Mutants impaired in derepressible alkaline phosphatase activity in Chlamydomonas reinhardtii

Plant Science 119 (1996) 93- 101

Mutants impaired in derepressible alkaline phosphatase in Chlamydomonas reinhardtii Fatima

Bachir”, Etienne

Baiseb, Roland

activity

Loppes””

“Laborutoire de GCnPtique des Microorganismes, Dipartement de Botanique, B2, 7%Universiti de Li2ge. Sart Tilman, B-4000 Liige, Belgium bLuboratoire de Biochimie, DPpartement de Chimie, B6, UniversitP de LiGge, Sart Tilman, B-4000 Ligge, Belgium

Received 2 January 1996; revised 17 May 1996; accepted 17 May 1996

Abstract In the green alga Chlamydomonas reinhardtii, inorganic phosphate starvation results in the synthesis and secretion of two classes of derepressible phosphatases, DN (pH optimum about 7.0) and DA (pH optimum 9.5). Mutants specifically impaired in DA phosphatase activity were isolated after UV treatment of the phoN6 strain lacking the DN phosphatase. Four mutants (phoA3, phoA4, phoA5, phoA6) were genetically analyzed. Mutations were allelic, non-complementing and recessive to the wild-type allele. The lack of DA phosphatase activity in phoA mutants was correlated with the absence of a high molecular weight (200 kDa) polypeptide (P2) present in the secretions of phoA + strains. In addition, partial purification of the DA phosphatase by anion-exchange chromatography in the presence of urea resulted in a concomitant enrichment in this polypeptide. These results indicate that P2 is likely a component of the DA phosphatase. Since the only detectable change in all phoA mutants was the disappearance of P2. it is proposed that phoA is a regulatory gene specifically involved in the control of the DA phosphatase. Keywords:

Chlamydomonas;

Derepressible phosphatases;

1. Introduction The presence of non-specific phosphatases in most living cells makes them able to utilize various phosphorylated organic substrates as sources

of phosphorus. The occurrence of these enzymes is actually essential when the cells have to face an environment poor in or devoid of inorganic phos* Corresponding 3241663840.

author,

Tel.:

+ 3241663823; fax:

+

Alkaline phosphatase;

Mutants

phate (Pi). In lakes, for example, Pi is often considered as a limiting nutrient [l]. In response to Pi starvation, most organisms readily synthesize high amounts of derepressible phosphatases. The regulation of these enzymes has been widely and carefully investigated in Saccharomyces cerevisiae. In this yeast, the most abundant phosphatases are encoded by PH05 (acid phosphatase) and PH08 (alkaline phosphatase) genes, respectively. The study of a large number of mutants made it possible to elaborate a genetic hierarchy of the

0 168-9452 96 5 15.00 C 1996 Elsevier Science Ireland Ltd. All rights reserved PI/ SO304-3835(96)04447-O

regulatory genes which organize the response to P, deficiency [2,3]. PHO5 expression is under the control of at least 5 genes. 4 of which are also involved in the regulation of PH08 expression. PH02 and PH09 specifically act on PH05 and PHOS expression, respectively. Many of these genes have now been cloned and are being investigated at the molecular level [4&7]. In the unicellular green alga C’/~/~rn~~~t/on~ono.s winhwdtii, two derepressible phosphatases prevail, one showing optimal activity at pH 7.0 (DN phosphatase). the other one at pH 9.5 (DA phosphatase) [8]. These enzymes are partially excreted into the growth medium. A large number of phoN mutants (conditional or not) with altered DN phosphatase activity have been isolated using a staining procedure revealing phosphatase activities at the level of colonies growing on agar plates [9]. The mutations were distributed in three loosely linked or unlinked genes and shown to be recessive to their wild-type alleles [IO]. On the basis of their properties. the phosphatases produced by the revertants as well as by the thermosensitive mutants grown under permissive conditions were indistinguishable from the wild-type enzyme, suggesting that all the phoN genes are involved in the regulation of the DN phosphatase [I 1,121. It must be stressed that in all of the phoN mutants the DA phosphatase activity was minimally affected, which suggests that the regulation pathways of the two phosphatases are independent. To date, no pho mutant with altered DA phosphatase activity has been isolated. Such mutants would be interesting for several reasons: (I) being a source of non-contaminated DN phosphatase, they would be convenient for the study of this enzyme; (2) they would provide information about the number and function of genes involved in DA phosphatase synthesis and, in this connection, it could be established whether some phoA mutations also alter production of the DN phosphatase; (3) wild-type cells of C. reinhardtii secrete several glycoproteins specific to Pi-deficiency in the surrounding medium. One of these (73 kDa) has been identified as a probable subunit of the DN phosphatase since it is missing in all phoN mutants [l3]. The comparative analysis of

proteins secreted by the phoA mutants and the wild-type strain should indicate which polypeptide(s) correspond(s) to the DA phosphatase. In this study, alkaline phosphatase-negative mutants have been induced by UV treatment. The electrophoretic analysis of the proteins secreted by these mutants revealed the loss of a 200-kDa polypeptide which is specifically produced by the wild-type strain under P,-deficient conditions and which probably constitutes a subunit of the DA phosphatase.

2. Materials

and methods

All strains of c’lllrrn~~v~~o,nu~lrrs rrinhrrrdtii used throughout this work were derived from the wildtype (WT) strain no. 137~. The phoN6 mutant, previously named PD6 [IO], is impaired in DN phosphatase activity. Arg7-7 and arg7-8 are arginine auxotrophs lacking argininosuccinate lyase; arg7-7 complements arg7-8 in diploids [l4]. Cells were cultured in minimal medium, Trisacetate-phosphate medium (TAP) [I 51. Tris-minimal-phosphate medium containing 5 mM Pi ( + P) or Tris-minimal-phosphate-free medium ( - P) [9] under continuous illumination (6000 lux, 25°C). Cells transferred from ( + P) to ( - P) medium undergo 2-3 residual divisions and accumulate the derepressible phosphatases. 2.2 Prepurcrtion

oj’ cell secretions

and extructs

For preparing extracellular proteins, cells were grown for 4 days on cellophane membranes (6.5 x 6.5 cm, IO6 cells per membrane) placed on solid medium ( 15 g Difco agar/l) as described [ 161. This allowed the collection of concentrated solutions of secreted phosphatases. For preparation of crude cell extracts, cells were pelleted by centrifugation, washed twice with distilled water, resuspended in water and then disrupted ultrasonically (Vibra Cell Sonics Materials). Cell debris was discarded following centrifugation (12 000 x g, 5 min, 4°C).

F. Bachir et al.

95

Plant Science 119 (1996) 93-101

Table I Phosphatase specific activity (icmol naphthol mg protein- ’ h-’ value is the mean of at least 3 independent determinations Mutants

phoN6 (control) phoN6phoA3 phoN6phoA4 phoN6phoAS phoN6phoA6 phoN6phoA7

Phosphatase activities were assayed at 37°C as described [17] using x-naphthylphosphate as a substrate and tetrazotized-o-dianisidine as a postcoupling reagent. Protein was determined according to Bradford [18] using bovine serum albumin as a standard. Activity units are expressed as pmol naphthol produced per h. Staining of colonies for phosphatase activity at pH 9.5 was performed as follows. A drop (2 ~1) of 1 mg ml ~ ’ a-naphthylphosphate in 0.2 M glycine-NaOH buffer, pH 9.5, was applied to each colony. Five min later, the plate was flooded with 10 ml of 2 mg ml - ’ tetrazotized-o-dianisidine in 0.2 M Na acetate buffer, pH 4.8, which resulted in the immediate appearance of purple staining at the level of the colony. 2.4. Mutagenic treatment and genetic unul~~sis Exponentially growing phoN6 cells (TAP medium, 2 x lo6 ml -- ‘) were pelleted, resuspended in fresh TAP medium ( lo7 cells ml ~ ‘) and exposed to UV light (254 nm, 0.2 erg mm - 2 s ’ with continuous agitation) for 4 min as described [19]. Under these conditions, the survival rate was about 1%. The irradiated cells were maintained for 12 h in the dark to avoid photoreactivation and then plated on ( + P) medium. Colonies were replica-plated onto ( - P) medium and after a 4-day incubation in the light screened for the lack of phosphatase activity at pH 9.5 (phoN6 colonies stain purple).

at 37°C pH 9.5) in cells and secretions of phoA mutants. Each

Specific activity of phosphatase at pH 9.5 Secretions

Cell extracts

201 7.10 3.30 8.10 14.3 0.01

4.10 0.05 0.09 0.07 0.17 0

The genetic analysis of the mutants was carried out according to standard procedures [15]. Diploids generated by interallelic complementation of arg7-7 and arg7-8 were selected on minimal medium. Diploidy was confirmed by verifying the increase in cell volume, the dominance of mt over mt +, and the segregation of arg- clones after crossing diploids to wild-type.

2.5. Electrqhoresis

and chronzcrtograph_v

Denatured proteins were resolved by electrophoretic separation in 8.5% polyacrylamide gels in the presence of SDS according to Laemmli [20]. The gels were stained for protein in 0.25% (w/v) Coomassie Brilliant Blue G250 in methanolacetic acid-water (5: 1:4 by volume) at 4°C for 20 min. The protein fraction precipitating between 40 and 60% ammonium sulfate saturation and containing the major part of the DA phosphatase activity was used for chromatography. A DEAE-cellulose column (3 x 23 cm) was equilibrated in 20 mM Tris-HCl, pH 8.0, and proteins were eluted (flow rate: 0.8 ml min ‘) with a NaCl gradient (O.l- 1.0 M in Tris buffer). In denaturation experiments, the same solutions containing 6 M urea were used. Gel filtration on a Sepharose4B column (1.5 x 100 cm) was carried out in 20 mM Tris-HCl, pH 8.0 (flow rate: 0.4 ml min ‘), using thyroglobulin (699 kDa) and apoferritin (440 kDa) as molecular weight standards.

F. Bachir et al. 1 Plant Science I19 (1996) 93-101

96 Table 2 Crosses of phoA

mutants with phoA + and allelism tests

Cross phoN6phoA3mt phoN6phoA4mt phoN6phoA3mt phoN6phoA5mt phoN6phoA6mt

-

x x + x + x + x

phoN6mt + phoN6mt + phoN6phoA4mtphoN6phoA4mt phoN6phoA4mt -

Total no. of clones

phoA +

phoA -

296 360 660 360 780

143 174 0 0 0

153 186 660 360 780

3. Results

3.1. Isolation and genetic analysis of phoA mutants

Attempts were made to isolate alkaline phosphatase mutants from the WT strain. These experiments were unsuccessful, probably due to the fact that the DN phosphatase present in this strain retained sufficient activity at pH 9.5 to stain all colonies, including phoA mutants. Thus, subsequent mutagenic treatments were carried out using phoN6, a mutant in which the DN phosphatase activity is 40 times lower than in the wild-type strain [lo]. Out of 12 400 clones issued from the treatment of phoN6 cells with UV light, 5 did not stain for phosphatase activity at pH 9.5. The phosphatase activities measured in crude cell extracts and in secretions from cells grown on cellophane membranes (Table 1) were much lower in the mutants than in the phoN6 control. The enzyme activity was far higher in secretions than in cell extracts, both in control (about 50-fold) and mutant samples (range 40-loo-fold). All mutants were able to mate except phoN6phoA7 which showed paralyzed flagella and was not further investigated. The results of the crosses presented in Table 2 indicate that phoA3 and phoA4 mutations are of nuclear origin (1:l segregation of wild and mutant alleles in the first two crosses) and that phoA3, phoA4, phoA5 and phoA6 are all allelic (no wild recombinant in phoA - x phoAcrosses). Arg7 derivatives of phoA mutants were constructed and then crossed with each other in combinations. Arg7 + diploid all possible

colonies isolated from all crosses were phoA-, which demonstrates that phoA mutants do not complement in diploids and confirms that the 4 mutations belong to a unique locus. In addition, 2 of the 4 mutant alleles (phoA3 and phoA4) were shown to be recessive to their wild counterpart . The double mutant phoN6phoA4 mt- was crossed to WT mt + to isolate the phoA4 single mutant. However, phoN + phoA - and phoN + phoA + colonies recovered from the random spore plating were indistinguishable from each other when stained for phosphatase activity at pH 9.5. Several tetrads issued from the same cross were thus dissected and one tetratype tetrad containing one phoN - phoA -, one phoN phoA + and two phoN + products was found. Given that the DA phosphatase is inactive at pH 4.8 [9], measurements of the phosphatase activities at pH 4.8 and pH 9.5 allowed the disfrom crimination of phoN + phoA phoN+phoA+ (WT) (Table 3). The results confirm that the DN phosphatase retains significant activity at pH 9.5, which explains why it is impossible to isolate phoA- mutants from the wild-type strain. It must also be pointed out that the DN phosphatase activity measured at pH 4.8 is identical whatever the phoA background (phoA + or phoA -), which indicates that phoA is not involved in the expression of the DN phosphatase. Using phoN6 and phoA4 single mutants, it has been possible to investigate separately the properties of DA and DN, respectively, notably the effect of pH on their activity (Fig. 1). Whereas DN displayed measurable activity in a broad range of pII values, DA was active only at alkaline pH, which confirms the deduced genotypes in Table 3.

F. Bucizir et al. / Plunt Sciente

Table

I19 (1996)

91

9?- 101

3

Extracellular 4 products Products

phosphatase of a tetratype

h-

’ at

tetrad resulting from the cross phoN6phoA4

x

activities (pmol

naphthol

mg protein _

’

37°C.

pH 4.8 or pH 9.5) and deduced genotypes of the

WT Deduced

Specific activity at pH 4.8

genotype

9.5

I

98

135

phoN + phoA + (WT)

2

99

37

phoN + phoA -

3

0.7

73

phoN

phoA + (phoN6)

4

1.9

2.5

phoN

phoA -

3.2. Biochemicul analysis of phoA mutants Dumont et al. [13] have observed that WT cells deprived of Pi secrete a characteristic set of proteins into the surrounding medium. Since most of the DA phosphatase activity is extracellular, we have examined the electrophoretic patterns of proteins secreted by the different mutants. Fig. 2 shows one high molecular weight polypeptide (P2) of about 200 kDa missing from all phoA mutants. Proteins secreted by the 4 tetrad products (see Table 3) were also analyzed by SDS-PAGE. Here too, the occurrence of P2 was strictly correlated with the presence of active DA phosphatase (data not shown). It can, thus, be hypothesized that P2 corresponds to a DA phosphatase subunit. Attempts were then made to purify the DA phosphatase from the secreted proteins. This step should result in concomitant P2 enrichment. The experiments were carried out using the phoN6 strain which does not produce the DN phosphatase (Table 3). The proteins were dialyzed against the loading buffer and then applied to a DEAE-cellulose column. Elution with a linear, 0.1-l M NaCl gradient resulted in one peak of phosphatase activity corresponding to the sole peak of protein (data not shown). Gel filtration on a Sepharose-4B column was also unsuccessful; the eluted DA phosphatase activity coincided with a unique protein peak at an elution volume very close to that of thyroglobulin (699 kDa). These results suggest that the secreted DA phosphatase is associated with other proteins in a high-molecular-weight aggregate or complex. Preliminary experiments showed that treatment of secreted proteins with 6 M urea did not com-

(phoA4) (phoN6phoA4)

pletely abolish DA phosphatase activity. Forty phosphatase units were passed through a DEAEcellulose column pre-equilibrated with 20 mM Tris-HCl, pH 8.0, plus 6 M urea and eluted with a linear NaCl gradient. Three major peaks of phosphatase activity were resolved (Fig. 3) and the corresponding fractions were analyzed by SDSPAGE (Fig. 4). A 200-kDa poiypeptide was prominent in peaks 2 and 3, which could represent two products resulting from the partial disaggregation of the secreted high molecular complex by 6 M urea (see above). These results support the proposal that the 200-kDa polypeptide likely corresponds to the subunit of the DA phosphatase (or to the holoenzyme). The 58-kDa polypeptide present in peak 1 could correspond to a hitherto unknown phosphatase. 4. Discussion All phoN mutants isolated to date in C. reinha&ii still produce a derepressible alkaline (DA) phosphatase [9]. In this study, we have generated and characterized phoA mutants impaired in the activity of this enzyme. The four mutants (phoA3, phoA4, phoA5 and phoA6) isolated after UVtreatment of the phoN6 strain and capable of mating were shown to be allelic and recessive to the wild-type strain. The use of single phoN + phoA and phoN - phoA + mutants made it possible to determine the relative activity of each phosphatase as a function of assay pH (Fig. 1). In phoN6phoA mutants, the phosphatase activity at pH 9.5 was almost completely abolished in both cell extracts and secretions (Table 1). This loss of activity was accompanied by the disap-

98

F. Bacllir

3

4

5

6

7

8

et al. / Pianr

9

loll

PH

Scierwr

119 (1996)

93- IO1

3

5

4

6

7

8

QlOll!

PH

Fig. 1. Phosphatase activity as a function of assay pH in secretions of cells grown on ( - P) medium. A: phoA4, B: phoN6 (see Table 3). Activities were measured in acetate (0). Tris-maleate (A) or glycine-NaOH ( n ) buffer; a.u.: activity units (ymol naphthol h ’ at 37°C).

pearance of a 200 kDa polypeptide in the secretions (Fig. 2). In another connection, peaks 2 and 3 of phosphatase activity observed after DEAE-cellulose chromatography of phoN6 secreted proteins in the presence of urea both contained a major 200 kDa polypeptide (Figs. 3 and 4). Finally, in some SDS-PAGE experiments carried out with phoN6 secretions (showing high DA phosphatase activity), the gel was extensively washed with 0.2 M Trismaleate buffer, pH 7.0, and then stained for phosphatase activity in the same buffer. A unique, but faint band was observed at a position corresponding to 200 kDa suggesting that some renaturation of the DA phosphatase can take place. Taken k Da 1 2 3 4 5 _

Fig. 2. SDS-PAGE analysis of proteins secreted by cells grown on ( -P) medium. Lanes 1: phoN6 (control); 2: phoA3; 3: phoA4: 4: phoA5: 5: phoA6. Coomassie Blue staining. Polypeptide P2 is located at the level of myosm (205 kDa).

together, the results of these experiments support the idea of P2 being a subunit of the DA phosphatase or the phosphatase holoenzyme itself. P2 has a very high apparent molecular weight compared to that of other phosphatases: 66 and 121 kDa for alkaline [21] and acid [22] phosphatases of S. cerevisiae, respectively, 60 kDa for the acid phosphatase of 5. licheniformis [23], and 82 kDa for the alkaline phosphatase of N. crassa [24]. The 200-kDa value should, however, be considered as tentative since P2 is known to be glycosylated [13], as are most of the extracellular proteins in Chlumydomonas [25]. Because glycoproteins, bind SDS less efficiently than standard proteins [26], their apparent molecular weight can be overestimated [27]. With respect to the 58-kDa polypeptide found in the first elution peak after DEAE-cellulose chromatography (Figs. 3 and 4) it is present in all strains (phoA + or phoA - ) and possibly corresponds to a hitherto unknown phosphatase. Experiments are in progress to explore this hypothesis. As the four phoA mutants lack the secreted 200-kDa polypeptide, it can be assumed that phoA is a regulatory gene controlling the expression of the structural gene encoding alkaline phosphatase. If phoA was a structural gene, one would expect to find mutants producing an inactive DA phosphatase with unmodified (missense mutation) or decreased (nonsense mutation) molecular weight. Alternatively, a mutation affecting the process of

99

1.0 --

- 1.0

0.250

0.9

0.225

Applied NaCl gradient

__

Phosphatase

...-..

activity (AsdO)

/

A280

0.8

/

0.8 0.7

0.200 0.175 0.150

0.6

0.6 s

Es a?

G 0.5 2

0.12s

s

0.100

0.4

0.075 0.050

0.2

0.025 0.0 .

0.000

40

0

60

Fraction number Fig. 3. DEAE-cellulose chromatography (in the presence of 6 M urea) of proteins secreted by phoN6. Fractions of 2 ml were collected. Phosphatase activity at pH 9.5 [I 71 and AzSo were recorded.

142

3

kDa zg -

Fig. 4. peaks I, proteins presence tionated phy.

97 78

SDS-PAGE analysis of fractions corresponding to 2 and 3 (lanes I. 2, 3) recovered from secreted phoN6 following chromatography on DEAE-cellulose in the of 6 M urea (see Fig. 3). Lane 4: sample of unfracproteins (I 5 ~8) before anion-exchange chromatogra-

maturation and/or secretion of the enzyme could also lead to the lack of P2 in the extracellular fluid. In S. crrerisiae, the regulation of both alkaline and acid phosphatases is mediated by the products of 6 genes, 4 of which are common to both pathways [3,28]. These conclusions were drawn from the study of mutants, some of which showed pleiotropic effects. In C. reinhardtii, all phoA (this work) and phoN [IO] mutants have been shown to be deficient in the accumulation of one phosphatase only which suggests that DN and DA are controlled by separate pathways. From an evolutionary viewpoint, the independence of the regulation circuits might be beneficial since one mutation altering one enzyme activity leaves the other enzyme fully functional.

100

F. Bachir et al. /Plant Science 119 (1996) 93-101

Progress in the understanding of phosphatase regulation in Chlamydomonas awaits further developments at the molecular level. In a first step, the structural genes coding for DN and DA should be isolated. The identification of the corresponding polypeptides in secretions will permit their microsequencing and provide the necessary probes for cloning the genes of interest from a genomic or cDNA library made from mRNAs isolated from Pi-depleted WT cells [29].

Acknowledgements This work was supported in part by grants from the Belgian F.R.F.C. (2.4582.93), from EEC (Human Capital and Mobility), from the University of Liege (Special Funds for Research), and from ARC (93/98-170). We thank Dr. R.F. Matagne for helpful comments on the manuscript, M. Hayet for expert typing and P. Parkinson and J. Vaassen for illustrations. F.B. is a Fellow of the Moroccan government and R.L. is Research Associate of the National Foundation for Scientific Research (Belgium).

References

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