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Research ArticleResearch Article
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Sac3, an Snf1-like Serine/Threonine Kinase That Positively and Negatively Regulates the Responses of Chlamydomonas to Sulfur Limitation

John P. Davies, Fitnat H. Yildiz, Arthur R. Grossman
John P. Davies
aDepartment of Botany, 353 Bessey Hall, Iowa State University, Ames, Iowa 50011-1020
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  • For correspondence: jdavies@iastate.edu
Fitnat H. Yildiz
bDepartment of Microbiology and Immunology, Stanford Medical School, Stanford University, Stanford, California 94305
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Arthur R. Grossman
cDepartment of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305
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Published June 1999. DOI: https://doi.org/10.1105/tpc.11.6.1179

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  • © 1999 American Society of Plant Physiologists

Abstract

The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system—the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.

INTRODUCTION

To survive in a dynamic environment, organisms must be able to sense changes in their environment and respond to those changes by altering their metabolism. Signal transduction mechanisms involved in controlling these responses may interact to form a network that links perception of the environment to physiological processes in the cell; this network may be required for survival of organisms in a dynamic environment that is often resource limited. We are using the genetically tractable, unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii) as a model system to investigate how photosynthetic organisms acclimate to changes in nutrient availability (de Hostos et al., 1988, 1989; Davies et al., 1994, 1996; Yildiz et al., 1994, 1996; Quisel et al., 1996; Wykoff et al., 1998). Several responses of Chlamydomonas to nutrient limitation are similar to those exhibited by vascular plants and soil-dwelling microbes (Marzluf and Metzenberg, 1968; Harder and Dijkhuizen, 1983; Hawkesford and Belcher, 1991; Tsay et al., 1993; Smith et al., 1995; Trueman et al., 1996; Davies and Grossman, 1998; Wykoff et al., 1998). Much of our work has focused on the ways in which Chlamydomonas adjusts to limiting sulfur levels.

Sulfur is a macronutrient that is required in relatively high concentrations by all organisms. It is a constituent of proteins, lipids, carbohydrates, electron carriers, and numerous cellular metabolites. For most organisms, the preferred source of sulfur is the sulfate anion (Uria-Nickelsen et al., 1993, 1994; Beil et al., 1996). However, the level of available inorganic sulfate in the soil may be low (David et al., 1982; Autry and Fitzgerald, 1990; Whalen and Warman, 1996). Many soil-dwelling microbes have developed mechanisms to scavenge sulfur from their environment by accessing organic sulfate esters, sulfamates, and sulfonates (Marzluf, 1970; Scott and Metzenberg, 1970; Apte et al., 1974; Lien and Schreiner, 1975; de Hostos et al., 1988; Murooka et al., 1990; Yildiz et al., 1994; Beil et al., 1996), which are abundant in the soil (Fitzgerald et al., 1988; Autry et al., 1990; Dhamala et al., 1990; Dhamala and Mitchell, 1995). In addition, both microbes and plants exhibit an increased efficiency for the import of sulfate when sulfate supplies are limiting (Breton and Surdin-Kerjan, 1977; Clarkson et al., 1983; Biedlingmaier and Schmidt, 1988; Green and Grossman, 1988; Hawkesford et al., 1993; Yildiz et al., 1994; Lappartient and Touraine, 1996).

Chlamydomonas cells exhibit a suite of responses when transferred from sulfate-replete medium to medium lacking sulfur. Cells stop dividing, photosynthetic oxygen evolution declines as a consequence of reduced photosystem II activity (Wykoff et al., 1998), novel proteins, including an arylsulfatase (Ars polypeptide and Ars gene), are synthesized and exported to the periplasm or cell wall (de Hostos et al., 1988), and a high-affinity sulfate transport system accumulates (Yildiz et al., 1994). Extracellular Ars releases the sulfate anion from esterified organic sulfates, allowing Chlamydomonas to access sulfur stores in the soil (Lien and Schreiner, 1975; de Hostos et al., 1988; Yildiz et al., 1994; Davies et al., 1996). To elucidate the mechanisms used to regulate the responses of Chlamydomonas to sulfur limitation, we have developed a colorimetric screen to isolate mutants that display aberrant regulation of Ars gene expression; the mutants are designated sac (for sulfur acclimation). After transfer to medium devoid of sulfur, the sac1 and sac2 mutants do not synthesize Ars or accumulate the high-affinity sulfate transport system to the same extent that wild-type cells do (Davies et al., 1994). In addition, the sac1 mutant is unable to decrease photosynthetic electron transport during sulfur-limited growth, which results in rapid cell death in the light (Davies et al., 1996). Unlike sac1 and sac2, the sac3 mutant exhibits constitutive, low-level synthesis of the extracellular Ars in the presence of sulfate. Furthermore, although the sac3 mutant is unable to produce elevated levels of the high-affinity sulfate transport system, it does accumulate Ars (Davies et al., 1994) and downregulates photosynthetic electron transport (J.P. Davies and A.R. Grossman, unpublished data) during sulfur-limited growth.

To determine the regulatory relationships among the Sac polypeptides, we are cloning, sequencing, and characterizing the Sac genes. The Sac1 gene encodes a polypeptide similar to a class of ion transporters present in cell membranes (Davies et al., 1996; Davies and Grossman, 1998). Despite the similarity of Sac1 to ion transporters, the phenotype of the sac1 mutant suggests that this protein either functions in sensing the sulfur status of the environment or participates in the signal transduction pathway that controls the responses of Chlamydomonas to sulfur deprivation. The Sac3 gene product appears to be required for the repression of Ars during sulfur-replete growth and for the full induction/activation of the sulfate transport system during sulfur starvation. Here, we report that the Sac3 gene encodes a polypeptide that exhibits similarities to Snf1, a protein kinase of Saccharomyces cerevisiae known to function in the signal transduction pathway controlling the expression of genes induced during glucose deprivation (Carlson et al., 1981; Carlson and Botstein, 1982). Interestingly, several putative vascular plant Snf1-related kinases have an even higher degree of similarity to Sac3.

RESULTS

Identification of Polymorphic Region in the sac3 Mutants

Chlamydomonas strain CC425 (cw15 arg7-8) was transformed with linearized plasmid pJD67 (containing the Arg7 gene [Debuchy et al., 1989] in the pBluescript KS+ vector [Stratagene, La Jolla, CA]), and transformants were selected as arginine prototrophs. This procedure generates mutants because the linearized plasmid integrates randomly into the Chlamydomonas genome. Mutants that exhibited constitutive expression of Ars were identified by growing the transformants on nutrient-replete solid medium and then spraying the colonies with the chromogenic Ars substrate 5-bromo-4-chloro-3-indolyl sulfate (Davies et al., 1994). The constitutive Ars-producing strains, designated are9-1, are10-1, and are16-1 (are10-1 was described as the sac3 mutant in Davies et al. [1994]), were obtained at a frequency of approximately one in 5000 transformants.

To determine whether the mutant phenotype cosegregated with the DNA that was introduced during transformation, we crossed strain are10-1 (sac3 mt+) with strain CC2677 (nit1 mt−). Twenty random progeny were tested for the mutant phenotype by scoring individuals for constitutive Ars expression and for the presence of the introduced plasmid by using DNA gel blot hybridization with probes specific for both Arg7 and pBluescript KS+ sequences. Cosegregation of the mutant phenotype and a single copy of the introduced DNA was observed (data not shown), suggesting that the insertion of pJD67 caused the mutant phenotype. The are10-1 strain was backcrossed several times with CC2677 to generate a new mutant strain, are10-12 (sac3 nit1), with a homogeneous genetic background. This strain was used for many of the experiments described below.

To identify the gene altered in are10-12, we isolated Chlamydomonas DNA flanking the integrated vector sequences by plasmid rescue. The strategy for plasmid rescue is diagrammed in Figure 1A. Briefly, genomic DNA isolated from are10-12 was digested with BamHI (which cuts between the Arg7 gene and pBluescript KS+ sequence in linearized pJD67) to generate a >12-kb fragment containing both pBluescript KS+ sequences and flanking Chlamydomonas DNA (based on DNA gel blot hybridizations using pBluescript KS+ as a probe; data not shown). The BamHI fragments were ligated and introduced into Escherichia coli, and transformants containing pBluescript KS+ sequences were selected on ampicillin-containing medium. All transformants harbored an identically sized >12-kb plasmid, designated pFY1, that contained both pBluescript KS + and Chlamydomonas genomic DNA. The insert DNA in pFY1 was mapped, and a 1.7-kb SalI fragment adjacent to the pBluescript KS+ sequence was subcloned into pBluescript KS+ to yield the plasmid designated pFY1a.

Figure 2 shows that hybridization of the 1.7-kb SalI fragment from pFY1a to genomic DNA from the different mutants that constitutively express Ars (i.e., are9-1, are16-1, and are10-12) yielded a polymorphic restriction pattern. A 7.0-kb SalI genomic fragment detected in the untransformed parental strain CC425 (Figure 2, lane 1) was missing in the mutants, and a new 1.7-kb SalI fragment was detected in are10-12 and are9-1 (Figure 2, lanes 2 and 4, respectively), as was a new 1.9-kb SalI fragment in are16-1 (Figure 2, lane 3). The observed polymorphisms indicate that the three independently isolated mutants that constitutively express Ars are altered in the same region of the Chlamydomonas genome. Furthermore, the 1.7-kb SalI fragment was detected in all of the mutant progeny of the cross of are10-12 with CC2677, whereas the 7-kb fragment was detected in all of the wild-type progeny (data not shown). Together, these results suggest that the three sac3 mutants are allelic and that the region of the genome defined by the 1.7-kb SalI fragment either contains or is very close to the coding region of the Sac3 gene.

Figure 1.
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Figure 1.

Genomic and cDNA Clones of Sac3.

(A) Schematic of the isolation of a genomic DNA adjacent to the introduced Arg7 gene in the sac3 mutant are10-12. Genomic DNA from are10-12 was digested with BamHI, ligated, and introduced into E. coli. Transformants were selected for growth in the presence of ampicillin, and a plasmid, designated pFY1, containing pBluescript KS+ sequences and a 9-kb fragment of Chlamydomonas genomic DNA, was isolated. A 1.7-kb SalI fragment adjacent to the pBluescript KS+ sequences was purified and cloned into pBluescript KS+ to form pFY1a. This 1.7-kb SalI fragment was used to isolate both the DNA that complemented the sac3 lesion and the cDNA for the Sac3 gene.

(B) Maps of fragments used to test complementation. The complementation frequencies for the plasmids pJD291, pJD292, and pJD293 are given as [number complemented]/[number of transformants tested].

(C) Map of the Sac3 cDNA, designated pJD290.

Complementation of the sac3 Mutants

Intact copies of the chromosomal region disrupted in are10-12 were isolated by screening a cosmid genomic library of a wild-type Chlamydomonas strain with the 1.7-kb SalI fragment from pFY1a. Three unique clones were isolated and introduced into Chlamydomonas strain are10-12 (sac3 nit1) via cotransformation with the pMN24 plasmid (containing the Nit1 gene; Fernandez et al., 1989) by using particle gun bombardment. To determine whether any of these cosmids complemented the mutant phenotype, we tested transformants for Ars activity on medium containing 5 mM sulfate. Three of 34 cell lines cotransformed by Nit1 and the cosmid cosFY2 showed no Ars activity on Tris–acetate–phosphate (TAP) medium, which was similar to wild-type cells. Fifty-two cell lines transformed with only pMN24 were tested, and all continued to express Ars activity.

Figure 2.
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Figure 2.

DNA Gel Blot Analysis of Genomic DNA from Wild-Type Cells, sac3 Mutants, and Complemented sac3 Mutants.

Genomic DNA from wild-type (CC425), three strains constitutively expressing Ars (are9-1, are16-1, and are10-12), and two complemented strains (are10-12-C1 and are10-12-C10) was digested with SalI, separated by agarose gel electrophoresis, and transferred to nitrocellulose membranes. Hybridization was performed with the 1.7-kb SalI insert of pFY1a. The positions of the molecular length markers are given at left in kilobases.

One of the apparently complemented strains, are10-12-C1, was further analyzed to determine whether it contained DNA from cosFY2. Genomic DNA from are10-12-C1 was isolated and digested with SalI, and the resolved genomic fragments were hybridized with the 1.7-kb SalI insert from pFY1a. In addition to the original 1.7-kb SalI genomic fragment present in the mutant strain, are10-12-C1 harbored a second fragment migrating at 7 kb (Figure 2, lane 5). The 7-kb species is identical in size to the wild-type SalI fragment that hybridizes with the pFY1a insert (Figure 2); it was also shown to be present on the cosFY2 DNA (data not shown). To determine whether the complemented phenotype cosegregated with the 7-kb SalI fragment, we crossed are10-12-C1 with are10-11 (sac3 mt−), a strain containing the same sac3 allele but of the opposite mating type. All of the progeny that exhibited the complemented phenotype contained both the 7- and 1.7-kb SalI fragments, whereas the mutant progeny contained only the 1.7-kb SalI fragment (data not shown).

Figure 3A shows that cultures of are10-12 grown in nutrient-replete medium exhibit significant levels of Ars activity, whereas cultures of both wild-type cells and the complemented strain exhibited essentially background levels of Ars activity. In addition, RNA gel blot analysis was used to measure the accumulation of the Ars transcript in wild-type cells, are10-12, and are10-12-C1. The loading of the RNA was normalized to the level of ribulose 1,5-bisphosphate carboxylase small subunit (RbcS1) transcript. Figure 3B shows that significant levels of the Ars transcript accumulated in are10-12 cells grown in nutrient-replete medium, whereas Ars mRNA was not detected in either CC125 or are10-12-C1 cells.

When the wild-type strain CC125 was starved for sulfate for 6 hr, the K1/2 for sulfate transport (the concentration of sulfate at which import is half-maximal) decreased from ~17 to 2 μM, and the Vmax increased from 20 to 200 fmol of sulfate sec−1 (105 cells)−1 (Yildiz et al., 1994). Whereas the sac3 mutant exhibited a decreased K1/2 for sulfate transport upon sulfur starvation (similar to wild-type cells), the Vmax did not increase to the same extent as in wild-type cells (Davies et al., 1994). As shown in Figure 4, the Vmax for sulfate transport in sulfur-starved, wild-type cells and are10-12 cells was 239 and 70 fmol of sulfate sec−1 (105 cells)−1, respectively. The complemented sac3 mutant, are10-12-C1, exhibited a Vmax for sulfate transport of 224 fmol of sulfate sec−1 (105 cells)−1, which is similar to the value measured in wild-type cells.

Figure 3.
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Figure 3.

Ars Activity and Transcript Accumulation in Wild-Type Cells, the sac3 Mutant, and the Complemented Strains.

(A) Strains CC125 (wild type), are10-12 (sac3), and are10-12-C1 (complemented sac3) were grown to mid-log (2 to 5 × 106 cells mL−1) phase, and Ars activity was measured. p-Nitrophenyl sulfate was used as the chromogenic substrate, and the activity is expressed as micrograms of p-nitrophenol produced by 105 cells in 1 hr. The data represent averages of three experiments. The error bars indicate one standard deviation.

(B) Poly(A)+ mRNA from strains CC125 (wild type), are10-12 (sac3), and are10-12-C1 (complemented sac3) was isolated, resolved on agarose gels by electrophoresis, transferred to nitrocellulose membranes, and hybridized with the 2.4-kb insert from pJD240 (Ars2 cDNA; J.P. Davies and A.R. Grossman, unpublished data), and the 1.1-kb SalI-BamHI fragment of p2.02 (Goldschmidt-Clermont and Rahire, 1986) containing a portion of the RbcS1 gene. Positions of the molecular length markers are given at left in kilobases.

Together, the results presented above demonstrate that are10-12-C1 is complemented for the phenotype of the sac3 mutant and that complementation is linked to the newly introduced 7-kb SalI fragment present on cosFY2.

Complementation of the Mutant Phenotype with the 7.0-kb SalI Fragment

To determine whether the 7-kb SalI fragment (Figure 2) from cosFY2 could complement the phenotype of the sac3 mutant, we subcloned it into pBluescript KS+, yielding pJD291 (Figure 1B), and introduced it into are10-12 (sac3 nit1) via cotransformation with the Nit1 gene by using particle bombardment (Klein et al., 1987). Random transformants were tested for complementation by assaying for Ars activity in TAP medium. Six of 62 transformants showed no Ars activity, indicating complementation of the mutant phenotype. Subclones of the 7-kb SalI fragment were also generated and introduced into the sac3 mutant, and transformants were tested for constitutive expression of Ars (Figure 1B). The plasmid pJD292, containing a 6-kb SalI-XhoI fragment of pJD291, was able to complement the sac3 mutation (three complemented lines in 11 transformants tested), whereas pJD293, containing the 4.7-kb SalI-KpnI fragment of pJD291, was not (zero complemented lines in 46 transformants tested). These data suggest that the Sac3 gene is located on the 6-kb SalI-XhoI fragment of pJD292 and that it spans the KpnI site located 1.3 kb from the XhoI site.

Figure 4.
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Figure 4.

Sulfate Uptake in Wild-Type Cells, the sac3 Mutant, and a Complemented Strain.

The Vmax for sulfate uptake in CC125 (wild type), are10-12 (sac3), and are10-12-C1 (complemented sac3) is given as femtomoles of sulfate per second per 105 cells. The values are averages of three experiments, and the bars represent one standard deviation.

DNA gel blot analysis of genomic DNA from one of the transformants complemented with pJD291, are10-12-C10, confirmed that plasmid sequences had integrated into the genome. As shown in Figure 2, are10-12-C10 has, in addition to the 1.7-kb SalI fragment present in the original mutant line, multiple copies of the introduced DNA. Some of these copies are detected as a 7-kb SalI fragment, indicating that the plasmid recombined into the genome within the vector sequences of pJD291, whereas others are either larger or smaller than 7 kb, indicating that the plasmid recombined within the 7-kb insert. To confirm that the introduced 7-kb fragment was responsible for the complemented phenotype, we crossed are10-12-C10 with are10-11 (sac3 mt−). All of the progeny analyzed showed cosegregation of the complemented phenotype and this introduced fragment. In addition, none of the progeny with the mutant phenotype contained any of the introduced fragments. These data indicate that the 7-kb SalI fragment in pJD291 contains the Sac3 gene and includes all the sequences necessary and sufficient to complement the sac3 lesion.

Isolation of the Sac3 cDNA and Expression and Sequence Analyses

The 1.7-kb SalI fragment of pFY1a was used to isolate pJD290, a cDNA containing a 1.9-kb insert (Figure 1C). The 0.8-kb PstI fragment of pJD290 containing the 5′ end of the Sac3 cDNA hybridized with pJD291, pJD292 (clones that complement the sac3 lesion), and pJD293 (a clone that did not complement the sac3 lesion). The 1.0-kb KpnI fragment of pJD290 containing the 3′ end of the Sac3 cDNA hybridized with the complementing plasmids pJD291 and pJD292 but not with pJD293, the plasmid that did not complement the sac3 mutation. These data indicate that the sequences coding for the Sac3 cDNA are on plasmids pJD291 and pJD292 and that the 3′ portion of the cDNA includes the KpnI site in pJD291 and pJD292. Hence, this cDNA spans the region of the genomic sequence required for complementation.

The cDNA was hybridized with poly(A)+ mRNA from wild-type, mutant, and complemented mutant strains. Figure 5 shows that the 1.9-kb cDNA insert hybridized with a 1.9-kb mRNA from wild-type cells. A 1-kb transcript was detected in the mutant strain are10-12. The complemented mutant, are10-12-C1, had both the truncated and full-length transcripts. Although the 1.9-kb transcript was more abundant in wild-type cells than in are10-12-C1 cells (normalized to either total RNA or the level of RbcS1 transcript), the low level of transcript in the complemented strain was sufficient to rescue the mutant phenotype.

To determine whether expression of the Sac3 gene is regulated by sulfate availability, we compared transcript accumulation in wild-type cells grown in complete medium with that of cells exposed for 2 hr to sulfur deprivation, which is sufficient time to observe elevated levels of Ars mRNA. Figure 6 shows that there was no significant difference in the level of the Sac3 transcript in starved and unstarved cells.

Figure 5.
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Figure 5.

Sac3 Transcript Accumulation in Wild-Type Cells, the sac3 Mutant, and the Complemented Strain.

Poly(A)+ RNA from CC125 (wild type), are10-12 (sac3), and are10-12-C1 (complemented sac3) was isolated, resolved on agarose gels by electrophoresis, blotted onto nitrocellulose, and hybridized with the 1.9-kb insert from pJD290 (Sac3) and the 1.1-kb SalI-BamHI fragment of p2.02 (RbcS1) (Goldschmidt-Clermont and Rahire, 1986). Positions of the molecular length markers are given at left in kilobases.

The Sac3 cDNA was sequenced, and an open reading frame of 1064 nucleotides flanked by 5′ and 3′ untranslated regions of 165 and 692 nucleotides, respectively, was detected. The derived amino acid sequence of the open reading frame was compared with sequences in the GenBank database. As shown in Figure 7A, significant similarity was detected between Sac3 and Snf1, an S. cerevisiae serine/threonine kinase involved in signaling glucose deprivation. However, Sac3 is also similar to a large number of open reading frames encoding presumed (based on sequence similarity) serine/threonine kinases from plants. All of the plant sequences also have significant identity with the catalytic domain of Snf1 of S. cerevisiae and are considered Snf1-related kinases (SnRKs) (Halford and Hardie, 1998). The N-terminal catalytic domain of Sac3 is 36% identical to that of Snf1 and between 50 and 56% identical to that of the plant serine/threonine kinases. All of the sequences presented in Figure 7 have the 11 kinase-specific subdomains (I to XI; Hanks et al., 1988). However, the plant sequences have two additional C-terminal subdomains, which we have designated A and B, and an acidic region, designated Neg, in common with Sac3. The functions of the putative kinases from vascular plants are not known, although many may be involved in the acclimation of plants to various environmental stresses (Halford and Hardie, 1998).

To determine the relationship of Sac3 to other serine/threonine kinases, we performed a phylogenetic analysis of the kinase domain sequences of these proteins (Figure 7B). Sac3 is most closely related to the vascular plant SnRK2 subfamily (Halford and Hardie, 1998). The proteins in this subfamily are characterized by an N-terminal catalytic domain similar to that of the S. cerevisiae Snf1 protein and a short C-terminal domain rich in acidic residues. Based on sequence divergence, the SnRK2 subfamily has been divided into SnRK2a and SnRK2b. Although Sac3 clearly groups with the SnRK2 subfamily, it falls outside of both of the SnRK2 subgroupings.

To determine where the Sac3 gene was interrupted, we sequenced the DNA adjacent to the pBluescript KS+ vector in the plasmid-rescued fragment. This sequence indicated that pJD67 integrated within an intron in the 5′ portion of Sac3 that separates nucleotides 340 and 341 of the cDNA. This site of integration allowed translation of only the first 58 amino acids of the Sac3 polypeptide and produced a protein lacking nine of the 11 subdomains important for kinase activity. On the basis of these data and the fact that the lesion in are10 is a recessive mutation, it is likely that the phenotype of this strain is the result of a complete lack of kinase function. This indicates that the Sac3 kinase positively regulates sulfate uptake and negatively regulates Ars activity.

Figure 6.
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Figure 6.

Sac3 Transcript Accumulation in Wild-Type Cells in Sulfur-Replete and Sulfur-Deficient Media.

Poly(A)+ RNA was isolated from CC125 cells grown in sulfur-replete (+S; lanes 1 and 3) or sulfur-deficient (−S; lanes 2 and 4) medium for 2 hr, resolved by agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized with the 1.9-kb insert from pJD290 (Sac3) (lanes 1 and 2) and the 1.1-kb SalI-BamHI fragment of p2.02 (RbcS1) (lanes 3 and 4) (Goldschmidt-Clermont and Rahire, 1986). Molecular length markers are given at left in kilobases.

Figure 7.
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Figure 7.

Comparison of Sac3, the Catalytic Domain of Snf1, and Serine/Threonine Kinases of Vascular Plants.

(A) Amino acids 1 to 314 of Snf1 are shown; no similarity between the C-terminal domain of Snf1 and the other proteins was observed. The 11 conserved subdomains of serine/threonine kinases are identified by roman numerals. Additional conserved regions are identified as A, B, and Neg. The dashes underneath the residues in the Neg region designate negatively charged amino acids in Sac3; the locations of negatively charged amino acids in the other proteins may differ. Identical amino acid residues are boxed in black. The name of each protein or the accession number is given at the left border. Dots were introduced to optimize alignment.

(B) Dendogram showing relative evolutionary distances between the catalytic domains of Sac3 and the other SnRKs. The name of each protein or the accession number is given. The alignment was produced using CLUSTALW (Thompson et al., 1994). The dendogram was constructed using Neighbor Joining analysis (Saitou and Nie, 1987) and was midpoint rooted using PAUP* 4.01B1 (Sinauer Press, Sunderland, MA). The GenBank accession numbers of the sequences presented are as follows: NPK5, D26602; BKIN12, X65606; SNF1, M13971; WPK4, D21204; PKABA1, M94726; ASK1, M91548; SPK1, L01453; and SAC3, AF100162. S56718 is the GenBank accession number for a cDNA sequence from Arabidopsis. The same result was obtained when more members of the SnRK2 family were used in the analysis.

DISCUSSION

Structure of Sac3 and Its Relationship to Other Serine/Threonine Kinases

The deduced amino acid sequence of Sac3 contains a domain that is similar to the catalytic domain of a large family of serine/threonine kinases (Figure 7) related to Snf1 of S. cerevisiae and the 5′ AMP-activated protein kinases in mammals. These kinases contain two domains, an N-terminal catalytic domain of 250 to 300 amino acids and a C-terminal regulatory domain of 250 to 380 amino acids (Hanks et al., 1988; Celenza and Carlson, 1989; Jiang and Carlson, 1996; Halford and Hardie, 1998). Two of the Snf1-like serine/threonine kinases in plants, RKIN1 from Secale cereale (Alderson et al., 1991) and NPK5 from tobacco (Muranaka et al., 1994), can functionally complement an snf1 mutation in S. cerevisiae.

In plants, there are at least three subfamilies of SnRKs (Halford and Hardie, 1998). The members of all of these subfamilies share significant sequence identity with the N-terminal catalytic domains of Snf1 and the AMP-activated protein kinases. The best-described subfamily, SnRK1, is composed of proteins with significant sequence identity to Snf1 and the AMP-activated protein kinases in both the N- and C-terminal domains. Members of this subfamily, which include the RKINI and NPK5 proteins, have 62 to 64% sequence identity at their N-terminal catalytic domains and 29 to 34% sequence identity at their C-terminal domains (Halford and Hardie, 1998). The C-terminal domain of Snf1 interacts with at least two other proteins and is involved in regulating the kinase activity (Stapleton et al., 1994; Jiang and Carlson, 1996; Hardie and Carling, 1997; Halford and Hardie, 1998). Members of SnRK3 are approximately the same size as Snf1 and the AMP-activated kinases, but the sequence of their C-terminal domains exhibits no similarity to either (Halford and Hardie, 1998).

Sac3 is a member of the SnRK2 subfamily, as are at least 10 proteins of vascular plants (deduced from cDNA sequences in GenBank). The plant kinases in this subfamily include PKABA1 from wheat (Anderberg and Walker-Simmons, 1992), SPK-3 and SPK-4 from soybean (Yoon et al., 1997), and ASK1 and ASK2 from Arabidopsis (Park et al., 1993). These proteins have a molecular mass of ~40 kD, with an N-terminal kinase domain linked to a short C-terminal sequence of unknown function. The C-terminal domain has two subdomains, which we have designated A and B, and a patch of acidic amino acids (designated Neg in Figure 7). Of these, only subdomain A is present in Snf1. The proteins in the SnRK2 subfamily have 57 to 61% amino acid identity in their N-terminal catalytic domains and 41 to 51% identity in their C-terminal domains. However, the plant kinases shown in Figure 7 are more similar to each other than to Sac3.

Sac3 regulates the response of Chlamydomonas to sulfur limitation. The functions of the other SnRK2 polypeptides have not been elucidated. However, the accumulation of transcripts encoding specific SnRK2 polypeptides under defined environmental conditions has led to speculation concerning their functions (Anderberg and Walker-Simmons, 1992; Park et al., 1993; Yoon et al., 1997). For example, PKABA1, SPK-3, and SPK-4 (Anderberg and Walker-Simmons, 1992; Yoon et al., 1997) transcripts accumulate in response to dehydration and elevated salt levels, suggesting that these kinases may be involved in the responses of plants to hypoosmotic conditions.

Often, the abundance of transcripts for specific regulators does not change under conditions in which the regulator functions to alter gene expression. Indeed, glucose-deprived yeast cells and sulfur-deprived Chlamydomonas do not exhibit altered levels of the SNF1 (Celenza and Carlson, 1984) and Sac3 (Figure 6) transcripts, respectively. The in vitro kinase activity of Snf1, the AMP-activated kinases, and two SnRKs from cauliflower (HRK-A and HRK-B) is controlled by the phosphorylation state of these proteins (Weekes et al., 1994; Woods et al., 1994; Ball et al., 1995). It is not known whether Sac3 kinase activity is regulated by phosphorylation.

Regulation of Sulfur Limitation Responses in Chlamydomonas

The sac3 mutants of Chlamydomonas exhibit low-level constitutive Ars activity when grown in nutrient-replete medium and do not fully activate sulfate uptake when deprived of sulfur. They respond normally to both phosphorus and nitrogen deprivation. These characteristics indicate that the Sac3 protein is specifically involved in controlling the acclimation of Chlamydomonas to sulfur deprivation.

Sulfur limitation triggers the production of Ars and an increase in the rate of sulfate transport. The inability of sac3 mutants to fully repress Ars activity during growth in nutrient-replete medium or to fully activate sulfate uptake upon sulfur starvation suggests that Sac3 is part of a phosphorylation-driven signal transduction chain that can positively and negatively regulate the expression of genes associated with the acclimation of Chlamydomonas to sulfur limitation. Several S. cerevisiae regulatory proteins can have both positive and negative affects on gene expression. Some of these proteins, such as Paf1, Gal11, Sin4, and Rgr1, form a mediator complex with RNA polymerase and influence gene expression by altering chromatin structure or associating with specific DNA binding proteins (Fassler and Winston, 1989; Jiang and Stillman, 1992; Jiang et al., 1995; Li et al., 1995; Shi et al., 1996). Others, such as Rap1, may modify gene activity by directly binding DNA (Kurtz and Shore, 1991; Shore, 1994).

Mutations in three genes have been shown to affect the responses of Chlamydomonas to sulfur limitation. Two genetic loci, Sac1 and Sac2, positively regulate responses to sulfur limitation, whereas one locus, Sac3, appears to both positively and negatively regulate these responses (Davies et al., 1994). Sac1 (Davies et al., 1996) controls the expression of Ars and other sulfur stress–induced genes, and it is required for the decrease in photosynthetic activity that accompanies sulfur starvation. It may be involved in sensing the level of sulfate in the medium (Davies and Grossman, 1998). The Sac1 gene has been cloned, and the derived amino acid sequence has similarity to ion transporters. The Sac2 gene has not been cloned, but the phenotype of the sac2 mutant suggests that it is either directly or indirectly involved in the transcriptional regulation of Ars genes and possibly other genes induced during sulfur limitation (Davies et al., 1994). In contrast, Sac3 is a protein kinase that appears to have both positive and negative effects on gene expression.

Genetic analysis of the mutants indicates that Sac1 and Sac2 function in a linear pathway to regulate Ars expression and that Sac3 may function in an independent manner. The phenotype of the sac1 mutant is epistatic to sac2 (Davies et al., 1994), but no clear epistatic relationship exists between sac1 and sac3 or sac2 and sac3 (Davies et al., 1994). Both pathways probably control Ars expression and the elevation of sulfate transport.

Distinct signal transduction pathways have been observed for the control of glucose deprivation responses in S. cerevisiae. One signaling pathway includes signaling through Snf1 (Trumbly, 1992) and controls the expression of invertase and the ability of the cells to use sugars other than glucose. The other signaling pathway controls expression of the high-affinity hexose transporters and is thought to be initiated by Snf3, which is believed to sense the levels of glucose in the medium (Liang and Gaber, 1996; Ozcan et al., 1996). There are parallels between carbon catabolite repression in S. cerevisiae and the responses of Chlamydomonas to sulfur deprivation. First, Sac1 and Snf3 are similar in sequence to integral membrane proteins involved in transporting nutrients but act as positive regulators (Celenza et al., 1988; Davies et al., 1996; Davies and Grossman, 1998). Second, Sac3 and Snf1 are both serine/threonine kinases (Celenza and Carlson, 1986). However, there are also significant differences between mechanisms involved in the acclimation of Chlamydomonas to sulfur limitation and the acclimation of S. cerevisiae to glucose limitation. Sac1 and Sac3 appear to be in distinct signaling pathways that regulate the same responses and genes (i.e., Ars expression and sulfate uptake), whereas Snf1 and Snf3 are components of distinct regulatory pathways that govern different sets of genes. Furthermore, whereas Sac3 negatively regulates Ars expression and positively regulates sulfate uptake, Snf1 appears to exclusively regulate genes in a positive manner. Finally, whereas the sac3 mutant affects only responses to sulfur deprivation (carbon-, nitrogen-, and phosphorus-deficient conditions were also tested), the snf1 mutant affects the cell's responses to different types of nutrient stress (Thompson-Jaeger et al., 1991). For example, snf1 mutants are unable to survive glucose, phosphorus, sulfur, and nitrogen starvation and do not accumulate high levels of glycogen duing glucose or phosphorus deprivation (Thompson-Jaeger et al., 1991).

Continued definition of the factors that regulate sulfur stress acclimation processes in Chlamydomonas will elucidate a control circuit that enables photosynthetic, eukaryotic microbes to interface successfully with a dynamic environment. This control may be similar to systems used by vascular plants. The extensive homology between Sac3 of Chlamydomonas and vascular plant serine/threonine kinases thought to be involved in environmental sensing raises the possibility that the mechanisms for communicating environmental cues to the biosynthetic machinery of the cell are similar in vascular plants and Chlamydomonas.

METHODS

Cell Growth and Mating

Chlamydomonas reinhardtii cells were grown in nutrient-replete or sulfate-deficient Tris–acetate–phosphate (TAP) medium, as described previously (Davies et al., 1994). When appropriate, the medium was supplemented with arginine at 50 μg/mL. SGII/NO2 and SGII/NO3 media were identical to Sager-Granick II (SGII; Kindle, 1990), except that they contained 0.43 mM nitrite and 0.35 mM nitrate, respectively, as their sole nitrogen sources. Matings were performed as described by Harris (1989).

Chlamydomonas Mutagenesis and Transformation

Strain CC425 (cw15 arg7) was mutagenized by introducing HindIII-digested pJD67 (Davies et al., 1994) by glass bead transformation (Kindle, 1990). Transformants were selected on a nutrient-replete solid medium lacking arginine. Strains constitutively synthesizing arylsulfatase (Ars) were identified by spraying the plates containing colonies of transformants with 300 to 500 μL of 10 mM 5-bromo-4-chloro-3-indolyl sulfate and screening for the formation of a blue halo around colonies.

Strain are10-12 (sac3 nit1) was transformed with pMN24, which contains the Nit1 gene (Fernandez et al., 1989), by using particle bombardment (Klein et al., 1987). The strain was grown in SGII/NO2 to mid-log phase, and 108 cells were collected by centrifugation (5000g for 5 min), resuspended in SGII/NO3, and spread on solid SGII/NO3 medium (0.8% agarose). Thirty milligrams of tungsten particles (M-17; Bio-Rad) was prepared by vortexing them in 100% ethanol for 3 to 5 min, pelleting them by centrifugation for 1 min in an Eppendorff (Brinkman Instruments, Westbury, NY) microcentrifuge at full speed, removing the supernatant, and resuspending the particles in 0.5 mL of sterile water. The particles were then washed twice by vortexing them for 1 min, pelleting them by centrifugation (as before), and resuspending them in 0.5 mL of sterile water. DNA was precipitated onto the particles by combining 5 μg of DNA, 50 μL of 2.5 M CaCl2, 20 μL of 0.1 M spermidine, and 50 μL of the tungsten particles, while continuously vortexing the mixture. After an additional minute of vortexing, the particles were allowed to settle for 10 min, the supernatant was removed, and the particles were resuspended by vortexing in 250 μL of 100% ethanol for 1 min. The particles were again allowed to settle (3 min) and, after removing the supernatant, were resuspended in 60 μL of 100% ethanol. Particle bombardment was performed with the Bio-Rad Biolistic PDS-1000/He system, with the target plate at 6 cm and using 900 psi rupture disks. After bombardment, the plates were moved to growth conditions, and colonies appeared after 7 to 9 days. Transformants were streaked onto SGII/NO3 plates and individual colonies, and restreaked twice on SGII/NO3 before assaying Ars activity.

Ars Activity, Sulfate Uptake, and DNA and RNA Gel Blotting

Ars activity was assayed as described by de Hostos et al. (1988) and Davies et al. (1994). Sulfate uptake was performed as described by Yildiz et al. (1994), except that the Vmax for sulfate uptake in sulfur-deprived cells was determined by measuring the velocity of sulfate import at 100 μM sulfate. DNA and RNA gel blot analyses were performed as described previously (Davies et al., 1994, 1996).

Isolation of Cosmid Clones

The cosmid library of Purton and Rochaix (1994) was obtained from Saul Purton (University College, London, UK) and screened by colony hybridization (Maniatis et al., 1982). Purified cosmid DNA was obtained using a Qiagen (Valencia, CA) DNA purification kit.

ACKNOWLEDGMENTS

We thank Randall Small for help with the phylogenetic analysis, Saul Purton for providing us with the cosmid library, and Dennis Wykoff and Dafna Elrad for reading the manuscript and making helpful suggestions. This work was supported by U.S. Department of Agriculture Grant No. 96351003142 to A.R.G. This is Carnegie Institution of Washington publication No. 1402. It is also Journal Paper No. J-18197 of the Iowa Agriculture and Home Economics Experiment Station (Ames, Iowa), Project No. 0-100, and was supported by Hatch Act and State of Iowa funds.

Footnotes

  • ↵1 These authors contributed equally to this work.

  • Received December 7, 1998.
  • Accepted March 29, 1998.
  • Published June 1, 1999.

REFERENCES

  1. ↵
    1. Alderson A.,
    2. Sabelli P.A.,
    3. Dickinson J.R.,
    4. Cole D.,
    5. Richardson M.,
    6. Kreis M.,
    7. Shewry P.R.,
    8. Halford N.G.
    (1991). Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA. Proc. Natl. Acad. Sci. USA 88, 8602–8605.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Anderberg R.J.,
    2. Walker-Simmons M.K.
    (1992). Isolation of a wheat cDNA clone for an abscisic acid–inducible transcript with homology to protein kinases. Proc. Natl. Acad. Sci. USA 89, 10183–10187.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Apte B.,
    2. Bhavsar P.,
    3. Siddiqi O.
    (1974). The regulation of arylsulphatase in Aspergillus nidulans. J. Mol. Biol. 86, 637–648.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Autry A.,
    2. Fitzgerald J.
    (1990). Sulfonate S: A major form of forest soil organic sulfur. Biol. Fertil. Soils 10, 50–56.
    OpenUrl
  5. ↵
    1. Autry A.,
    2. Fitzgerald J.,
    3. Caldwell P.
    (1990). Sulfur fractions and retention mechanisms in forest soils. Can. J. For. Res. 20, 337–342.
  6. ↵
    1. Ball K.L.,
    2. Barker J.,
    3. Halford N.G.,
    4. Hardie D.G.
    (1995). Immunological evidence that HMG–CoA reductase kinase-A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases. FEBS Lett. 377, 189–192.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Beil S.,
    2. Kertesz M.,
    3. Leisinger T.,
    4. Cook A.
    (1996). The assimilation of sulfur from multiple sources and its correlation with expression of the sulfate-starvation-induced stimulon in Pseudomonas putida S-313. Microbiology 142, 1989–1995.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Biedlingmaier S.,
    2. Schmidt A.
    (1988). Sulfate transport in normal and S-deprived Chlorella fusca. Z. Naturforsch. 44c, 495–503.
    OpenUrl
  9. ↵
    1. Breton A.,
    2. Surdin-Kerjan Y.
    (1977). Sulfate uptake in Saccharomyces cerevisiae: Biochemical and genetic study. J. Bacteriol. 132, 224–232.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Carlson M.,
    2. Botstein D.
    (1982). Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase. Cell 28, 145–154.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Carlson M.,
    2. Osmond B.C.,
    3. Botstein D.
    (1981). Genetic evidence for a silent SUC gene in yeast. Genetics 98, 41–54.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Celenza J.L.,
    2. Carlson M.
    (1984). Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 49–53.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Celenza J.L.,
    2. Carlson M.
    (1986). A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233, 1175–1180.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Celenza J.L.,
    2. Carlson M.
    (1989). Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. Mol. Cell. Biol. 9, 5034–5044.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Celenza J.L.,
    2. Marshall-Carlson L.,
    3. Carlson M.
    (1988). The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc. Natl. Acad. Sci. USA 85, 2130–2134.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Clarkson D.,
    2. Smith F.,
    3. Vanden Berg P.
    (1983). Regulation of sulphate transport in a tropical legume, Macroptilium atropurpureum cv. Siratro. J. Exp. Bot. 34, 1463–1483.
  17. ↵
    1. David M.,
    2. Mitchell M.,
    3. Nakas J.
    (1982). Organic and inorganic sulfur constituents of a forest soil and their relationship to microbial activity. Soil Sci. Soc. Am. J. 46, 847–852.
    OpenUrlCrossRef
  18. ↵
    1. Rochaix J.-D.,
    2. Goldschmidt-Clermont M.,
    3. Merchant S.
    1. Davies J.P.,
    2. Grossman A.R.
    (1998). Survival during macronutrient limitation. In The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, Rochaix J.-D., Goldschmidt-Clermont M., Merchant S., eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 613–635.
  19. ↵
    1. Davies J.P.,
    2. Yildiz F.H.,
    3. Grossman A.R.
    (1994). Mutants of Chlamydomonas with aberrant responses to sulfur deprivation. Plant Cell 6, 53–63.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Davies J.P.,
    2. Yildiz F.H.,
    3. Grossman A.
    (1996). Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J. 15, 2150–2159.
    OpenUrlPubMed
  21. ↵
    1. Debuchy R.,
    2. Purton S.,
    3. Rochaix J.D.
    (1989). The argininosuccinate lyase gene of Chlamydomonas reinhardtii: An important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J. 8, 2803–2809.
    OpenUrlPubMed
  22. ↵
    1. de Hostos E.L.,
    2. Togasaki R.K.,
    3. Grossman A.
    (1988). Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii. J. Cell Biol. 106, 29–37.
    OpenUrlAbstract/FREE Full Text
    1. de Hostos E.L.,
    2. Schilling J.,
    3. Grossman A.R.
    (1989). Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii. Mol. Gen. Genet. 218, 229–239.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Dhamala B.,
    2. Mitchell M.
    (1995). Sulfur speciation, vertical distribution and seasonal variation in a northern hardwood forest soil, U.S.A. Can. J. For. Res. 25, 234–243.
    OpenUrlCrossRef
  24. ↵
    1. Dhamala B.,
    2. Mitchell M.,
    3. Stam A.
    (1990). Sulfur dynamics in mineral horizons of two northern hardwood soils. A column study with 35S. Biogeochemistry 10, 143–160.
    OpenUrl
  25. ↵
    1. Fassler J.S.,
    2. Winston F.
    (1989). The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription. Mol. Cell. Biol. 9, 5602–5609.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Fernandez E.,
    2. Schnell R.,
    3. Ranum L.P.,
    4. Hussey S.C.,
    5. Silflow C.D.,
    6. Lefebvre P.A.
    (1989). Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 86, 6449–6453.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Fitzgerald J.,
    2. Hale D.,
    3. Swank W.
    (1988). Sulphur-containing amino acid metabolism in surface horizons of a hardwood forest. Soil Biol. Biochem. 20, 825–831.
    OpenUrlCrossRef
  28. ↵
    1. Goldschmidt-Clermont M.,
    2. Rahire M.
    (1986). Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. J. Mol. Biol. 191, 421–432.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Green L.,
    2. Grossman A.
    (1988). Changes in sulfate transport characteristics and protein composition of Anacystis nidulans R2 during sulfur deprivation. J. Bacteriol. 170, 583–587.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Halford N.G.,
    2. Hardie D.G.
    (1998). SNF1-related protein kinases: Global regulators of carbon metabolism in plants? Plant Mol. Biol. 37, 735–748.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Hanks S.K.,
    2. Quinn A.M.,
    3. Hunter T.
    (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Harder W.,
    2. Dijkhuizen L.
    (1983). Physiological responses to nutrient limitation. Annu. Rev. Microbiol. 37, 1–23.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Hardie D.G.,
    2. Carling D.
    (1997). The AMP-activated protein kinase—Fuel gauge of the mammalian cell? Eur. J. Biochem. 246, 259–273.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Harris E.H.
    (1989). The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. (San Diego, CA: Academic Press).
  35. ↵
    1. Hawkesford M.,
    2. Belcher A.
    (1991). Differential protein synthesis in response to sulfate and phosphate deprivation: Identification of possible components of plasma-membrane transport systems in cultured tomato roots. Planta 185, 323–329.
    OpenUrlPubMed
  36. ↵
    1. Hawkesford M.,
    2. Davidian J.-C.,
    3. Grignon C.
    (1993). Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L.: Increased transport in membranes isolated from sulphur-starved plants. Planta 190, 297–304.
    OpenUrl
  37. ↵
    1. Jiang R.,
    2. Carlson M.
    (1996). Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10, 3105–3115.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Jiang Y.W.,
    2. Stillman D.J.
    (1992). Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 4503–4514.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Jiang Y.W.,
    2. Dohrmann P.R.,
    3. Stillman D.J.
    (1995). Genetic and physical interactions between yeast RGR1 and SIN4 in chromatin organization and transcriptional regulation. Genetics 140, 47–54.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Kindle K.L.
    (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87, 1228–1232.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Klein T.,
    2. Wolf E.,
    3. Wu R.,
    4. Samford J.
    (1987). High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327, 70–73.
    OpenUrlCrossRef
  42. ↵
    1. Kurtz S.,
    2. Shore D.
    (1991). RAP1 protein activates and silences transcription of mating-type genes in yeast. Genes Dev. 5, 616–628.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Lappartient A.,
    2. Touraine B.
    (1996). Demand-driven control of root ATP sulfurylase activity and sulfate uptake in intact canola. Plant Physiol. 111, 147–157.
    OpenUrlAbstract
  44. ↵
    1. Li Y.,
    2. Bjorklund S.,
    3. Jiang Y.W.,
    4. Kim Y.J.,
    5. Lane W.S.,
    6. Stillman D.J.,
    7. Kornberg R.D.
    (1995). Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. USA 92, 10864–10868.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Liang H.,
    2. Gaber R.F.
    (1996). A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6. Mol. Biol. Cell. 7, 1953–1966.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Lien T.,
    2. Schreiner O.
    (1975). Purification of a derepressible arlysulfatase from Chlamydomonas reinhardtii. Biochem. Biophys. Acta 384, 168–179.
    OpenUrlPubMed
  47. ↵
    1. Maniatis T.,
    2. Fritsch E.,
    3. Sambrook J.
    (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
  48. ↵
    1. Marzluf G.A.
    (1970). Genetic and metabolic controls for sulfate metabolism in Neurospora crassa: Isolation and study of chromate-resistant and sulfate transport–negative mutants. J. Bacteriol. 102, 716–721.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Marzluf G.A.,
    2. Metzenberg R.L.
    (1968). Positive control by the cys-3 locus in regulation of sulfur metabolism in Neurospora. J. Mol. Biol. 33, 423–437.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Muranaka T.,
    2. Banno H.,
    3. Machida Y.
    (1994). Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae. Mol. Cell. Biol. 14, 2958–2965.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Murooka Y.,
    2. Ishibashi K.,
    3. Yasumoto M.,
    4. Sasaki M.,
    5. Sugino H.,
    6. Azakami H.,
    7. Yamashita M.
    (1990). A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene. J. Bacteriol. 172, 2131–2140.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Ozcan S.,
    2. Dover J.,
    3. Rosenwald A.G.,
    4. Wolfl S.,
    5. Johnston M.
    (1996). Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc. Natl. Acad. Sci. USA 93, 12428–12432.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Park Y.S.,
    2. Hong S.W.,
    3. Oh S.A.,
    4. Kwak J.M.,
    5. Lee H.H.,
    6. Nam H.G.
    (1993). Two putative protein kinases from Arabidopsis thaliana contain highly acidic domains. Plant Mol. Biol. 22, 615–624.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Purton S.,
    2. Rochaix J.-D.
    (1994). Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol. Biol. 24, 533–537.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Quisel J.D.,
    2. Wykoff D.D.,
    3. Grossman A.R.
    (1996). Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii. Plant Physiol. 111, 839–848.
    OpenUrlAbstract
  56. ↵
    1. Saitou N.,
    2. Nie M.
    (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
    OpenUrlAbstract
  57. ↵
    1. Scott W.,
    2. Metzenberg R.
    (1970). Location of aryl sulfatase in conidia and young mycelia of Neurospora crassa. J. Bacteriol. 104, 1254–1265.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Shi X.,
    2. Finkelstein A.,
    3. Wolf A.J.,
    4. Wade P.A.,
    5. Burton Z.F.,
    6. Jaehning J.A.
    (1996). Paf1p, an RNA polymerase II–associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol. Cell. Biol. 16, 669–676.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Shore D.
    (1994). RAP1: A protein regulator in yeast. Trends Genet. 10, 408–412.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Smith F.W.,
    2. Ealing P.M.,
    3. Hawkesford M.J.,
    4. Clarkson D.T.
    (1995). Plant members of a family of sulfate transporters reveal functional subtypes. Proc. Natl. Acad. Sci. USA 92, 9373–9377.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Stapleton D.,
    2. Gao G.,
    3. Michell B.J.,
    4. Widmer J.,
    5. Mitchelhill K.,
    6. Teh T.,
    7. House C.M.,
    8. Witters L.A.,
    9. Kemp B.E.
    (1994). Mammalian 5′-AMP–activated protein kinase non-catalytic subunits are homologs of proteins that interact with yeast Snf1 protein kinase. J. Biol. Chem. 269, 29343–29346.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Thompson J.D.,
    2. Higgins D.G.,
    3. Gibson T.J.
    (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Thompson-Jaeger S.,
    2. Francois J.,
    3. Gaughran J.P.,
    4. Tatchell K.
    (1991). Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics 129, 697–706.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Trueman L.J.,
    2. Richardson A.,
    3. Forde B.G.
    (1996). Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene 175, 223–231.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Trumbly R.J.
    (1992). Glucose repression in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 6, 15–21.
    OpenUrlCrossRefPubMed
  66. ↵
    1. Tsay Y.F.,
    2. Schroeder J.I.,
    3. Feldmann K.A.,
    4. Crawford N.M.
    (1993). The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72, 705–713.
    OpenUrlCrossRefPubMed
  67. ↵
    1. Uria-Nickelsen M.R.,
    2. Leadbetter E.R.,
    3. Godchaux W. III.
    (1993). Sulfonate-sulfur assimilation by yeasts resembles that of bacteria. FEMS Microbiol. Lett. 114, 73–77.
    OpenUrlCrossRefPubMed
  68. ↵
    1. Uria-Nickelsen M.R.,
    2. Leadbetter E.R.,
    3. Godchaux W. III.
    (1994). Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12. Arch. Microbiol. 161, 434–438.
    OpenUrlCrossRefPubMed
  69. ↵
    1. Weekes J.,
    2. Hawley S.A.,
    3. Corton J.,
    4. Shugar D.,
    5. Hardie D.G.
    (1994). Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues. Eur. J. Biochem. 219, 751–757.
    OpenUrlPubMed
  70. ↵
    1. Whalen J.,
    2. Warman P.
    (1996). Arylsulfatase activity in soil and soil extracts using natural and artificial substrates. Biol. Fert. Soils 22, 373–378.
    OpenUrlCrossRef
  71. ↵
    1. Woods A.,
    2. Munday M.R.,
    3. Scott J.,
    4. Xiaolu Y.,
    5. Carlson M.,
    6. Carling D.
    (1994). Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo. J. Biol. Chem. 269, 19509–19515.
    OpenUrlAbstract/FREE Full Text
  72. ↵
    1. Wykoff D.D.,
    2. Davies J.P.,
    3. Melis A.,
    4. Grossman A.R.
    (1998). The regulation of photosynthetic electron transport during nutrient deprivation of Chlamydomonas reinhardtii. Plant Physiol. 117, 129–139.
    OpenUrlAbstract/FREE Full Text
  73. ↵
    1. Yildiz F.H.,
    2. Davies J.P.,
    3. Grossman A.R.
    (1994). Characterization of sulfate transport in Chlamydomonas reinhardtii during sulfur-limited and sulfur-sufficient growth. Plant Physiol. 104, 981–987.
    OpenUrlAbstract
  74. ↵
    1. Yildiz F.H.,
    2. Davies J.P.,
    3. Grossman A.
    (1996). Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii. Plant Physiol. 112, 669–675.
    OpenUrlAbstract
  75. ↵
    1. Yoon H.W.,
    2. Kim M.C.,
    3. Shin P.G.,
    4. Kim J.S.,
    5. Kim C.Y.,
    6. Lee S.Y.,
    7. Hwang I.,
    8. Bahk J.D.,
    9. Hong J.C.,
    10. Han C.,
    11. Cho M.J.
    (1997). Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus. Mol. Gen. Genet. 255, 359–371.
    OpenUrlCrossRefPubMed
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Sac3, an Snf1-like Serine/Threonine Kinase That Positively and Negatively Regulates the Responses of Chlamydomonas to Sulfur Limitation
John P. Davies, Fitnat H. Yildiz, Arthur R. Grossman
The Plant Cell Jun 1999, 11 (6) 1179-1190; DOI: 10.1105/tpc.11.6.1179

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Sac3, an Snf1-like Serine/Threonine Kinase That Positively and Negatively Regulates the Responses of Chlamydomonas to Sulfur Limitation
John P. Davies, Fitnat H. Yildiz, Arthur R. Grossman
The Plant Cell Jun 1999, 11 (6) 1179-1190; DOI: 10.1105/tpc.11.6.1179
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The Plant Cell Online: 11 (6)
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