overexpressing Mxr1p, Mxr1p N400 , or GDH2 myc -P. pastoris ⌬mxr1 strain in which MXR1 coding region was replaced by a Zeocin expression cassette has been described (13). P. pastoris ⌬mxr1 strain overexpressing full-length Mxr1p (MXR1-OE) or a truncated Mxr1p containing 400 N-terminal amino acids (MXR1 N400 -OE) has been described (13). To generate ⌬mxr1 strain expressing Myc-tagged GDH2 (GDH2 Myc ) from the GAPDH promoter, the gene encoding GDH2 was amplified by PCR from P. pastoris genomic DNA using the primer pair 5Ј-CCGGAA-TTCATTATGGTCGACAGATCCAAG-3Ј and 5Ј-CGGGGT-ACCCAATCCCCGAGACTTGTAC-3Ј. EcoRI and KpnI sites FIGURE 8. A, schematic representation of key enzymes involved in the utilization of amino acids as the sole source of carbon in P. pastoris whose expression is regulated by Mxr1p. B, schematic diagram depicting Mxr1p as a global regulator of multiple metabolic pathways. Genes regulated by Mxr1p in various metabolic pathways are indicated. Mxr1p target genes in methanol and acetate utilization pathways have been described (5,13). C, model for the translational regulation of GDH2 by Mxr1p in cells cultured in YP or YNB ϩ Glu media. We hypothesize that Mxr1p regulates the transcription of a gene encoding protein X, which is required for the translation of GDH2 mRNA. See under "Discussion" for details.
in the primers are underlined. The gene was cloned at the EcoRI and KpnI sites of the pGAPZA vector, and the recombinant plasmid thus obtained (pGAPDH-GDH2 myc ) was transformed into ⌬mxr1 strain to obtain ⌬mxr1-GDH2-OE1. The pCUP1-GDH2 expression cassette containing S. cerevisiae copper-inducible promoter (pCUP1) (15) was generated by a series of overlapping PCRs. The S. cerevisiae CUP1 promoter region between Ϫ460 and Ϫ1 bp was amplified by PCR from S. cerevisiae genomic DNA using the primer pair 5Ј-CCGGAATTCG-CCGATCCCATTACCGAC-3Ј (Ϫ460 to Ϫ443 bp of CUP promoter, EcoRI site is underlined) and 5Ј-GACACTTGGAG-TCTGTCGACCATTTTATGTGATGATTGATTGATTGA-TTG-3Ј (region from ϩ1 to ϩ23 bp of GDH2 and Ϫ32 to Ϫ1 bp of CUP1 promoter (italics)). In another PCR, GDH2 was amplified from P. pastoris genomic DNA using the primer pair 5Ј-CAATCAATCAATCAATCATCACATAAATGGTCGACAG-ACTCCAAGTGTC-3Ј (Ϫ32 to Ϫ1 bp of CUP promoter, ϩ1 to ϩ23 bp of GDH2 (italics)) and 5Ј-CGGGGTACCCAATCCC-CGAGACTTGTAC-3Ј (BamHI site is underlined). The products of the two PCRs were used as templates in the final PCR and amplified using the primer pair 5Ј-CCGGAATTCGCCG-ATCCCATTACCAC-3Ј and 5Ј-CGGGGTACCCAATCCCC-GAGACTTGTAC-3Ј (EcoRI and BamHI sites are underlined). The pCUP1-GDH2 expression cassette thus obtained was digested with EcoRI and KpnI and cloned into pGAPZA vector to obtain pCUP-GDH2 myc plasmid. The recombinant plasmid was linearized with AvrII and transformed to P. pastoris ⌬mxr1 strain by electroporation to obtain ⌬mxr1-GDH2-OE2.
Construction of P. pastoris ⌬gdh2, ⌬aat1, ⌬aat2, ⌬mdh1, and ⌬mdh2 Strains-The coding regions of genes encoding GDH2, AAT1, AAT2, MDH1, and MDH2 were deleted by homologous recombination method, using resistance to Zeocin (Zeocin R ) as selection marker. The gene deletion constructs for each of these genes consisted of ϳ1 kb of promoter, ϳ1.2 kb of Zeocin expression cassette, and ϳ1 kb of 3Ј-flanking sequences. The promoters and 3Ј-flanking regions of respective genes were amplified by PCR from P. pastoris GS115 genomic DNA, and the Zeocin expression cassette was amplified from pGAPZA (Invitrogen).
In the third PCR, the 3Ј-flanking region of MDH1 was amplified using the primer pair 5Ј-GCTGGAGACCAACATGTGAGCTC-CTGGGTGACGATATTGAACG-3Ј (ϩ3130 to ϩ3151 bp of pGAPZA, ϩ1060 to ϩ1081 bp of 3Ј-flanking region of MDH1) and 5Ј-CGTGGGGATGGACATGATCGTGG-3Ј (ϩ1997 to ϩ2019 bp in the reverse complement of 3Ј-flanking region of MDH1). All three PCR products were purified and used as templates in the final PCR along with the primer pair 5Ј-CGTGGGGATGGA-CATGATCGTGG-3Ј and 5Ј-TGTGTAGCTCTGAACTC-GTTG-3Ј to obtain a 3.2-kb product consisting of Zeocin expression cassette flanked by ϳ1 kb of MDH1 promoter and 1 kb of 3Ј-flanking region of MDH1 that was transformed into P. pastoris GS115. Zeocin-resistant colonies were selected, and deletion of MDH1 was confirmed by PCR using gene-specific primers.
MDH1 was amplified by PCR with the primer pair 5Ј-CGC-GGATCCTGTGTAGCTCTGAACTCGTTG-3Ј (EcoRI site is underlined) and 5Ј-CCGCTCGAGCTAATGATGATGAT-GATGATGTGGGTTTTGCTTAACAAACTC-3Ј (XhoI site underlined, DNA encoding His tag is shown in italics). MDH2 was amplified by PCR using the primer pair 5Ј-CGCGGA-TCCGAGGACGTGTGGGATTAGAAAG-3Ј (EcoRI site is underlined) and 5Ј-CCGCTCGAGCTAATGATGATGATGA-TGATGGTTGCCAGCAATGAAGGCAGTTC-3Ј (XhoI site is underlined; DNA encoding His tag is shown in italics). P. pastoris genomic DNA was used as the template. The MDH1 and MDH2 expression cassettes were digested with EcoRI and XhoI and cloned into pIB3 vector to obtain pIB3-MDH1 His and pIB3-MDH2 His , respectively. These plasmids were transformed into P. pastoris GS115 to obtain MDH1 His and MDH2 His strains, respectively.
GLN1 along with Ϫ1000 bp of its own promoter was amplified by PCR with primer pairs 5Ј-CCGCTCGAGTCAGTATT-AATCTTGTCACATGACCTAC-3Ј and 5Ј-TAAGAATGCG-GCCGCTATCAGATTCTCTCTTGTACTCCTTG-3Ј from P. pastoris genomic DNA. KpnI and NotI restriction sites are underlined. PCR product was digested with KpnI and NotI and cloned into pGAPZA vector. The recombinant plasmid (pGAPZA-GLN1 Myc ) was linearized and transformed to GS115 to obtain GLN1 Myc strain. P. pastoris strains and plasmids used in this study are listed in Tables 1 and 2, respectively. Western Blotting-Total protein extracts were prepared from P. pastoris by vortexing with glass beads, and cell lysates containing 50 g of proteins were resolved by PAGE in the presence of SDS. The resolved proteins were transferred to a PVDF membrane using an electroblotting apparatus in transfer buffer (39 mM glycine, 48 mM Tris-HCl (pH 8.0), 20% methanol). The membrane was blocked overnight in 5% nonfat milk (HiMedia) prepared in 1ϫ TBS (25 mM Tris-HCl, 125 mM NaCl (pH 8.0)). The protein of interest was detected by using antibodies raised against the specific protein or anti-epitope tag antibodies. Primary antibodies were detected by peroxidase-conjugated antirabbit/anti-mouse IgG secondary antibody (1:10,000 dilutions; Bangalore Genei, India). The blots were developed with chemiluminescence plus reagents (PerkinElmer Life Sciences) according to the manufacturer's instructions.
Subcellular Localization-Subcellular localization of epitopetagged proteins was studied by immunofluorescence using a fluorescent microscope (Leica) and Zeiss confocal microscope (Zeiss 510 Meta). Immunofluorescence was carried out essentially as described (8).
Statistical Analysis-Statistical tests were carried out by oneway analysis of variance followed by Tukey's multiple comparison. GraphPad Prism 5 software was used. Data are presented as mean Ϯ S.D. p value summary is mentioned on the bar of each figure as follows: *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.0005, ns, not significant.
DNA-Protein Interactions-Recombinant Mxr1p N150 consisting of 150 N-terminal amino acids of Mxr1p was expressed as His-tagged protein in E. coli and purified essentially as described (5). The ability of Mxr1p N150 to bind to 32 P-labeled oligonucleotides containing MXREs of promoters encoding AAT2, MDH2, and GLN1 was examined by electrophoretic