Mar 24, 1989 - strain of yeast characterized by the ability to secrete gluco- amylase, an ...... flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol.
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1989, p. 3992-3998 0270-7306/89/093992-07$02.00/0 Copyright C 1989, American Society for Microbiology
Vol. 9, No. 9
Regulation of STAI Gene Expression by MAT during the Life Cycle of Saccharomyces cerevisiae A. M. DRANGINIS Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Buiilding 6, Room Bl-lO, Bethesda, Maryland 20892 Received 24 March 1989/Accepted 12 June 1989
STAl encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and a haploid yeast cells secrete high levels of the enzyme, but a/a diploid cells produce undetectable amounts. It has been suggested that STAl is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA) regulation but that deletion of MATal sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MA Tal and a product of the MATa locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulationspecific glucoamylase of S. cerevisiae. A diploid strain with a MATal deletion showed normal induction of STAl in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STAl induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.
Saccharomyces cerevisiae var. diastaticus is a variant strain of yeast characterized by the ability to secrete glucoamylase, an enzyme that catalyzes the digestion of starch. Three unlinked homologous genes, STA1, STA2, and STA3, encode the secreted glucoamylase isozymes I, II, and III (33). These genes are absent in S. cerevisiae, but a related gene, SGA (37) or SAG (5), which encodes an intracellular, sporulation-specific glucoamylase, is present. S. cerevisiae and the variant diastaticus mate readily and produce viable spores (36). Laboratory strains of S. cerevisiae var. diastaticus are generally the products of backcrossing to S. cerevisiae strains. STA1, STA2, and STA3 are known to be regulated by the mating-type locus MAT, which specifies cell type in yeasts. Glucoamylase is secreted by a and at haploid cells but not by a/a diploid cells (24, 35). Yamashita et al. (40) have proposed a role for the MATa2 gene product in mating-type repression of STAI. They mapped a mutation that results in loss of MAT regulation to MATa and reported that MAT regulation could be restored in the mutant by cloned MA Ta2 sequences but not by MATal sequences. They further reported that this regulation by MATa2 is posttranscriptional. Although MATa2 produces a transcript, it has been a gene of unknown function. The other three transcripts from the MA T locus (MA Tal, MA Totl, and MA Ta2) have been found to regulate the expression of genes that define cell type in yeasts (reviewed in references 19 and 29), but no function has been reported for the MATa2 transcript (34). A number of genes that share with STAI the property of haploidspecific expression are known. Transcription of several of these haploid-specific genes, including HO (13), RMEJ (18), and the Tyl elements (7), has been shown to be repressed in diploids by the combined action of the MATal and MATa2 gene products. MATa2 encodes a DNA-binding protein (14)
whose sequence specificity is modified by the presence of MATal (10). In this paper, an analysis is presented of the regulation of STA1 gene expression by MAT throughout the yeast life cycle and of its regulation by nutritional conditions. I show that a strain with a complete deletion of MATa2 is unaltered in mating-type regulation of STAI, whereas deletion of MATal sequences completely abolishes diploid-specific repression. Regulation by MATal is shown to occur at the level of RNA accumulation. STAI therefore appears to be a haploid-specific gene, regulated by MATal and a product of MA Tt, presumably ox2. SGA, which encodes the sporulation-specific nonsecreted glucoamylase responsible for the degradation of glycogen during development of the ascospore, has been shown to be induced at the level of RNA accumulation (20, 37). SGA is closely linked to STA1 (22). It has been proposed that STAl originated as the product of a gene duplication of SGA, followed by a recombination event to give STAJ different 5'-flanking sequences (22, 39). I demonstrate that STA1 mRNA, like that of SGA, is induced during sporulation but by an apparently different mechanism than that used by SGA. Deletion of MATal abolishes SGA induction but does not decrease induction of STAl. Therefore, these two linked and homologous glucoamylase genes are regulated in quite different ways. STAI induction may be a response to the starvation conditions necessary for sporulation, while the induction of SGA is controlled by MAT. The expression of STAI in haploids is shown to be glucose repressible, but cells cultured in rich medium containing glucose still express appreciable amounts of glucoamylase. MATERIALS AND METHODS Yeast strains, media, and genetic methods. The yeast strains used and their relevant genotypes are listed in Table 3992
REGULATION OF STAI IN S. CEREVISIAE
VOL. 9, 1989 TABLE 1. Strains used Strain
M1-800D
Ml-800Da2 M1-800Dal YlY319
Genotype
Source
MA Ta leii2-3,112 arg4 STAI rnata2A: :LEU2 leui2-3, 112 arg4 STAI
Sporulation of YYT1 (from ATCC") This work
inatalA::LEU2 leiu2-3,112 arg4 STAI MATot ura3 STAI
This work
ATCC"
" ATCC. American Type Culture Collection, Rockville. Md. Strain was deposited by 1. Yamashita (38. 40).
1. Standard genetic methods of mating, purifying diploids, sporulation, and tetrad dissection were used as described by Sherman et al. (28). YPGE medium contained 1% yeast extract, 2% Bacto-Peptone (Difco Laboratories, Detroit, Mich.), 3% (vol/vol) glycerol, and 2% (vol/vol) ethanol. Synthetic minimal (SM) medium containing 0.67% yeast nitrogen base without amino acids (Difco) was supplemented with either 2% glucose or 3%c soluble starch (Sigma Chemical Co., St. Louis, Mo.) or both as carbon source. Synthetic complete (SC) medium had the same constituents as SM medium, with the addition of a mixture of amino acids and other growth supplements (25). YEP medium contained 1% yeast extract and 2% Bacto-Peptone; it was supplemented with either 2% glucose or 3% soluble starch or both. Unless otherwise indicated, yeast cells were grown in YEP plus 2% glucose. Presporulation medium contained 0.67% yeast nitrogen base, 0.1% yeast extract, 1% potassium acetate, and 50 mM potassium phthalate buffer (pH 5.5). Sporulation medium contained 0.3% potassium acetate and 0.02% raffinose. Cells were grown to mid-logarithmic phase in YEP plus glucose. transferred to presporulation medium at a density of 105/ml, and allowed to grow for six or seven generations (24 h). The cells were washed with water, suspended in sporulation medium at the same density, and incubated at 30°C (24). Samples were collected for RNA extraction, beginning with suspension in sporulation medium. Construction of MATal and MATa2 deletions. Figure 1 shows the extent of the DNA deletions in relation to the transcriptional units of MATa. I used the XIzoI linker insertion mutations of the Hindlll DNA fragment containing MATa that were constructed in vitro by Tatchell et al. (34). In matax 7, the linker is inserted downstream of the 3' end of the MATa2 coding sequences; in matax 12, the XhoI linker is inserted just upstream of the 5' end of MATa2 (34). When these two constructions were combined by Tatchell et al. (34) at the XhoI site to produce matax 7-12, the entire MATa2 gene was deleted and replaced with a unique XhoI site. Deletion of MATa2 was performed by using plasmid
mata2A MATa2
_--
A
400, which was generously provided by K. Tatchell. This plasmid has matax 7-12 cloned into the HindlIl site of pBR322. I verified the location of the XhoI linkers relative to the coding sequences of MATal and MATa2 in plasmids 196 (described below) and 400 by restriction analysis. A 2.2kilobase (kb) XhoI-SalI fragment containing the LEU2 gene from YEp13 (1) was inserted into the unique XhoI site of plasmid 400, and a HindIII digest of this construction was used to transform strain M1-800D by the lithium acetate method of Ito et al. (12). Leucine prototrophs were selected, and integration at MAT was confirmed by a series of restriction digests and Southern blots of genomic DNA. In all cases, care was taken to distinguish integration at MAT from integration at the silent copies HML and HMR; the latter event never occurred. The MATa2 deletion strain was designated M1-800Da2. When crossed to a MATot leu2 strain, it mated readily, sporulated normally, and produced tetrads in which the parental ditype/nonparental ditype/tetratype ratio was 16:0:0, thus demonstrating cosegregation of the LEU2 marker with MATa. Plasmid 196, which consists of matax 50 in the HindlIl site of vector YRp7 (32), was also received from K. Tatchell. In matax 50, the XhoI linker insertion is near the 5' end of MATal (34). Two adjacent EcoRI fragments were deleted from the vector to eliminate the BgII site in the TRPI ARSI sequences, leaving a unique BglII site at the 3' end of the MA Tal gene. The XhoI-BglII fragments of MA Tal was then replaced by a 2.5-kb XlioI-BglII fragment containing the LEU2 gene. The resulting construct retained enough MATal sequences to encode 28 amino acids at the N terminus and 3 amino acids at the C terminus, representing 29% of the MATal coding sequences. The plasmid was digested with Pill and HindlIl, which cut in sequences unique to MAT. The restriction digest was used to transform strain M1-800D to leucine prototrophy. The transformants were screened as described above except that tetrad analysis was not performed because diploids made from the strain would not sporulate. The resulting MATal deletion strain was designated M1-800Dal. Standard procedures were used throughout for the preparation, modification, and cloning of plasmid DNA molecules (16). Glucoamylase assay. Cells were grown at 30°C for 2 days and then pelleted by centrifugation. The culture supernatants were dialyzed extensively against 10 mM sodium acetate (pH 5.2) at 4°C. The supernatants were incubated in 100 mM sodium acetate (pH 5.2)-4% soluble starch in a total volume of 0.5 ml at 50°C for 30 min. The reaction was terminated by boiling for 10 min and then allowed to cool. Glucose produced by action of glucoamylase on soluble starch was assayed by using a coupled glucose oxidaseperoxidase assay kit (Sigma Diagnostics no. 510). Blanks for matalA MATal ---
I
HindUl I
Okb
3993
BglII I
I
I I lkb
I
I
I 2kb
I
I
I 3kb
HindlIH I Il I I I
4kb
FIG. 1. Map of MATa showing the extent of the chromosomal deletions made. Positions of the MATal and MATa2 transcriptional units are indicated. Arrows indicate direction of transcription. Brackets show locations of endpoints of the sequences that were deleted and replaced by the LEU2 gene for the construction of strains Ml-800Da2 and M1-800Dal by homologous gene replacement.
3994
MOL. CELL. BIOL.
DRANGINIS
each sample were prepared by boiling a portion of the dialyzed culture supernatant before the assay and subtracting this value as background from that obtained for the nonboiled samples. Only in the case of cells grown in medium containing soluble starch did this boiled blank show any appreciable background in the glucose assay. Amylase activity is expressed as micrograms of glucose released per 100 ,u1 of supernatant per A60 cells. RNA extraction, electrophoresis, and gel transfer. Total RNA was prepared by a method based on that of Carlson and Botstein (3), as follows. Miniprep cultures (10 ml) were grown to mid-logarithmic phase. The cells were collected by centrifugation, transferred in 1 ml of water to microfuge tubes, and collected again. To the cell pellet was added 0.4 g of washed glass beads (0.45 to 0.5 mm; Thomas Scientific, Swedesboro, N.J.), followed rapidly by 0.3 ml of cracking buffer (0.5 M NaCl, 0.2 M Tris hydrochloride [pH 7.4], 10 mM EDTA, 1% sodium dodecyl sulfate) and 0.3 ml of 25:24:1 phenol-chloroform-isoamyl alcohol. The samples were vortexed at top speed for 1 min. Another 0.3 ml of cracking buffer and 0.3 ml of phenol-chloroform-isoamyl alcohol was added, and the mixture was vortexed for 30 s. The samples were centrifuged for 5 min in a microfuge, and the aqueous phase was again extracted with 0.3 ml of phenol-chloroform-isoamyl alcohol. The sample was precipitated with 1 ml of ethanol at -20°C and then centrifuged for 1 min. The pellet was washed with 70% ethanol and dissolved in sterile water. Formaldehyde-agarose gels (16) were blotted to GeneScreen nylon membrane (Dupont, NEN Research Products, Boston, Mass.) according to the instructions of the manufacturer. Hybridization of RNA blots. Amylase sequences were detected by hybridization to a 72-base-pair oligonucleotide complementary to bases 1891 to 1962 of the STAI DNA sequence (39). This sequence is in a domain that is homologous to the SGA gene. A model 380B DNA synthesizer (Applied Biosystems, Foster City, Calif.) was used to make the oligonucleotide as well as a complementary 27-base-pair primer. The oligonucleotides were annealed, and the primer was extended by using Klenow enzyme (16) and [a32P]dATP. Hybridizations were performed at 37°C in 50% formamide buffer and washed as specified by the manufacturer of GeneScreen. The URA3 gene (26) was used as a probe to provide an internal quantitative RNA standard on transfers of RNA from vegetative cells; an oligonucleotide probe to 18S rRNA was used as the internal quantitative standard on transfers of RNA from sporulating cells. The 18S oligonucleotide probe was complementary to nucleotides 1496 to 1537 of the 18S rRNA gene sequence (27). Autoradiography was done by exposure to X-ray film at -70°C. Quantitation of RNA was performed on a Hoeffer GS300 scanning densitometer under conditions in which response was linear with respect to band intensity. RESULTS MAT control of glucoamylase secretion. The glucoamylase encoded by STA1 is secreted from a and ao haploid yeast cells but not from a/a. diploid cells. To analyze the basis of this diploid-specific repression, a set of isogenic MATa strains with deletions of either MATal (M1-800Dal) or MATa2 (M1-800Da2) was constructed by homologous gene replacement. Figure 1 shows the extent of the deletions made in MATa. The selectable marker LEU2
was
inserted at the site
TABLE 2. MAT control of glucoamylase secretion Expt
Strain
MAT genotype
Glucoamylase activity'
1
YIY319 M1-800D M1-800D x YIY319 M1-800Da2 M1-800Da2 x YIY319
MA Tot MATa MA TaMA Tot mata2A::LEU2 mata2A::LEU21MATot
46.4 26.8
