Oe etios
71
,
© Springer-Verlag 1983
Isolation and Characterization of a Maltose Transport Mutant in the Yeast
Saccharomyces cerevisiae Michael J. Goldenthal, Joe D. Cohen, and Julius Marmur Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Abstract. Yeast strains carrying a functional MAL locus are inducible for the co-ordinate synthesis of both maltase and maltose permease when grown in the presence of maltose. Whether the maltose permease is encoded by a gene at the MAL loci has remained unclear due to the lack of mutants in this function. To isolate mutants defective in maltose transport, a positive selection strategy was employed in which a number of Malmutants were obtained. Among these one Mal- mutant was isolated which had normal levels of wild-type maltase in cell free extracts. This isolate, designated MGT1, has a defect in maltose transport (malT1), detected by its markedly lower uptake of [14C]maltose, and by its growth on media containing 10% but not 2% maltose. Since the K m of maltose uptake is altered 10-fold in this mutant and the Vmax remains unchanged, it is suggested that the mutation alters the structure of the maltose permease involved in transport of the disaccharide into the cell rather than its regulation. A genetic analysis of the malT1 mutation shows that it is in a gene allelic to one at the MALl locus. Transformation of this mutant to the Mal + phenotype using a chimeric yeast/E, coli shuttle plasmid containing a subcloned fragment of the MAL6 locus suggests that the presence of a functional analogue of the gene encoding the maltose transport function is an integral part of the MAL6 locus as well.
Key words: Maltose permease - Transport m u t a n t Yeast MAL loci
Introduction
Maltose utilization in Saccharomyces cerevisiae (or the closely related strain S. carlsbergenesis) depends on the
Offprint requests to: J. Marmur
presence of any one of 5 unlinked chromosomal MAL loci; these have been designated M A L l - 4 and MAL6. Strains carrying a functional MAL locus are inducible by maltose for the synthesis of both maltase and maltose permease (Kroon and Kroningsberger 1970). Naumov (1971, 1976) showed by complementation analysis of naturally occurring maltose-negative (Mal-) strains of Saccharomyces that a MAL locus consisted of at least two linked genes, MALp and MALg. In complementation studies of the MALp gene with maltase regulatory gene mutants isolated by ten Berge et al. (1973), Naumov (1976) could equate MALp with the maltase regulatory gene. Maltase structural gene information has recently been shown to be present at each of the five MAL loci examined (Goldenthal et al. submitted for publication). This was made possible by genetic and by Southern blot analyses, using a subcloned fragment of the cloned MAL6 maltase structural gene (Federoff et al. 1982) as a probe. Whether maltose permease is encoded by a gene at a MAL locus has remained unclear due to the lack of mutants in this function. The only report of yeast strains defective in maltose uptake (dsf) showed that the lesions were in genes unlinked to MAL loci (Zimmermann and Eaton 1973). Since maltose permease is apparently regulated in a similar fashion to maltase, its possible linkage to regulatory and structural genes at the MAL locus appeared an attractive possibility. Physiologically, the presence of a maltose permease has been inferred from kinetic analysis of maltose transport which is typical of carrier mediated transport (Harris and Thompson 1961; de la Fuente and Sols 1962; Kroon and Koningsberger 1970; Siro and Lovgren 1979). There is good evidence that inducible maltose uptake is an active transport process accompanied by a concomitant uptake of protons (Seaston et al. 1973), is inhibited by energy uncouplers and is coupled to a
196 p r o t o n g r a d i e n t even w h e n a c c u m u l a t i o n against a conc e n t r a t i o n g r a d i e n t does n o t take place ( S e r r a n o 1977). N e i t h e r t h e a c t u a l carrier p r o t e i n or m a l t o s e p e r m e a s e , n o r t h e c o m p o n e n t s i n v o l v e d in e n e r g y - c o u p h n g have b e e n i d e n t i f i e d . One possible c a n d i d a t e i.e., a m a l t o s e b i n d i n g a c t i v i t y l o c a t e d in t h e y e a s t p l a s m a m e m b r a n e , h a s b e e n p r e l i m i n a r i l y c h a r a c t e r i z e d b y Siro et al. ( 1 9 8 1 ) . T h e p r e s e n t s t u d y was u n d e r t a k e n t o isolate m u t a n t s defective in m a l t o s e u p t a k e b u t w h i c h still r e t a i n a f u n c t i o n a l m a l t a s e s t r u c t u r a l gene. T h e u l t i m a t e goal is t o i d e n t i f y t h e gene(s) i n v o l v e d in m a l t o s e u p t a k e a n d in p a r t i c u l a r t h e o n e ( s ) c o d i n g for m a l t o s e p e r m e a s e . This p a p e r p r e s e n t s t h e m u t a n t i s o l a t i o n s c h e m e a n d t h e preliminary characterization of one such mutant.
Materials a n d M e t h o d s
Strains. The relevant genotypes of the S. eerevisiae strains used in these studies are JM 1820, a M A L l c his4 leu2 pdcl, a strain with constitutive expression of maltase and maltose uptake; JM 1820R, a PDC revertant of JM 1820; MGT1, his4 Ieu2PDC, a maltose transport mutant derived from JM 1820; JC-2C, a M A L l his4 lys. Media. YEP (1% Difco yeast extract, 2% Difco peptone) was supplemented with either 2% dextrose (D), 2% each of glycerol and ethanol (GE), 2% maltose (M) or 1.5% raffinose (R). Minimal media (S) consisted of 0.67% Difco Yeast Nitrogen Base without amino acids supplemented with amino acids and nitrogenous bases as required and with the appropriate carbon source. Solid media had 2% agar added. Tests for maltose utilization were performed on indicator plates (YEPM) containing either eosin and methylene blue (EMB) or bromcreosol purple (BCP) (ten Berge 1972). YEPM-EMB plates were supplemented with the auxotrophic requirements of the strains being examined. Mal + phenotypes were also detected by growth on SM plates supplemented with auxotrophic requirements. Maltose fermentation was monitored using Durham tubes containing 5 ml YEP supplemented with varying concentrations of maltose. Gas production was scored during 7 days incubation at 25 °C. For induction and assay of maltase and maltose permease, cells were innoculated in YEPM and harvested at mid logarithmic phase. To test for constitutive maltase synthesis cells were grown to mid logarithmic phase in YEPR and harvested. Positive Selection of Mal- Mutants. Strain JM 1820 carries a functional MAL locus and is constitutive for both maltase synthesis and maltose uptake. This constitutivity makes it convenient to analyze and distinguish among Mal- isolates those mutants affecting the synthesis or structure of maltase from those defective in the maltose uptake; the synthesis of maltase in this strain is independent of a functional maltose permease. JM 1820 also carries the pdcl mutation which affects pyruvate decarboxylase (Lam and Marmur 1977). This strain will only grow on media containing a carbon source obligatorily metabolized via the mitochondria (e.g. YEPGE). The cells however will not grow on YEPD, YEPM or YEPGE plus D or M since either dextrose or maltose apparently causes pyruvate accumulation and excretion leading to a drop in pH which inhibits cell growth (Schmitt
M.J. Goldenthal et al.: Yeast Maltose Transport Mutant and Zimmermann 1982). Mutants defective in maltose metabolism will thus allow cells to grow in YEPGE plus M. Strain JM 1820 was mutagenized with ethylmethane sulfonate (EMS) under conditions resulting in approximately 50% survival. Treated cells were plated on YEPGE plus M at a density of 106 cells/plate, and incubated 4 - 6 days at 30 °C. Colonies that appeared were picked, purified and checked to ensure that did not grow on YEPD (to eliminate possible pdel revertants). Those isolates that did not grow on YEPD were then reverted with respect to the pdcl mutation by selection on YEPD. Revertants that grew on YEPD were selected and tested for maltose utilization of YEPM-BCP and YEPM-EMB. Presumptive Mal- mutants were also examined in Durham tubes for maltose fermentation. Out of 700 mutants examined, 90 were Malby all three criteria.
Other Genetic Methods. Standard genetic methods of mating, selection and sporulation of diploids, and tetrad analysis were done according to Sherman et al. (1972). Measurement of Maltose Uptake. Maltose uptake was measured by the method of Serrano (1977). Yeast cells suspended at a concentration of 20 mg/ml in 0.1 M tartaric acid adjusted to pH 4.3 with Tris. The transport assay was started by the addition of 0.1 uCi of [14C]maltose to 1 ml of cell suspension, with the concentration of maltose ranging from 0 . 1 - 1 0 raM. After mixing, aliquots of 100 ul were removed at 20 s intervals and transport was stopped by dilution in 10 ml ice cold water. The cells were immediately collected on glass-fiber filters, washed twice with 10 ml ice cold water, filters dried, Aquasol added and radioactivity counted in a liquid scintillation counter. The activities reported are the average of 2 to 4 determinations. The data presented has been corrected for background and non-specific binding by subtraction of counts obtained in control experiments in which boiled cells were used. Enzyme Assays. Washed yeast cells suspended in a buffer of 50 mM potassium phosphate, pH 6.8, containing 1 mM phenylmethane sulfonyl fluoride were disrupted in a Braun homogenizer with glass beads for 2 min with cooling. Broken cells were centrifuged at 12,000 x g for 10 min and the supernatant used in the following assays. PNPGase activity (PNPG is hydrolyzed by a-methylgincosidase as well as maltase) was assayed in a reaction mix containing PM buffer (50 mM potassium phosphate, pH 6.8, 1 mM ~-mercaptoethanol) and a final concentration of 0.3 mg/ml of p-nitrophenyl ~-D-glucoside (PNPG). The increase in absorption at 400 nm due to the release of p-nitrophenol was measured after the reaction was stopped by the addition of an equal volume of 1 M sodium carbonate. Enzyme activities are expressed as nmoles substrate split per min per mg protein at 25 °C. Maltase activity was determined by using a coupled assay procedure. The assay mixture contained PM buffer, 4 mM MgC12, 0.45 mg/ml NADP, 10 mM ATP, 2 mM maltose, 1.4 U hexokinase (140 U/mg) and 0.35 U glucose 6-phosphate dehydrogenase (350 U/mg) (both enzymes obtained from Boehringar Mannheim) The reduction of NADP to NADPH was measured at 340 nm using a Gilson recording spectrophotometer after the addition of a disrupted yeast cell extract dialyzed overnight against PM buffer. Protein determinations were carried out by the method of Bradford (1976). Transformation of Yeast with Plasmicl DNA. Transformation of malT1 to a Mal + phenotype was carried out using the LiC1 procedure described by Ito et al. (1983). Plasmid DNA was used at a concentration of 5 - 1 0 #g for each transformation.
M. J. Goldenthal et al.: Yeast Maltose Transport Mutant
197
Table 1. Comparison of kinetic properties of parental JM 1820R and mutant MGT1 strains with respect to PNPGase and maltase activities
Table 2. Characteristics of [14C]maltose uptake: Effects of various pretreatments and of other carbohydrates on [14Clmaltose uptake by strain JM 1820R
Strain
(A) Treatment
JM 1820R MGT1
Km PNPG (raM)
Vmax PNPG
0.3 0.285
9 9.2
Km Maltose (raM)
Vmax Maltose
15.6 16.5
11.2 11.8
Calculation of the kinetic parameters were done using doublereciprocal plots of data representing three experiments with different cell batches. Vmax for PNPGase and maitase activity are expressed in arbitrary activity units
Control Dinitrophenol Nystatin Cold (4 °C) Boiled Cells
Final Conc.
0.1 mM 0.5 mM 250 ~g/ml -
(B) Carbohydrate Added Selection of transformants was on SM medium supplemented with the appropriate auxotrophic requirements. Mitotic instability of plasmids in transformed cells was checked after growth on YPD for 10 generations.
Results A positive selection scheme was used to isolate yeast mutants defective in maltose metabolism as described in Materials and Methods. Strain JM 1820 was mutagenized with EMS and mutants were selected for their ability to grow on YEPGE plates in the presence o f 2% maltose. Putative mutants were checked for their ability to ferment maltose after reversion o f the pdcl mutation. Among these was a class o f mutants which were unable to utilize maltose on SM or on YEPM-BCP or YEPMEMB indicator plates or to ferment maltose in Durham tubes. The majority o f the M a l - mutants obtained had nondetectable or greatly diminished levels o f both maltase and PNPGase activities. Among the M a l - isolates one, designated MGT1, when characterized biochemically still synthesized maltase constitutively (as did the parent strain). That it still had a functional maltase structural gene was confirmed b y the analysis o f the maltase from PDC revertants of both m u t a n t and parent (JM 1820R) strains. The enzymes from both sources were found to be identical with respect to the kinetic properties o f K m and Vma x (see Table 1). The presence of constitutive maltase activity, indistinguishable from that found in the wild-type, suggested that this M a l - mutant might be defective in maltose uptake. A further indication that this was indeed the case was obtained by analyzing the growth and fermentation characteristics o f this m u t a n t in Durham tubes in media with maltose concentrations varying from 2 to 10%. After a 7 day incubation period, MGT1 fermented maltose only at the highest maltose concentrations. Other M a l - mutants tested did n o t show a similar fermentation response. Fermentation at high, but not at
Control Fructose Raffinose Sucrose ce-Methylglucoside Dextrose 5-Thiomaltose Maltose 10 mM Maltose 20 mM
Maltose Uptake (cpm/min mg cells)
% Control Uptake
2,190 710 400 325 300 200
33 18 18 17 9
Maltose Uptake (cpm/min/ mg cells)
% Control Uptake
2,190 2,295 2,210 2,125 1,950 1,880 1,350 790 285
105 101 97 88 85 62 36 13
A) Cells of the strain were preincubated for 10-20 min at 20 °C with the concentration of inhibitors indicated in the table. At the end of the preincubation maltose transport was measured by the uptake of [14Clmaltose (2 raM). The specific activity of [14C]maltose was 200 cpm/nmol: B) The uptake of [14C]maltose (2 mM) by strain JM 1820R was measured in the presence of other carbohydrates added at a concentration of 20 mM
low concentrations of maltose therefore provides an initial diagnostic test for putative maltose permease mutants. The growth in media with high concentrations o f maltose might occur b y passive diffusion o f the sugar into the cells, thus bypassing the carrier-mediated mode o f transport. A similar observation was made in the case o f galactose permease mutants (Douglas and Condie 1954). Direct evidence that MGT1 was affected in maltose uptake was obtained as follows. Maltose uptake was assayed directly by measuring the initial velocity o f [14C]maltose uptake according to the m e t h o d o f Serrano (1977). Preliminary experiments, described in Table 2, demonstrate the validity, of the assay procedure and describe some of the characteristics displayed b y the maltose uptake system in wild-type yeast. Maltose uptake is essentially linear for 2 - 3 min. To reduce the level o f maltose hydrolysis during the course of the assay, short
198
M.J. Goldenthal et al.: Yeast Maltose Transport Mutant
E,-,3 E O. o
< iJJ (1)
71
,
© Springer-Verlag 1983
Isolation and Characterization of a Maltose Transport Mutant in the Yeast
Saccharomyces cerevisiae Michael J. Goldenthal, Joe D. Cohen, and Julius Marmur Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Abstract. Yeast strains carrying a functional MAL locus are inducible for the co-ordinate synthesis of both maltase and maltose permease when grown in the presence of maltose. Whether the maltose permease is encoded by a gene at the MAL loci has remained unclear due to the lack of mutants in this function. To isolate mutants defective in maltose transport, a positive selection strategy was employed in which a number of Malmutants were obtained. Among these one Mal- mutant was isolated which had normal levels of wild-type maltase in cell free extracts. This isolate, designated MGT1, has a defect in maltose transport (malT1), detected by its markedly lower uptake of [14C]maltose, and by its growth on media containing 10% but not 2% maltose. Since the K m of maltose uptake is altered 10-fold in this mutant and the Vmax remains unchanged, it is suggested that the mutation alters the structure of the maltose permease involved in transport of the disaccharide into the cell rather than its regulation. A genetic analysis of the malT1 mutation shows that it is in a gene allelic to one at the MALl locus. Transformation of this mutant to the Mal + phenotype using a chimeric yeast/E, coli shuttle plasmid containing a subcloned fragment of the MAL6 locus suggests that the presence of a functional analogue of the gene encoding the maltose transport function is an integral part of the MAL6 locus as well.
Key words: Maltose permease - Transport m u t a n t Yeast MAL loci
Introduction
Maltose utilization in Saccharomyces cerevisiae (or the closely related strain S. carlsbergenesis) depends on the
Offprint requests to: J. Marmur
presence of any one of 5 unlinked chromosomal MAL loci; these have been designated M A L l - 4 and MAL6. Strains carrying a functional MAL locus are inducible by maltose for the synthesis of both maltase and maltose permease (Kroon and Kroningsberger 1970). Naumov (1971, 1976) showed by complementation analysis of naturally occurring maltose-negative (Mal-) strains of Saccharomyces that a MAL locus consisted of at least two linked genes, MALp and MALg. In complementation studies of the MALp gene with maltase regulatory gene mutants isolated by ten Berge et al. (1973), Naumov (1976) could equate MALp with the maltase regulatory gene. Maltase structural gene information has recently been shown to be present at each of the five MAL loci examined (Goldenthal et al. submitted for publication). This was made possible by genetic and by Southern blot analyses, using a subcloned fragment of the cloned MAL6 maltase structural gene (Federoff et al. 1982) as a probe. Whether maltose permease is encoded by a gene at a MAL locus has remained unclear due to the lack of mutants in this function. The only report of yeast strains defective in maltose uptake (dsf) showed that the lesions were in genes unlinked to MAL loci (Zimmermann and Eaton 1973). Since maltose permease is apparently regulated in a similar fashion to maltase, its possible linkage to regulatory and structural genes at the MAL locus appeared an attractive possibility. Physiologically, the presence of a maltose permease has been inferred from kinetic analysis of maltose transport which is typical of carrier mediated transport (Harris and Thompson 1961; de la Fuente and Sols 1962; Kroon and Koningsberger 1970; Siro and Lovgren 1979). There is good evidence that inducible maltose uptake is an active transport process accompanied by a concomitant uptake of protons (Seaston et al. 1973), is inhibited by energy uncouplers and is coupled to a
196 p r o t o n g r a d i e n t even w h e n a c c u m u l a t i o n against a conc e n t r a t i o n g r a d i e n t does n o t take place ( S e r r a n o 1977). N e i t h e r t h e a c t u a l carrier p r o t e i n or m a l t o s e p e r m e a s e , n o r t h e c o m p o n e n t s i n v o l v e d in e n e r g y - c o u p h n g have b e e n i d e n t i f i e d . One possible c a n d i d a t e i.e., a m a l t o s e b i n d i n g a c t i v i t y l o c a t e d in t h e y e a s t p l a s m a m e m b r a n e , h a s b e e n p r e l i m i n a r i l y c h a r a c t e r i z e d b y Siro et al. ( 1 9 8 1 ) . T h e p r e s e n t s t u d y was u n d e r t a k e n t o isolate m u t a n t s defective in m a l t o s e u p t a k e b u t w h i c h still r e t a i n a f u n c t i o n a l m a l t a s e s t r u c t u r a l gene. T h e u l t i m a t e goal is t o i d e n t i f y t h e gene(s) i n v o l v e d in m a l t o s e u p t a k e a n d in p a r t i c u l a r t h e o n e ( s ) c o d i n g for m a l t o s e p e r m e a s e . This p a p e r p r e s e n t s t h e m u t a n t i s o l a t i o n s c h e m e a n d t h e preliminary characterization of one such mutant.
Materials a n d M e t h o d s
Strains. The relevant genotypes of the S. eerevisiae strains used in these studies are JM 1820, a M A L l c his4 leu2 pdcl, a strain with constitutive expression of maltase and maltose uptake; JM 1820R, a PDC revertant of JM 1820; MGT1, his4 Ieu2PDC, a maltose transport mutant derived from JM 1820; JC-2C, a M A L l his4 lys. Media. YEP (1% Difco yeast extract, 2% Difco peptone) was supplemented with either 2% dextrose (D), 2% each of glycerol and ethanol (GE), 2% maltose (M) or 1.5% raffinose (R). Minimal media (S) consisted of 0.67% Difco Yeast Nitrogen Base without amino acids supplemented with amino acids and nitrogenous bases as required and with the appropriate carbon source. Solid media had 2% agar added. Tests for maltose utilization were performed on indicator plates (YEPM) containing either eosin and methylene blue (EMB) or bromcreosol purple (BCP) (ten Berge 1972). YEPM-EMB plates were supplemented with the auxotrophic requirements of the strains being examined. Mal + phenotypes were also detected by growth on SM plates supplemented with auxotrophic requirements. Maltose fermentation was monitored using Durham tubes containing 5 ml YEP supplemented with varying concentrations of maltose. Gas production was scored during 7 days incubation at 25 °C. For induction and assay of maltase and maltose permease, cells were innoculated in YEPM and harvested at mid logarithmic phase. To test for constitutive maltase synthesis cells were grown to mid logarithmic phase in YEPR and harvested. Positive Selection of Mal- Mutants. Strain JM 1820 carries a functional MAL locus and is constitutive for both maltase synthesis and maltose uptake. This constitutivity makes it convenient to analyze and distinguish among Mal- isolates those mutants affecting the synthesis or structure of maltase from those defective in the maltose uptake; the synthesis of maltase in this strain is independent of a functional maltose permease. JM 1820 also carries the pdcl mutation which affects pyruvate decarboxylase (Lam and Marmur 1977). This strain will only grow on media containing a carbon source obligatorily metabolized via the mitochondria (e.g. YEPGE). The cells however will not grow on YEPD, YEPM or YEPGE plus D or M since either dextrose or maltose apparently causes pyruvate accumulation and excretion leading to a drop in pH which inhibits cell growth (Schmitt
M.J. Goldenthal et al.: Yeast Maltose Transport Mutant and Zimmermann 1982). Mutants defective in maltose metabolism will thus allow cells to grow in YEPGE plus M. Strain JM 1820 was mutagenized with ethylmethane sulfonate (EMS) under conditions resulting in approximately 50% survival. Treated cells were plated on YEPGE plus M at a density of 106 cells/plate, and incubated 4 - 6 days at 30 °C. Colonies that appeared were picked, purified and checked to ensure that did not grow on YEPD (to eliminate possible pdel revertants). Those isolates that did not grow on YEPD were then reverted with respect to the pdcl mutation by selection on YEPD. Revertants that grew on YEPD were selected and tested for maltose utilization of YEPM-BCP and YEPM-EMB. Presumptive Mal- mutants were also examined in Durham tubes for maltose fermentation. Out of 700 mutants examined, 90 were Malby all three criteria.
Other Genetic Methods. Standard genetic methods of mating, selection and sporulation of diploids, and tetrad analysis were done according to Sherman et al. (1972). Measurement of Maltose Uptake. Maltose uptake was measured by the method of Serrano (1977). Yeast cells suspended at a concentration of 20 mg/ml in 0.1 M tartaric acid adjusted to pH 4.3 with Tris. The transport assay was started by the addition of 0.1 uCi of [14C]maltose to 1 ml of cell suspension, with the concentration of maltose ranging from 0 . 1 - 1 0 raM. After mixing, aliquots of 100 ul were removed at 20 s intervals and transport was stopped by dilution in 10 ml ice cold water. The cells were immediately collected on glass-fiber filters, washed twice with 10 ml ice cold water, filters dried, Aquasol added and radioactivity counted in a liquid scintillation counter. The activities reported are the average of 2 to 4 determinations. The data presented has been corrected for background and non-specific binding by subtraction of counts obtained in control experiments in which boiled cells were used. Enzyme Assays. Washed yeast cells suspended in a buffer of 50 mM potassium phosphate, pH 6.8, containing 1 mM phenylmethane sulfonyl fluoride were disrupted in a Braun homogenizer with glass beads for 2 min with cooling. Broken cells were centrifuged at 12,000 x g for 10 min and the supernatant used in the following assays. PNPGase activity (PNPG is hydrolyzed by a-methylgincosidase as well as maltase) was assayed in a reaction mix containing PM buffer (50 mM potassium phosphate, pH 6.8, 1 mM ~-mercaptoethanol) and a final concentration of 0.3 mg/ml of p-nitrophenyl ~-D-glucoside (PNPG). The increase in absorption at 400 nm due to the release of p-nitrophenol was measured after the reaction was stopped by the addition of an equal volume of 1 M sodium carbonate. Enzyme activities are expressed as nmoles substrate split per min per mg protein at 25 °C. Maltase activity was determined by using a coupled assay procedure. The assay mixture contained PM buffer, 4 mM MgC12, 0.45 mg/ml NADP, 10 mM ATP, 2 mM maltose, 1.4 U hexokinase (140 U/mg) and 0.35 U glucose 6-phosphate dehydrogenase (350 U/mg) (both enzymes obtained from Boehringar Mannheim) The reduction of NADP to NADPH was measured at 340 nm using a Gilson recording spectrophotometer after the addition of a disrupted yeast cell extract dialyzed overnight against PM buffer. Protein determinations were carried out by the method of Bradford (1976). Transformation of Yeast with Plasmicl DNA. Transformation of malT1 to a Mal + phenotype was carried out using the LiC1 procedure described by Ito et al. (1983). Plasmid DNA was used at a concentration of 5 - 1 0 #g for each transformation.
M. J. Goldenthal et al.: Yeast Maltose Transport Mutant
197
Table 1. Comparison of kinetic properties of parental JM 1820R and mutant MGT1 strains with respect to PNPGase and maltase activities
Table 2. Characteristics of [14C]maltose uptake: Effects of various pretreatments and of other carbohydrates on [14Clmaltose uptake by strain JM 1820R
Strain
(A) Treatment
JM 1820R MGT1
Km PNPG (raM)
Vmax PNPG
0.3 0.285
9 9.2
Km Maltose (raM)
Vmax Maltose
15.6 16.5
11.2 11.8
Calculation of the kinetic parameters were done using doublereciprocal plots of data representing three experiments with different cell batches. Vmax for PNPGase and maitase activity are expressed in arbitrary activity units
Control Dinitrophenol Nystatin Cold (4 °C) Boiled Cells
Final Conc.
0.1 mM 0.5 mM 250 ~g/ml -
(B) Carbohydrate Added Selection of transformants was on SM medium supplemented with the appropriate auxotrophic requirements. Mitotic instability of plasmids in transformed cells was checked after growth on YPD for 10 generations.
Results A positive selection scheme was used to isolate yeast mutants defective in maltose metabolism as described in Materials and Methods. Strain JM 1820 was mutagenized with EMS and mutants were selected for their ability to grow on YEPGE plates in the presence o f 2% maltose. Putative mutants were checked for their ability to ferment maltose after reversion o f the pdcl mutation. Among these was a class o f mutants which were unable to utilize maltose on SM or on YEPM-BCP or YEPMEMB indicator plates or to ferment maltose in Durham tubes. The majority o f the M a l - mutants obtained had nondetectable or greatly diminished levels o f both maltase and PNPGase activities. Among the M a l - isolates one, designated MGT1, when characterized biochemically still synthesized maltase constitutively (as did the parent strain). That it still had a functional maltase structural gene was confirmed b y the analysis o f the maltase from PDC revertants of both m u t a n t and parent (JM 1820R) strains. The enzymes from both sources were found to be identical with respect to the kinetic properties o f K m and Vma x (see Table 1). The presence of constitutive maltase activity, indistinguishable from that found in the wild-type, suggested that this M a l - mutant might be defective in maltose uptake. A further indication that this was indeed the case was obtained by analyzing the growth and fermentation characteristics o f this m u t a n t in Durham tubes in media with maltose concentrations varying from 2 to 10%. After a 7 day incubation period, MGT1 fermented maltose only at the highest maltose concentrations. Other M a l - mutants tested did n o t show a similar fermentation response. Fermentation at high, but not at
Control Fructose Raffinose Sucrose ce-Methylglucoside Dextrose 5-Thiomaltose Maltose 10 mM Maltose 20 mM
Maltose Uptake (cpm/min mg cells)
% Control Uptake
2,190 710 400 325 300 200
33 18 18 17 9
Maltose Uptake (cpm/min/ mg cells)
% Control Uptake
2,190 2,295 2,210 2,125 1,950 1,880 1,350 790 285
105 101 97 88 85 62 36 13
A) Cells of the strain were preincubated for 10-20 min at 20 °C with the concentration of inhibitors indicated in the table. At the end of the preincubation maltose transport was measured by the uptake of [14Clmaltose (2 raM). The specific activity of [14C]maltose was 200 cpm/nmol: B) The uptake of [14C]maltose (2 mM) by strain JM 1820R was measured in the presence of other carbohydrates added at a concentration of 20 mM
low concentrations of maltose therefore provides an initial diagnostic test for putative maltose permease mutants. The growth in media with high concentrations o f maltose might occur b y passive diffusion o f the sugar into the cells, thus bypassing the carrier-mediated mode o f transport. A similar observation was made in the case o f galactose permease mutants (Douglas and Condie 1954). Direct evidence that MGT1 was affected in maltose uptake was obtained as follows. Maltose uptake was assayed directly by measuring the initial velocity o f [14C]maltose uptake according to the m e t h o d o f Serrano (1977). Preliminary experiments, described in Table 2, demonstrate the validity, of the assay procedure and describe some of the characteristics displayed b y the maltose uptake system in wild-type yeast. Maltose uptake is essentially linear for 2 - 3 min. To reduce the level o f maltose hydrolysis during the course of the assay, short
198
M.J. Goldenthal et al.: Yeast Maltose Transport Mutant
E,-,3 E O. o
< iJJ (1)
