Mks1p Is a Regulator of Nitrogen Catabolism Upstream of Ure2p in Saccharomyces cerevisiae

Corresponding author: Reed B. Wickner, Bldg. 8, Rm. 225, National Institutes of Health, 8 Center Dr., MSC 0830, Bethesda, MD 20892-0830., wickner{at}helix.nih.gov (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation. Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation. Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa⁺) when cells grow on a poor nitrogen source such as proline. We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline. Overexpression of Mks1p does not affect cellular levels of Ure2p. An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline. Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia. These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH₃ Mks1p Ure2p Gln3p DAL5. Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth.

BOTH the abundance and the chemical nature of environmental nitrogen sources provide important cues regulating cellular events. Virulence of the fungal tomato pathogen Cladosporium fulvum and the rice blast fungus Magnaporthe grisea are closely connected to nitrogen regulation. Saccharomyces, among other microorganisms, can utilize multiple nitrogen sources and can discriminate among them, repressing the utilization of poor nitrogen sources when good sources are available (COOPER 1982 ; MAGASANIK 1992 ; MARZLUF 1997 ). This is referred to as nitrogen catabolite repression, nitrogen regulation, or nitrogen control. Nitrogen supply also regulates pseudohyphal formation, an asymmetric mode of growth that allows yeast to forage for nutrients (GIMENO et al. 1992 ). In addition, nitrogen starvation is a signal for entrance into meiosis and spore formation or a resting state.

In the presence of ammonia, a good nitrogen source, yeast turns off utilization of poor sources, such as allantoate and proline. This nitrogen catabolite repression is determined by several GATA transcription factors, each regulated somewhat differently by various nitrogen sources and each having a different spectrum of action on genes encoding proteins involved in assimilation of poor nitrogen sources (MITCHELL and MAGASANIK 1984 ; COOPER et al. 1990 ; CUNNINGHAM and COOPER 1991 ; MINEHART and MAGASANIK 1991 ; STANBROUGH et al. 1995 ; TABILI et al. 1995 ; XU et al. 1995 ; COFFMAN et al. 1996 , COFFMAN et al. 1997 ). Gln3p is a positively acting GATA transcription factor whose activity is negatively regulated by Ure2p (MITCHELL and MAGASANIK 1984 ; COURCHESNE and MAGASANIK 1988 ). Ure2p activity, in turn, is regulated by the availability of a good nitrogen source, including ammonia (DRILLIEN and LACROUTE 1972 ; DRILLIEN et al. 1973 ). Among the targets of Gln3p is DAL5, the allantoate permease gene (RAI et al. 1987 ; TUROSCY and COOPER 1987 ). The structural resemblance of ureidosuccinate (USA) to allantoate results in Dal5p being able to transport USA into cells. The imported USA can feed a uracil auxotroph blocked in aspartate transcarbamylase (ura2). The regulatory pathway is, formally, NH₃ Ure2p Gln3p DAL5 USA uptake. Thus, wild-type cells can take up USA when growing on a poor nitrogen source such as proline (Usa⁺), but cannot take up USA on ammonia (Usa^-). ure2 mutants are Usa⁺ even on ammonia-containing media. Among the outstanding questions concerning this pathway is the means by which the availability of ammonia is signaled to Ure2p.

[URE3] is a non-Mendelian genetic element (LACROUTE 1971 ; AIGLE and LACROUTE 1975 ) that is due to a prion (infectious protein) form of Ure2p (WICKNER 1994 ; MASISON and WICKNER 1995 ; MASISON et al. 1997 ; EDSKES et al. 1999 ; TAYLOR et al. 1999 ). In [URE3] cells, Ure2p is in an aggregated, protease-resistant form (MASISON and WICKNER 1995 ; EDSKES et al. 1999 ) that is inactive in nitrogen regulation, apparently because it has assumed an amyloid structure (TAYLOR et al. 1999 ). Thus, [URE3] strains, like ure2 mutants, can take up USA on ammonia-containing media. Overexpression of Ure2p leads to a 100-fold increase in the frequency with which [URE3] arises (WICKNER 1994 ).

multicopy kinase suppressor (MKS1) was first isolated in a screen aimed at detecting a negative regulator downstream of the Ras-cyclic AMP pathway (MATSUURA and ANRAKU 1993 ). MKS1 overexpression prevented TPK1, encoding a catalytic subunit of the cAMP-dependent protein kinase, from relieving the growth defect of ras1 ras2^ts cells deficient in cAMP (MATSUURA and ANRAKU 1993 ). Deletion of MKS1 partially relieved the growth defect of a cyr1^ts (adenylate cyclase) strain. However, MKS1 overexpression did not affect cAMP levels or expression of the A-kinase. The mks1 mutants were also found to be unable to grow on galactose or on glycerol media, indicating their pleiotropic nature (MATSUURA and ANRAKU 1993 ). The authors suggested that Mks1p was a negative regulator of growth, acting either downstream of TPK1 or in a parallel pathway (MATSUURA and ANRAKU 1993 ). MKS1 was later found to be identical to LYS80, a negative regulator of lysine biosynthesis (FELLER et al. 1997 ). The lys80 mutants had higher than normal pools of -ketoglutarate and glutamate, with elevated activities of several tricarboxylic acid cycle enzymes (FELLER et al. 1997 ); it was suggested that lysine overproduction resulted from elevated -ketoglutarate.

We sought to isolate genes, other than URE2, which, when overexpressed, could induce the appearance of [URE3], and thus make the cells Usa⁺ on ammonia. We isolated MKS1 in this screen and found that Mks1p overexpression made cells Usa⁺ without inducing the appearance of [URE3]. Our analysis indicates that Mks1p is involved in the control of nitrogen catabolism.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
Media were as described (SHERMAN 1991 ). Ureidosuccinate was added to minimal media at 100 µg/ml. When used as nitrogen sources, glutamate, glutamine, proline, and asparagine were added to the medium at 1 g/liter. Pseudohyphal induction was performed as described (GIMENO et al. 1992 ). Strains used are shown in Table 1. MAT spore clones of strains MLY61, MLY104, MLY108, MLY115, MLY128, MLY129, MLY130, and MLY131 were used to assess the role of ammonium permeases in growth inhibition by Mks1p overproduction.

Strain 12T7clys80 was crossed with strain 1278b and after sporulation and tetrad dissection two spore clones were obtained, YHE670 (73-6A: MATa ura3) and YHE672 (73-6C: MAT lys80 {LYS80 = MKS1}). YHE670 was crossed with 1278b and YHE672 was crossed with 12T7clys80, resulting in strains YHE676 (MKS1/MKS1) and YHE677 (mks1/mks1), respectively.

Strain YHE672 (73-6C: MAT lys80) was crossed with strain YHE678 (MATa ura2; this ura2 allele had been backcrossed 10 times with strain 1278b) and after sporulation and tetrad dissection ura2 and ura2 mks1 strains were obtained.

Subclones of B54:
Clone B54 contains a 4.8-kb fragment from chromosome 14 (Fig 1). Fusing the HindIII-BamHI sites of B54 removed 1656-bp encompassing YNL076w (MKS1) and the 5' region of YNL075w, resulting in clone pH64. YNL075w was subcloned as an EcoRV-StuI fragment into the SmaI site of pRS425 (CHRISTIANSON et al. 1992 ), resulting in clone pH79. The coding sequence of YNL075w was also amplified by PCR using Pfu polymerase and primers HE31 (5'-CAA AGA TCT CAA ATG CTA AGA AGA CAA GCC-3') and HE32 (5'-GTT CTC GAG CCT CAT CGG CCT TCT ATT-3') and placed under control of the ADH1 promoter in the high copy number vector pH7 (EDSKES et al. 1999 ), resulting in clone pH47. The sequence of the clones used in this study was determined and has been deposited in GenBank (accession no. bankit272474). At least two independently derived clones of each part of the MKS1 gene were sequenced. In addition to several silent changes, our sequence revealed the following differences from the genome project sequence (R. POEHLMANN and P. PHILIPPSEN, GenBank accession nos. Z71351 and Y13139): H55Y, P144A, D154E, P411T, E450G. The sequence of MATSUURA and ANRAKU 1993 lacks two G residues 149 and 150 nt 3' of the A of the ATG in the genome project sequence and our sequence. This frameshift led Matsuura and Anraku to assign the start of the open reading frame (ORF) at what is Met127 of our sequence.

View larger version (57K): In this window In a new window Download PPT slide   Figure 1. MKS1 overexpression allows uptake of USA on ammonium and also slows growth on ammonium medium. (A) Plasmid B54 induces bypass of nitrogen regulation in strain 3686 as cells containing it can grow on NH4/USA medium. (B) Map of B54 and the location of MKS1 (YNL076w). (C) Overexpression of fragments of MKS1 from centromere plasmids promotes growth on NH4/USA medium in strain 3686. The fragments overexpressed in centromere (CEN) or 2µ DNA (2µ) plasmids are shown as solid lines and the part deleted as dashed lines. (D) Slowing of growth by overexpression of fragments of MKS1 from CEN (top) and 2µ (bottom) plasmids in strain 3686 on different nitrogen sources.

Mks1p expression plasmids:
The 2µ vector pH7 (EDSKES et al. 1999 ) and the centromere vector pH62 carry LEU2. pH62 was created by cloning the ADH1 promoter as a blunted SphI fragment from pVT103 (VERNET et al. 1987 ) into PvuII-digested pRS315 (SIKORSKI and HIETER 1989 ). The ADH1 cassette is directed opposite to the LEU2 gene.

All PCR reactions used genomic DNA of yeast strain 1278b as template and were performed with Pfu polymerase (Stratagene, La Jolla, CA). PCR products were cloned as BamHI-XhoI fragments into the BamHI-XhoI window of the expression vectors pH7 and pH62 (Table 2).

Expression vectors pH230 and pH231, directing full-length Mks1p expression, were created by inserting the HindIII-XhoI fragment from the PCR product generated with oligos HE37/HE38 into pBCKS+ (Stratagene) carrying the PCR product obtained from oligos HE63/HE79 and then transferring the resulting MKS1 ORF as a BamHI-XhoI fragment into the expression vectors pH62 and pH7, respectively.

Vectors pH130 and pH131, containing URA3, were constructed by inserting the ADH1 cassette, amplified by PCR from pH7 using oligos HE66 (5'-ACA GCT AGC ATT ACG CCA GCA ACT TCT-3') and HE67 (5'-ACA AGA TCT TAA TGC AGC CGG TAG AG-3'), into PvuII-digested pRS316 (SIKORSKI and HIETER 1989 ) and pRS426 (CHRISTIANSON et al. 1992 ), respectively. In both pH130 and pH131 the ADH1 cassette is directed opposite to the URA3 gene. The MKS1 ORF present as a BamHI-XhoI fragment in the expression vectors pH320 and pH321 was cloned in the BamHI-XhoI window of pH130 and pH131, resulting in constructs pH319 and pH320, respectively.

Ure2p expression plasmids and DAL5-lacZ fusion plasmid:
The ADH1 cassette, amplified as above from pH7 using oligos HE66 and HE67, was ligated into PvuII-treated pRS424 (CHRISTIANSON et al. 1992 ), resulting in pH123 with the ADH1 cassette directed opposite to TRP1. URE2 and the URE2 C terminus (residues 66–354) were cloned as BamHI-XhoI fragments from pH14 and pH13 (EDSKES et al. 1999 ) into the BamHI-XhoI window of pH123, resulting in constructs pH239 and pH240, respectively. pRR29 is a TRP1-ARS1 shuttle plasmid expressing lacZ from the DAL5 promoter (RAI et al. 1989 ) and was the generous gift of Terry Cooper.

GFP-Mks1p expression vector:
The green fluorescent protein (GFP)-URE2C expression vector pH198 (EDSKES et al. 1999 ) contains a NotI site at the border between GFP and URE2C. This NotI site was changed into a BamHI site using oligo HE120 (5'-ggc cag gat cct-3'), resulting in pH314. The BamHI fragment of pH314 encompassing GFP was ligated into BamHI-digested pH230, creating pH315.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Overexpression of MKS1 bypasses nitrogen control:
Growth of a ura2 mutant on USA in the presence of ammonia selects for inactivity of Ure2p, including cells in which Ure2p has become the prion, [URE3]. We used a high copy plasmid library to screen for genes that when overexpressed directed the uptake of USA in the presence of ammonium.

A 2µ LEU2 genomic library (NASMYTH and REED 1980 ) was used to transform strains 3385 and 3686. Approximately 65,000 transformant colonies were replica plated onto minimal plates with both ammonium and USA. One positive colony contained a plasmid harboring GLN3. Another colony (B54) was identified that contained a plasmid directing uptake of USA with high efficiency (Fig 1A). However, colonies that had subsequently lost pB54 were no longer Usa⁺, indicating that [URE3] had not been generated.

Plasmid B54 contains a 4.8-kb fragment from chromosome 14 (Fig 1B). This fragment starts at nucleotide 649 3' of the ATG start codon of YNL076w (MKS1, multicopy compensator of A-kinase suppression) and ends in YNL073w (MSK1, mitochondrial lysine-tRNA synthetase). Between MKS1 and MSK1 are YNL075w and YNL074c, both with unknown functions. Thus pB54 does not contain any known nitrogen regulatory genes.

Deletion of a HindIII/BamHI fragment from pB54, encompassing all of MKS1 and 250 bp of the coding region of YNL075w, resulted in the inability to induce bypass of nitrogen regulation. This was not caused by the deletion of part of YNL075w, as was confirmed by creating two clones, pH79 and pH47. In pH79 the EcoRV/StuI fragment from pB54 (bp 955–2364) encompassing the promoter and the whole coding region of YNL075w was cloned into the LEU2 2µ vector pRS425. In pH47 the coding region of YNL075w was amplified by PCR and cloned under control of the ADH1 expression signals in a LEU2 2µ vector, pH7 (EDSKES et al. 1999 ). Neither of these clones allowed strain 3686 to take up USA in the presence of ammonium. Thus the ability of pB54 to direct bypass of nitrogen regulation is related to the presence of a fragment of MKS1.

A C-terminal fragment from MKS1 starting at methionine M127 (bp 379), corresponding to the ORF described by MATSUURA and ANRAKU 1993 , was obtained by PCR from strain 1278b. Expression of this MKS1 fragment from pH76 in strain 3686 resulted in bypass of nitrogen regulation as shown by the ability to take up USA in the presence of ammonium (Fig 1C).

Overexpression of MKS1 results in a slow-growth phenotype:
This same plasmid (pH76), expressing a C-terminal fragment of Mks1p from an ADH1 promoter on a centromeric plasmid, gave cells a mild slow-growth phenotype in either strain 3686 (Fig 1D, top) or in YHE371, congenic with 1278b (data not shown). This slow-growth phenotype became very severe when the Mks1p fragment was expressed from a high copy number plasmid (pH77; Fig 1D, bottom). However, pB54 directs efficient uptake of USA without causing a noticeable growth defect, perhaps because it expresses Mks1p at a lower level than the ADH1-promoted constructs (Fig 1A). In contrast to the slow growth produced on ammonia, when cells overexpressing the Mks1p C terminus are grown on proline as the sole nitrogen source little or no slow-growth phenotype was observed (pH77; Fig 1D). As with ammonia, overexpression of this Mks1p fragment substantially slows growth on the good nitrogen sources asparagine and glutamine and, to a lesser degree, on the intermediate source, glutamate (Fig 1D).

Mks1p domain that inhibits growth on ammonium and relieves nitrogen regulation:
To delineate the portion of MKS1 causing slow growth on ammonia and nitrogen deregulation, a series of MKS1 deletion constructs in both centromere and high copy plasmids was prepared (MATERIALS AND METHODS; Fig 1C and Fig D).

Deleting up to 244 amino acids from the N terminus (pH135, pH134) or to amino acid 346 from the C terminus (pH226, pH227) of Mks1p had no effect on its ability to slow growth on ammonium (Fig 1C and Fig D), but further deletions abolished this activity. Thus, a domain of Mks1p, from Met245 through Asn340 (base pairs 734 and 1019), is needed to induce a slow-growth phenotype on ammonium medium. A very similar, but weaker, sequence-dependent slow-growth phenotype is observed on asparagine medium (Fig 1D). The slow-growth induction pattern is similar for the centromeric and the high copy number expression vectors. However, on glutamate or glutamine medium, among the centromeric expression plasmids, only pH230 and pH76, expressing the whole Mks1p or the N-terminal truncated Mks1p that starts at Met127, respectively, reduced growth. Growth reduction is only slightly stronger when the Mks1p fragments are expressed from high copy number plasmids on these nitrogen sources. On medium containing proline as the sole nitrogen source, overexpression of Mks1p, or its derivatives, has only a very slight effect on growth and this only when a high copy plasmid was used (Fig 1D).

All the Mks1p fragments that reduce growth in the presence of good nitrogen sources also direct uptake of USA in the presence of ammonium (Fig 1C), although the two abilities compete with each other.

Deletion of MKS1 prevents expression of Dal5 permease on proline medium:
Neither strain YHE711 (ura2) nor an isogenic strain YHE710 in which MKS1 was deleted (ura2 mks1) could take up USA in the presence of ammonium (data not shown). When these two strains were plated on media containing proline as the sole nitrogen source only the wild-type strain could take up USA (data not shown). Thus deletion of MKS1 prevents appearance of the Dal5 permease activity under nitrogen-derepressing conditions.

ß-Galactosidase was expressed from a DAL5 promoter-lacZ fusion plasmid (pRR29; RAI et al. 1989 ) in an mks1 deletion mutant (YHE83-1B) and isogenic wild type (YHE83-2A), both grown on proline. ß-Galactosidase activities were 1.56 OD₄₂₀/20 min/OD₅₅₀ for the wild type, and 0.022 OD₄₂₀/20 min/OD₅₅₀ for the mutant. Therefore, Mks1p is a positive regulator of Dal5p expression.

ure2 is epistatic to mks1:
To distinguish whether Mks1p acts directly to activate DAL5 or through the established pathway involving Ure2p, we examined tetrads from a diploid heterozygous for both ure2 and for mks1 (Fig 2). As shown above, mks1 segregants were Usa^- on both ammonia and proline media, and ure2 segregants were Usa⁺. The mks1 ure2 double mutants were Usa⁺ on both ammonia and proline media, indicating that the ure2 mutation was epistatic. This suggests that Mks1p activates DAL5 through the Ure2p pathway, rather than through an alternate route, and that Ure2p is downstream of Mks1p in this path.

View larger version (112K): In this window In a new window Download PPT slide   Figure 2. An mks1 mutation prevents uptake of USA on proline, an effect suppressed by ure2. The ura2 mks1 strain 4657-1B was crossed with the ura2 ure2 strain 4657-1C and the segregation of mks1 and ure2 were scored by G418 resistance and complementation tests, respectively. A tetratype tetrad (4664-2ABCD) was streaked on the indicated media.

Overexpression of Ure2p and Mks1p:
When strain 3686 carrying the MKS1 expression plasmid pH230 was cotransformed with vectors directing either the expression of Ure2p or the C-terminal fragment of Ure2p, slow growth was still observed on ammonium medium. Likewise, overexpression of Mks1p from pH230 or pH231 in a strain with a ure2 deletion (YHE311) caused slow growth on ammonium medium. However, the ability to take up USA on ammonium medium induced by pH230 was blocked by overexpression of Ure2p or Ure2Cp when expressed from the ADH1 promoter on a high copy plasmid in strain YHE751 (Fig 3). This result again indicates that Mks1p acts through Ure2p to regulate nitrogen metabolism. Overexpression of Mks1p in a gln3 mutant (MLY139a) shows a growth-slowing effect similar to that seen in an isogenic wild-type strain (YHE732). This indicates that, unlike the effect on nitrogen catabolism, the effect of Mks1p on growth includes a component independent of the Ure2p-Gln3p pathway.

View larger version (74K): In this window In a new window Download PPT slide   Figure 3. Overexpression of Ure2p suppresses the Usa+ phenotype resulting from overexpression of Mks1p. Strain YHE751 transformed with the indicated plasmids was streaked on ammonium medium with USA in place of uracil.

Ammonium levels are not signaled to MKS1 through ammonium permeases:
Overexpression of MKS1 slows growth most dramatically when ammonium is the nitrogen source. The possibility that MKS1 receives a signal about ammonium in the environment through one of the three ammonium permeases, encoded by MEP1, MEP2, and MEP3, was tested. These experiments were motivated by the finding that Mep2p signals ammonium starvation to the pseudohyphal growth pathway (LORENZ and HEITMAN 1998 ). High and low copy URA3 plasmids in which Mks1p expression was directed by the ADH1 promoter were introduced into strains in which each of the three ammonium permeases was deleted individually or in combination. These plasmids still induced a severe growth inhibition on ammonium-containing medium (5 g/liter of ammonium sulfate; data not shown). Control plasmids without MKS1 sequences did not inhibit growth. Because all transformants were prototrophic, the slow-growth phenotype could not be due to inhibition of an amino acid permease needed for growth (SCHMIDT et al. 1998 ).

Bypass of nitrogen regulation by overexpressed Mks1p is not reversed by cAMP addition:
Since previous work suggested that Mks1p is downstream of the Ras-cAMP pathway and may be negatively regulated by cAMP (MATSUURA and ANRAKU 1993 ), we analyzed the effect of adding extracellular cAMP on the ammonium/USA growth phenotypes. Strains YHE711 and YHE371 were transformed with pH7, pH231, pH62, and pH230. In both strains overexpression of Mks1p directed by pH230 or pH231 resulted in the uptake of USA in the presence of ammonium, but neither of the two control vectors pH7 and pH62 conferred this ability (data not shown). Addition of 1, 3, or 10 mM cAMP did not change this growth pattern. Note that in the 1278b background, cAMP in the concentration range used here can be taken up and utilized when added to the growth medium (KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ).

These same strains were assayed for growth inhibition on ammonium medium. As previously noted, overexpression of Mks1p severely inhibited the growth of these cells on ammonium medium, and addition of 1, 3, or 10 mM cAMP did not change this growth pattern (data not shown).

Overexpression or deletion of MKS1 reduces pseudohyphae formation:
MKS1 was originally cloned as a negative regulator downstream of the RAS-cyclic AMP pathway (MATSUURA and ANRAKU 1993 ). Here we identified MKS1 by its ability to suppress nitrogen regulation resulting in expression of Dal5p when the good nitrogen source ammonium is present. When nitrogen in the medium is growth limiting, S. cerevisiae undergoes a dimorphic switch (GIMENO et al. 1992 ), enabling cells to forage for nutrients through growth as pseudohyphae. A MAP kinase pathway including many elements of the pheromone response cascade regulates pseudohyphal differentiation (LIU et al. 1993 ). The RAS-cyclic AMP pathway also regulates this filamentous growth (KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ). Because MKS1 is involved in both nitrogen regulation and the RAS-cyclic AMP pathway we considered the possibility that Mks1p also influences the dimorphic switch.

When cells carrying the expression vector controls were streaked onto nitrogen-starvation plates pseudohyphae developed readily (Fig 4A and Fig B). However, pseudohyphal development was severely impaired when either full-length Mks1p or a C-terminal fragment of Mks1p, starting at Met127, was expressed from a centromeric vector (Fig 4D and Fig F). No pseudohyphae developed when either was expressed from a high copy number vector (Fig 4C and Fig E). Thus expression of Mks1p reduces the ability of yeast cells to undergo a dimorphic switch in a concentration-dependent manner.

View larger version (58K): In this window In a new window Download PPT slide   Figure 4. Over- or underproduction of Mks1p impairs pseudohyphal growth. Strains were streaked to single colonies on nitrogen-starvation medium. Colonies were observed after 3 days. Strain YHE371 carried (A) the 2µ vector (pH7) or (C) pH76 expressing Mks1p127-584 or (E) pH230 expressing full-length Mks1p from CEN plasmids and (B) the CEN vector (pH62) or (D) pH77 expressing Mks1p127-584 or (F) pH231 expressing full-length Mks1p from 2µ DNA plasmids. (G) Strain YHE676 (MATa/MAT ura3/+) and (H) strain YHE710 (MATa/MAT mks1/mks1 ura3/+) were treated as above. For each strain, an isolated single colony and the border of the heavy streak of cells are shown.

Because overexpression of a C-terminal fragment of Mks1p resulted in reduced filamentation we expected that deletion of MKS1 would result in increased pseudohyphal development. However, when otherwise isogenic MKS1 and mks1 strains were streaked onto nitrogen-starvation medium only the wild-type cells formed pseudohyphae (Fig 4G and Fig H). Thus, both overexpression and depletion of Mks1p reduces the ability of S. cerevisiae to undergo filamentous growth.

Mks1p overexpressing strains express Ure2p normally:
Expression levels of Ure2p were compared in isogenic wild-type and Mks1p-overexpressing strains by immunoblot (Fig 5). The overexpression of Mks1p from either a CEN plasmid or a 2µ plasmid did not affect Ure2p levels in cells with the normal single-copy URE2 gene. This was true whether cells were grown on proline or on ammonia as the nitrogen source. Controls showed that the immunoblot detection was linear in the range used. Thus, the effects of MKS1 on nitrogen control are not due to effects on URE2 expression or stability.

View larger version (46K): In this window In a new window Download PPT slide   Figure 5. Mks1p overexpression does not affect expression of Ure2p. Immunoblot analysis of extracts was carried out using rabbit anti-Ure2p (WICKNER 1994 ) as described (MASISON and WICKNER 1995 ). Strain YHE371 carrying different plasmids was grown in synthetic complete medium minus leucine with proline (1 g/liter) as the nitrogen source to OD600 = 0.5. Part of each culture was harvested and extracts were made as described (MASISON and WICKNER 1995 ). Another aliquot was centrifuged and resuspended at OD600 = 0.1 in synthetic complete medium minus leucine with ammonium as the nitrogen source. After 6–8 hr of growth, cells were harvested and extracted. Twenty micrograms of protein of each extract was used. Another blot (not shown) with 40 µg of protein showed that these measurements were in the linear range for detection of Ure2p. Lane 1, pH62 (CEN vector) on proline; lane 2, pH230 (CEN MKS1) on proline; lane 3, pH7 (2µ vector) on proline; lane 4, pH231 (2µ MKS1) on proline; lane 5, pH62 on ammonium; lane 6, pH230 on ammonium; lane 7, pH7 on ammonium; lane 8, pH231 on ammonium.

Localization of Mks1p:
Because MKS1 interacts with the RAS-cAMP pathway and participates in nitrogen signaling, Mks1p could be associated with the plasma membrane. To localize Mks1p, GFP was fused to the N terminus of Mks1p. This fusion protein was evenly distributed in the cytoplasm of strain 3686 grown on proline medium (Fig 6). When cells were patched onto ammonium medium the distribution of the GFP-Mks1 fusion protein remained cytoplasmic (data not shown). The fusion protein still severely inhibited growth and induced USA uptake on ammonium-containing medium, indicating that the Mks1-GFP fusion protein was active.

View larger version (69K): In this window In a new window Download PPT slide   Figure 6. Localization of GFP-Mks1 fusion protein. Strain 3686 carrying either pH315 (GFP-Mks1p) or pH200 (GFP) was examined by fluorescence microscopy.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The role of Mks1p in nitrogen regulation:
We present three lines of evidence suggesting that MKS1 is an important component of nitrogen control in S. cerevisiae. First, overexpression of MKS1 activates nitrogen uptake systems under conditions when they are normally turned off, while underexpression inactivates the same system when it is normally turned on. Second, overexpression or depletion of MKS1 prevents the dimorphic switch induced by limited availability of nitrogen. Finally, overexpression of MKS1 results in poor growth specifically on good nitrogen sources. However, mks1 strains do sporulate normally (MATSUURA and ANRAKU 1993 ).

On proline medium, mks1 prevents expression of Dal5p, making cells unable to utilize USA, but this phenotype is suppressed by an additional ure2 mutation. This indicates that Mks1p does not act directly on DAL5 to activate it, but rather through the well-known Ure2p-Gln3p pathway. The same conclusion is supported by our finding that overexpression of Ure2p suppresses the Usa⁺ phenotype produced by overexpression of Mks1p on ammonia. The epistatic effects of ure2 over mks1 and of Ure2p overproduction over Mks1p overproduction indicate that Mks1p acts upstream of Ure2p. Since Mks1p has effects on Dal5p opposite those of Ure2p, Mks1p must be a (direct or indirect) inhibitor of Ure2p. Since Mks1p overexpression does not affect the level of expression of Ure2p, Mks1p must affect its activity. This suggests that the flow of control in this part of the nitrogen control pathway is as follows: NH₃ Mks1p Ure2p Gln3p DAL5 USA uptake.

A model in which Mks1p regulates Gln3p through a parallel pathway (not via Ure2p) is consistent with all the results, but does not predict as many of the results as the model above.

Mks1p could act either as a simple signal transducer, or by its overproduction alter metabolism so that cells are unable to utilize ammonia. The latter effect would also send the (false) signal to Ure2p that the nitrogen source was inadequate. However, it is not clear how deficiency of Mks1p could alter metabolism to provide an adequate nitrogen supply when cells were on proline, leading to failure of USA uptake. Only if Mks1p is a signal transducer would generation of this false signal be expected.

Slow-growth effect of Mks1p overproduction:
We found that the fragments of Mks1p that slow cell growth on ammonia (but not on proline) were the same as those that result in uptake of USA on ammonia. These results suggested that the role of Mks1p in these two processes is due to a single action of this protein, a model consistent with the growth-slowing action of Mks1p being conditional on the nitrogen source. Overproduction of Gln3p also dramatically slows growth on ammonia-containing media, but not on proline (MINEHART and MAGASANIK 1991 ), and this scheme predicts that overproduction of Mks1p should mimic overproduction of Gln3p. However, this model would predict that there would be no growth-slowing effect of Mks1p overproduction in a gln3 mutant. This proved not to be the case. Moreover, while overproduction of Ure2p overcomes the USA uptake effect of Mks1p, it does not prevent the growth-slowing effect. It thus seems likely that these effects of Mks1p work through different pathways.

Relation of Mks1p to pseudohyphal growth:
We find that either overproduction or undersupply of Mks1p interferes with pseudohyphal growth. These results can also be explained by the scheme shown above. Deletion of either URE2 or GLN3 results in loss of pseudohyphal growth (LORENZ and HEITMAN 1998 ). Overproduction of Mks1p should superinactivate Ure2p, mimicking deletion of URE2, while undersupply of Mks1p should allow overactivity of Ure2p and thus mimic deletion of GLN3. However, as discussed below, Mks1p appears to be involved in the Ras-cAMP pathway, and cAMP has also been implicated in control of pseudohyphal growth (KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ).

Mks1p and the Ras-cAMP system:
MKS1 was originally cloned by its ability to suppress the ability of TPK1 (one of the three catalytic subunits of A-kinase) to rescue growth of a ras1 ras2^ts mutant at the restrictive temperature. It was thus thought to be a negative regulator in the downstream part of the RAS-cyclic AMP pathway (MATSUURA and ANRAKU 1993 ). Indeed, in a cyr1^ts strain, deletion of mks1 partially suppresses the temperature sensitivity for growth, supporting this interpretation, and MKS1 overexpression did not affect cAMP levels or expression of the A-kinase. Interestingly, the mks1 deletion strain has a reduced level of cAMP (MATSUURA and ANRAKU 1993 ), and, perhaps as a result, mutation of MKS1 leads to caffeine resistance (LUSSIER et al. 1997 ). Caffeine inhibits the cAMP phosphodiesterase.

The observed cAMP-related effects of Mks1p could be explained if the A-kinase phosphorylated Mks1p, thereby inactivating it. However, using strains that can take up cAMP (1278b; KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ), we find that addition of cAMP to the growth medium does not affect the slow growth or USA uptake resulting from overproduction of Mks1p. Further work will be needed to relate the role of Mks1p in the Ras-cAMP path to that in nitrogen regulation.

The dual effect of Mks1p excess or deficiency on pseudohyphal growth may be similar to other dual effects of the Ras/cAMP pathway reported previously. For instance, of the three rate-limiting activators of cell division (the G1 cyclins Cln1, Cln2, and Cln3), Cln1 and Cln2 were found to be inhibited by cAMP and also to require cAMP for their expression (BARONI et al. 1994 ; TOKIWA et al. 1994 ; HALL et al. 1998 ).

MKS1 and lysine biosynthesis:
MKS1 was also found to be identical to LYS80, a negative regulator of lysine biosynthesis (FELLER et al. 1997 ). The lys8/mks1 mutants have increased pools of lysine, -ketoglutarate, and glutamate. Further work will be needed to determine whether the Mks1p effect on nitrogen catabolism involves alterations in cellular pools of these key nitrogen metabolites. In a screen for genes affecting cell wall structure and biogenesis, mks1 mutants were found to be hypersensitive to Calcofluor white and killer toxin, with altered cell wall composition (LUSSIER et al. 1997 ).

We cannot yet offer a unifying explanation of the various activities attributed to MKS1. Its cytoplasmic location is like that of Ure2p, its apparent target (EDSKES et al. 1999 ), but there is no evidence that Ure2p has a role either in lysine regulation or as an effector of cAMP. The fact that only residues 245–340 are needed for the effects seen here suggests that it may be a multifunctional protein.

	ACKNOWLEDGMENTS

We thank Gerry Fink, Evelyn Dubois, Mike Lorenz, and Joe Heitman for strains, Terry Cooper for pRR29, and Huei-Fung Tsai for sequence analysis.

Manuscript received April 14, 1999; Accepted for publication June 4, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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