Current Genetics
Curr Genet (1992)22:273-275
9 Springer-Verlag 1992
Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed Andrzej Paszewski * and Bun-Ichiro Ono 2
1 Institute of Biochemistryand Biophysics, Polish Academy of Sciences, 36. Rakowiecka str., PL-02-532 Warszawa, Poland 2 Laboratory of Environmental Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 700 Okayama, Japan Received November 5, 199i/April 12, 1992 Summary. cys4-1, a mutation in the reverse trans-sulphu-
ration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression. Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses. Key words: S. cerevisiae - Sulphur amino acids - Regula-
tory effectors
cysteine through the reverse trans-sulphuration pathway (Fig. 1). Therefore, it is far from certain that the repression (observed when methionine is present in the growth medium) is mediated by S-adenosylmethionine rather than by other metabolites derived from it. To resolve this question we have used a strain unable to synthesise cysteine from methionine due to the cys4-1 mutation resulting in cystathionine 13-synthase deficiency (Ono et al. 1988). This mutation renders the strain insensitive to sulphur metabolite repression. Therefore methionine repression in wild-type cells must be exerted by a metabolite derived from methionine through the reverse trans-sulphuration pathway, downstream from homocysteine (Fig. 1).
Introduction
Saccharomyces cerevisae, like other fungi, is able to utilize sulphate as a sole sulphur source; via the sulphate assimilation pathway sulphate is converted to sulphide which is a substrate for cysteine and homocysteine synthesis (Cherest et al. 1969; Yamagata 1971; see Fig. 1). The addition of methionine and/or S-adenosylmethionine to the growth medium results in almost complete repression of the sulphate assimilation pathway activities and, to a lesser extent, of some other enzymes involved in sulphur-containing amino-acid synthesis, such as homoserine O-transacetylase [EC 2.3.1.3I] and homocysteine synthase [EC 4.1.99.10] (Cherest etal. 1971, 1973; Piotrowska and Paszewski 1990). Both methionine and S-adenosylmethionine appear to be better sulphur sources than sulphate; therefore, their repressive effect on the energy-consuming sulphate assimilation system is a manifestation of sulphur metabolite repression. It has been postulated that S-adenosylmethionine, which is synthesized rapidly from methionine, acts as a low-molecular-mass effector in this regulatory system (Cherest et al. 1973; Thomas et al. 1989). This hypothesis however, has, a weak point as both methionine and S-adenosylmethionine are readily metabolized to homocysteine and
Correspondenceto: A. Paszewski
Results and discussion
Data in Table 1 show that exogenous methionine has no effect on sulphate assimilation in the cys4-1 mutant in stark contrast to the wild-type strain, where sulphate incorporation is almost eliminated, from which it is inferred that sulphate assimilation activities are repressed. It is worth noting that cys4-1 cells grown with and without methionine exhibit reciprocal levels of glutathione and homocysteine with the former compound predominating in minimal-medium-grown cells and the latter in methionine-supplemented cells. This suggests that normally in methionine-grown cells a high proportion of cysteine which is used for glutathione synthesis is made from homocysteine via the reverse trans-sulphuration pathway which is blocked in the mutant. In addition, methionine-grown cys4-1 cells contain more labelled methionine probably as a result of a high homocysteine pool. A similar mode of regulation to that observed for sulphate assimilation was found for homocysteine synthase: methionine in the growth medium led to strong repression of the enzyme in the wild-type strain while causing only slight repression in the mutant (Table 2). These resuits, together with the sulphate assimilation data, indi-
274 SO4
,,~""~""* SO4
) APS
AC-SERINE
> PAPS
.pS 2-
) S:zO~-
SERINE
~- METHIONINE eth2, eth3
Fig. 1. An outline of the sulphate assimilation pathway and sulphur amino-acid metabolism in fungi. The reverse transsulphuration pathway is indicated by heavy lines, cys4-1 and eth denote mutations blocking cystathionine fl-synthase and S-adenosylmethionine synthetase, respectively. HS, homocysteine synthase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; APS, adenosine 5-phosphosulphate; PAPS, adenosine 3-phospho 5-phosphosulfate
ISAM
SAH G
Table 1. Accumulation of a~S-labelled compounds in cells of cyst4-1 and wild-type strains grown in the presence of Strain and genotype a
35SO4
Amount of aSS-labelled compound [llmol (g dry wt)-1]
Medium
Total
Cysteine
Glutathione
Homocysteine
Cystathionine
Methionine
35sb
5-4SA (cys4-1)
GO GO + L-Met (3 mM)
60.0 65.4
0.56 0.88
26.4 7.6
3.40 21.50
4.2 7.1
0.41 4.20
AS4-5 (wild-type)
GO GO + L-Met (3 mM)
26.6 0.2
1.00 Trace
14.2 0.10
0.64 Trace
0.53 Tace
0.42 ND
AS4-5 is a haploid, prototrophic strain. The genotype of the 5-4SA strain is eys4-1, his3, leu2, trpl b retained on Dowex 50 (H +) ND, not detectable A modified GO medium (Swietlinska et al. 1978) in which the sulphate concentration was lowered to 2 mM (substituting the corresponding chlorides for the sulphates omitted) was used. Methionine was supplemented as indicated at the start of culture. The cells were grown on a rotary shaker (150 rpm) at 28 ~ for 6 h, then Na 235SO4 (5 x 108 cpm mmole- 1) was added and growth was continued. The
cells were harvested at early stationary phase and 35-labelled compounds isolated by water extraction (Paszewski and Grabski 1974). The extracts (10ml) were applied on Dowex 50(H +) columns and amino acids eluted with 2N NH 4 OH. The eluates were evaporated to dryness and the residues disolved in water. Separation and determination of labelled metabolites was achieved by thin-layer chromatography and high-voltage paper electrophoresis as described previously (Paszewski and Grabski 1974; Paszewski et al. 1984). The amounts of labelled metabolites were calculated from the specific activity of 35SO4
Table 2. The effect of exogenous methionine on the level of homo-
b o t h the m u t a n t and wild-type strains accumulate large a m o u n t s o f methionine and S-adenosylmethionine. However, only the wild-type strain efficiently m e t a b o lizes these c o m p o u n d s to cysteine. Therefore, the lack o f repression o f the sulphate assimilation p a t h w a y activities and o f homocysteine synthase in m e t h i o n i n e - g r o w n cys4I cells m u s t be attributed to the m u t a t i o n a l defect in the reverse trans-sulphuration p a t h w a y . This interpretation is also consistent with reduced repressibility o f m e t h i o n ine biosynthetic enzymes in eth m u t a n t s defective in Sadenosylmethionine synthetases [EC 2.5.1.6] (Cherest et al. 1973a and see Fig. 1). I f neither methionine n o r S-adenosylmethionine is the low-molecular-mass effector in the sulphur regulatory system, w h a t metabolite is? The position o f the metabolic block in cys4-1 (Fig. 1) also excludes h o m o c y s t e i n e (which is, in fact, greatly elevated in the m u t a n t g r o w n on methionine) and points to cysteine as a candidate for this role. Cysteine was s h o w n to exert repression o f h o m o c y s teine synthase in Aspergillus nidulans (Paszewski and G r a b s k i 1974), Yarrowia lipolytica ( M o r z y c k a and Paszewski 1979) and a newly isolated yeast designated P R G - 1 3 (Piotrowska and Paszewski 1990), which is naturally devoid o f cystathionine ~-synthase and cystathionine y-lyase. In the cys4-I m u t a n t we observed some repression o f h o m o c y s t e i n e synthase, but n o t o f sulphate
cysteine synthase in cys4-1 and wild-type strains. Growth conditions were as described in Table 1. Preparation of cell-free extracts and enzyme assays were as described by Cherest et al. (1969) Strain and relevent genotype
Medium
5-4SA
GO GO + L-Met (3 raM)
667 592
11
GO GO + L-Met (3 mM)
341 37
89
(cys4-1) AS4-5 (wild-type)
Activity [nmoles (rain mg prot)- 1]
Repression (%)
cate that S-adenosylmethionine-mediated repressibility, reported for the whole g r o u p o f coordinately regulated enzymes involved in sulphur amino-acid synthesis, is impaired in the cys4-1 m u t a n t . The absence o f methionine repression in the eys4-1 m u t a n t might be a consequence o f a reduction in methionine t r a n s p o r t a n d / o r its conversion to S-adenosylmethionine. T h e results in Table 3 rule o u t b o t h possibilities. W h e n g r o w n in a methionine-containing medium,
275 Table 3. Accumulation of 3SS-labelled compounds in cells of cys4-1
and wild-type strains grown in the presence of L-[35S]methionine Strain and genotype
Amount of 35S_labelled compound [Ixmol (g dry wt)- 1] Total 35Sb
5-4SA
277
Cysteine + glutathione 3.0
Homo- Methicysteine onine
S-adenosylmethionine
5.0
107
100
Acknowledgements. We wish to thank Dr. H.N. Arst for critical reading of the manuscript.
References
(cys4-1) AS4-5 a 181 (wild-type)
ine a n d S - a d e n o s y l m e t h i o n i n e are i n v o l v e d in this regulation, their role is likely to be as p r e c u r s o r s o f the effector(s) r a t h e r t h a n as effector(s) themselves.
22.0
4.7
66
43
a The data for AS4-5 strain are from Piotrowska and Paszewski (1990) b Retained on Dowex 50 (H +) The cells were grown in GO medium supplemented with 3 mM L-[35S]-methionine (3.6 x 10 s cpm nmole-1). Growth conditions and radioactive substance determination were as described in Table I. Because S-adenosylmethionine decomposes during water extraction at 100 ~ it was determined as 5-methylthioadenosine by thin-layer chromatography along with methionine. Since the radioactive reagent used in this experiment contained only 83 per cent of 3sS as methionine, it is possible that in the case of cys4-1 the radioactivity found in the cysteine and glutathione spots came from non-methionine sulphur present in the reagent
a s s i m i l a t i o n activities, b y cysteine. This effect was n o t f o u n d , h o w e v e r , in the w i l d - t y p e s t r a i n we used, p o s s i b l y as a result o f p o o r u p t a k e o f this a m i n o a c i d ( O n o et al. 1984, 1988; O n o a n d N a i t o 1991). Clearly, f u r t h e r w o r k is n e e d e d to i d e n t i f y the l o w - m o l e c u l a r - m a s s effector(s) in the r e g u l a t i o n o f s u l p h u r m e t a b o l i s m in S. cerevisiae. T h e results p r e s e n t e d here i n d i c a t e that, even if m e t h i o n -
Cherest H, Eihler F, de Robichon-Szulmajster H (1969) J Bacteriol 97:328-336 Cherest H, Surdin-Kerjan Y, de Robichon-Szulmajster H (1971) J Bacteriol 106:758-772 Cherest H, Surdin-Kerjan Y, Antoniewski J, de Robichon-Szulmajster H (1973) J Bacteriol 114:928-933 Cherest H, Surdin-Kerjan Y, Antoniewski J, de Robichon-Szulmajster H (1973 a) J Bacteriol 115:1084-1093 Morzycka E, Paszewski A (1979) Mol Gen Genet 174:33-38 Ono B, Naito K (1991) Yeast 7:849-855 Ono B-I, Suruga T, Yamamoto M, Yamamoto S, Murata K, Kimura A, Shinoda S, Ohmori S (1984) J Bacteriol 158:860-865 Ono B-I, Shirahige Y-I, Nanjoh A, Anodu N, Ohue H, Ishino-Arao Y (1988) J Bacteriol 170:5883-5889 Paszewski A, Grabski J (1974) Mol Gen Genet 132:307-320 Paszewski A, Prazmo W, Nadolska J, Regulski M (1984) J Gen Microbiol 130:1113-1121 Piotrowska M, Paszewski A (1990) J Gen Mierobiol 136:22832286 Swietlinska Z, Zaborowska D, Haladus E, Zuk J (1978) Mol Gen Genet 166:97-107 Thomas D, Cherest H, Surdin-Kerjan Y (1989) Mol Cell Biol 9: 3292- 3298 Yamagata S (1971) J Biochem 70:1035-1045 Communicated by C.P. Hollenberg
Curr Genet (1992)22:273-275
9 Springer-Verlag 1992
Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed Andrzej Paszewski * and Bun-Ichiro Ono 2
1 Institute of Biochemistryand Biophysics, Polish Academy of Sciences, 36. Rakowiecka str., PL-02-532 Warszawa, Poland 2 Laboratory of Environmental Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 700 Okayama, Japan Received November 5, 199i/April 12, 1992 Summary. cys4-1, a mutation in the reverse trans-sulphu-
ration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression. Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses. Key words: S. cerevisiae - Sulphur amino acids - Regula-
tory effectors
cysteine through the reverse trans-sulphuration pathway (Fig. 1). Therefore, it is far from certain that the repression (observed when methionine is present in the growth medium) is mediated by S-adenosylmethionine rather than by other metabolites derived from it. To resolve this question we have used a strain unable to synthesise cysteine from methionine due to the cys4-1 mutation resulting in cystathionine 13-synthase deficiency (Ono et al. 1988). This mutation renders the strain insensitive to sulphur metabolite repression. Therefore methionine repression in wild-type cells must be exerted by a metabolite derived from methionine through the reverse trans-sulphuration pathway, downstream from homocysteine (Fig. 1).
Introduction
Saccharomyces cerevisae, like other fungi, is able to utilize sulphate as a sole sulphur source; via the sulphate assimilation pathway sulphate is converted to sulphide which is a substrate for cysteine and homocysteine synthesis (Cherest et al. 1969; Yamagata 1971; see Fig. 1). The addition of methionine and/or S-adenosylmethionine to the growth medium results in almost complete repression of the sulphate assimilation pathway activities and, to a lesser extent, of some other enzymes involved in sulphur-containing amino-acid synthesis, such as homoserine O-transacetylase [EC 2.3.1.3I] and homocysteine synthase [EC 4.1.99.10] (Cherest etal. 1971, 1973; Piotrowska and Paszewski 1990). Both methionine and S-adenosylmethionine appear to be better sulphur sources than sulphate; therefore, their repressive effect on the energy-consuming sulphate assimilation system is a manifestation of sulphur metabolite repression. It has been postulated that S-adenosylmethionine, which is synthesized rapidly from methionine, acts as a low-molecular-mass effector in this regulatory system (Cherest et al. 1973; Thomas et al. 1989). This hypothesis however, has, a weak point as both methionine and S-adenosylmethionine are readily metabolized to homocysteine and
Correspondenceto: A. Paszewski
Results and discussion
Data in Table 1 show that exogenous methionine has no effect on sulphate assimilation in the cys4-1 mutant in stark contrast to the wild-type strain, where sulphate incorporation is almost eliminated, from which it is inferred that sulphate assimilation activities are repressed. It is worth noting that cys4-1 cells grown with and without methionine exhibit reciprocal levels of glutathione and homocysteine with the former compound predominating in minimal-medium-grown cells and the latter in methionine-supplemented cells. This suggests that normally in methionine-grown cells a high proportion of cysteine which is used for glutathione synthesis is made from homocysteine via the reverse trans-sulphuration pathway which is blocked in the mutant. In addition, methionine-grown cys4-1 cells contain more labelled methionine probably as a result of a high homocysteine pool. A similar mode of regulation to that observed for sulphate assimilation was found for homocysteine synthase: methionine in the growth medium led to strong repression of the enzyme in the wild-type strain while causing only slight repression in the mutant (Table 2). These resuits, together with the sulphate assimilation data, indi-
274 SO4
,,~""~""* SO4
) APS
AC-SERINE
> PAPS
.pS 2-
) S:zO~-
SERINE
~- METHIONINE eth2, eth3
Fig. 1. An outline of the sulphate assimilation pathway and sulphur amino-acid metabolism in fungi. The reverse transsulphuration pathway is indicated by heavy lines, cys4-1 and eth denote mutations blocking cystathionine fl-synthase and S-adenosylmethionine synthetase, respectively. HS, homocysteine synthase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; APS, adenosine 5-phosphosulphate; PAPS, adenosine 3-phospho 5-phosphosulfate
ISAM
SAH G
Table 1. Accumulation of a~S-labelled compounds in cells of cyst4-1 and wild-type strains grown in the presence of Strain and genotype a
35SO4
Amount of aSS-labelled compound [llmol (g dry wt)-1]
Medium
Total
Cysteine
Glutathione
Homocysteine
Cystathionine
Methionine
35sb
5-4SA (cys4-1)
GO GO + L-Met (3 mM)
60.0 65.4
0.56 0.88
26.4 7.6
3.40 21.50
4.2 7.1
0.41 4.20
AS4-5 (wild-type)
GO GO + L-Met (3 mM)
26.6 0.2
1.00 Trace
14.2 0.10
0.64 Trace
0.53 Tace
0.42 ND
AS4-5 is a haploid, prototrophic strain. The genotype of the 5-4SA strain is eys4-1, his3, leu2, trpl b retained on Dowex 50 (H +) ND, not detectable A modified GO medium (Swietlinska et al. 1978) in which the sulphate concentration was lowered to 2 mM (substituting the corresponding chlorides for the sulphates omitted) was used. Methionine was supplemented as indicated at the start of culture. The cells were grown on a rotary shaker (150 rpm) at 28 ~ for 6 h, then Na 235SO4 (5 x 108 cpm mmole- 1) was added and growth was continued. The
cells were harvested at early stationary phase and 35-labelled compounds isolated by water extraction (Paszewski and Grabski 1974). The extracts (10ml) were applied on Dowex 50(H +) columns and amino acids eluted with 2N NH 4 OH. The eluates were evaporated to dryness and the residues disolved in water. Separation and determination of labelled metabolites was achieved by thin-layer chromatography and high-voltage paper electrophoresis as described previously (Paszewski and Grabski 1974; Paszewski et al. 1984). The amounts of labelled metabolites were calculated from the specific activity of 35SO4
Table 2. The effect of exogenous methionine on the level of homo-
b o t h the m u t a n t and wild-type strains accumulate large a m o u n t s o f methionine and S-adenosylmethionine. However, only the wild-type strain efficiently m e t a b o lizes these c o m p o u n d s to cysteine. Therefore, the lack o f repression o f the sulphate assimilation p a t h w a y activities and o f homocysteine synthase in m e t h i o n i n e - g r o w n cys4I cells m u s t be attributed to the m u t a t i o n a l defect in the reverse trans-sulphuration p a t h w a y . This interpretation is also consistent with reduced repressibility o f m e t h i o n ine biosynthetic enzymes in eth m u t a n t s defective in Sadenosylmethionine synthetases [EC 2.5.1.6] (Cherest et al. 1973a and see Fig. 1). I f neither methionine n o r S-adenosylmethionine is the low-molecular-mass effector in the sulphur regulatory system, w h a t metabolite is? The position o f the metabolic block in cys4-1 (Fig. 1) also excludes h o m o c y s t e i n e (which is, in fact, greatly elevated in the m u t a n t g r o w n on methionine) and points to cysteine as a candidate for this role. Cysteine was s h o w n to exert repression o f h o m o c y s teine synthase in Aspergillus nidulans (Paszewski and G r a b s k i 1974), Yarrowia lipolytica ( M o r z y c k a and Paszewski 1979) and a newly isolated yeast designated P R G - 1 3 (Piotrowska and Paszewski 1990), which is naturally devoid o f cystathionine ~-synthase and cystathionine y-lyase. In the cys4-I m u t a n t we observed some repression o f h o m o c y s t e i n e synthase, but n o t o f sulphate
cysteine synthase in cys4-1 and wild-type strains. Growth conditions were as described in Table 1. Preparation of cell-free extracts and enzyme assays were as described by Cherest et al. (1969) Strain and relevent genotype
Medium
5-4SA
GO GO + L-Met (3 raM)
667 592
11
GO GO + L-Met (3 mM)
341 37
89
(cys4-1) AS4-5 (wild-type)
Activity [nmoles (rain mg prot)- 1]
Repression (%)
cate that S-adenosylmethionine-mediated repressibility, reported for the whole g r o u p o f coordinately regulated enzymes involved in sulphur amino-acid synthesis, is impaired in the cys4-1 m u t a n t . The absence o f methionine repression in the eys4-1 m u t a n t might be a consequence o f a reduction in methionine t r a n s p o r t a n d / o r its conversion to S-adenosylmethionine. T h e results in Table 3 rule o u t b o t h possibilities. W h e n g r o w n in a methionine-containing medium,
275 Table 3. Accumulation of 3SS-labelled compounds in cells of cys4-1
and wild-type strains grown in the presence of L-[35S]methionine Strain and genotype
Amount of 35S_labelled compound [Ixmol (g dry wt)- 1] Total 35Sb
5-4SA
277
Cysteine + glutathione 3.0
Homo- Methicysteine onine
S-adenosylmethionine
5.0
107
100
Acknowledgements. We wish to thank Dr. H.N. Arst for critical reading of the manuscript.
References
(cys4-1) AS4-5 a 181 (wild-type)
ine a n d S - a d e n o s y l m e t h i o n i n e are i n v o l v e d in this regulation, their role is likely to be as p r e c u r s o r s o f the effector(s) r a t h e r t h a n as effector(s) themselves.
22.0
4.7
66
43
a The data for AS4-5 strain are from Piotrowska and Paszewski (1990) b Retained on Dowex 50 (H +) The cells were grown in GO medium supplemented with 3 mM L-[35S]-methionine (3.6 x 10 s cpm nmole-1). Growth conditions and radioactive substance determination were as described in Table I. Because S-adenosylmethionine decomposes during water extraction at 100 ~ it was determined as 5-methylthioadenosine by thin-layer chromatography along with methionine. Since the radioactive reagent used in this experiment contained only 83 per cent of 3sS as methionine, it is possible that in the case of cys4-1 the radioactivity found in the cysteine and glutathione spots came from non-methionine sulphur present in the reagent
a s s i m i l a t i o n activities, b y cysteine. This effect was n o t f o u n d , h o w e v e r , in the w i l d - t y p e s t r a i n we used, p o s s i b l y as a result o f p o o r u p t a k e o f this a m i n o a c i d ( O n o et al. 1984, 1988; O n o a n d N a i t o 1991). Clearly, f u r t h e r w o r k is n e e d e d to i d e n t i f y the l o w - m o l e c u l a r - m a s s effector(s) in the r e g u l a t i o n o f s u l p h u r m e t a b o l i s m in S. cerevisiae. T h e results p r e s e n t e d here i n d i c a t e that, even if m e t h i o n -
Cherest H, Eihler F, de Robichon-Szulmajster H (1969) J Bacteriol 97:328-336 Cherest H, Surdin-Kerjan Y, de Robichon-Szulmajster H (1971) J Bacteriol 106:758-772 Cherest H, Surdin-Kerjan Y, Antoniewski J, de Robichon-Szulmajster H (1973) J Bacteriol 114:928-933 Cherest H, Surdin-Kerjan Y, Antoniewski J, de Robichon-Szulmajster H (1973 a) J Bacteriol 115:1084-1093 Morzycka E, Paszewski A (1979) Mol Gen Genet 174:33-38 Ono B, Naito K (1991) Yeast 7:849-855 Ono B-I, Suruga T, Yamamoto M, Yamamoto S, Murata K, Kimura A, Shinoda S, Ohmori S (1984) J Bacteriol 158:860-865 Ono B-I, Shirahige Y-I, Nanjoh A, Anodu N, Ohue H, Ishino-Arao Y (1988) J Bacteriol 170:5883-5889 Paszewski A, Grabski J (1974) Mol Gen Genet 132:307-320 Paszewski A, Prazmo W, Nadolska J, Regulski M (1984) J Gen Microbiol 130:1113-1121 Piotrowska M, Paszewski A (1990) J Gen Mierobiol 136:22832286 Swietlinska Z, Zaborowska D, Haladus E, Zuk J (1978) Mol Gen Genet 166:97-107 Thomas D, Cherest H, Surdin-Kerjan Y (1989) Mol Cell Biol 9: 3292- 3298 Yamagata S (1971) J Biochem 70:1035-1045 Communicated by C.P. Hollenberg
