Molecular Microbiology (1990) 4(4). 585-596
The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product E. Weber,^ C. Rodriguez, M. R. Chevallier^ and R.Jund I.B.M.C. du C.N.R.S., 15 rue R. Descartes, 67084 Strasbourg Cedex, Erance. Summary A 2.1kb DNA segment carrying the purine-cytosine permease gene {FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HISI gene.
Introduction In the yeast Saccharomyces cerevisiae, membrane transport is well documented by both physiological and genetic studies. Many phenomenological descriptions of specific transport systems are available {reviewed by Cooper, 1982) showing that most of the transport proteins in yeast (permeases) ensure active transport and function as profon symporters using the proton gradient created by the proton-translocating ATPase of the plasma membrane (for reviews on the plasma membrane ATPase of lower eukaryofes, see Goffeau and Slayman, 1981; Serrano, 1988). However, little is known at the molecular level about the recognition of substrate and the transport process itself. This requires structural information on the proteins which mediate the transport, elusive at the level of molecular analysis until recently. Indeed, several yeast permeases have been cloned and their sequences reported: the arginine pernnease (Hoffmann, 1986; Ahmad and Bussey, 1987), the histidine permease (Tanaka and Fink, 1987), the allantoate permease (Rai ef ai., '1988). the glucose carrier (Celenza et al., 1988), the uracil permease (Jund etal., 1988).
Three distinct permeases involved in pyrimidine uptake by the yeast S. cerevisiae have been described: (i) :he uracil permease, a very specific transport system which does not recognize other pyrimidines such as cytosine or thymine {Lacroute and Slonimski, 1964; Grenson, 1969; Jund and Lacroute, 1970; Jund efa/., 1977); {ii) fhe uridine permease, a permease with low affinity for uridine {Grenson, 1969; Losson et ai., 1977); and {iii) the purine-cytosine permease, a system with broad specifity towards purines and which also transports cytosine and 5-methylcytosine but neither uracil nor thymine {Grenson, 1969; Jund and Lacroute, 1970; Grenson and Polak, 1973; Reichert and Winter, 1974; Chevallier ef al., 1975). The broad specificity of this latter permease allowed the selection of mutants altered in affinity for one or several of its substrates as reported by Ghevallier et al. {1975). The uracil permease gene was cloned and sequenced (Chevallier, 1982; Jund ef al., 1988). The purine-cytosine permease gene (FCY2) was cloned and characterized, Northern blot analysis showed an mRNA band of 1.9kb which could encode a protein of up to 630 amino acids. By photoaffinity labelling of the permease with 8-azidoadenine in cells carrying a multicopy plasmid of FCY2, it was shown that this protein is extensively glyoosylated with an apparent molecular weight of about 120000 (Schmidt efa/.. 1984). Here we report the sequence of a DNA segment which encodes the FCY2 protein and the deduced primary structure of the protein (533 amino acids) whose calculated molecular weight is 58000 and which contains only two potential A/-glycosylation sites. Programs for secondary structure prediction designed for membrane proteins were applied to this sequence and lead us to propose a folding pattern in the membrane. Using the thermosensitive mutant seel8-1 blocked in the ER-Golgi transit (Schekman, 1985) we show that the expression of this protein requires a functional secretory pathway. Moreover, permease activity is significantly reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The ECY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene. Results
Received 20 September, 1989; revised 2 January, 1990, ''Present address: E.M.B.L., Meyerhofstrasse 1, 6900 Heidelberg 1, FRG. 'For correspondence. Tel. (6221) 88417039; Fax (6221) 88610680.
Nucleotide sequence analysis of the FCY2 region We have sequenced a 2.1 kb S. cerevisiae genomic DNA
586
E. Weber, C. Rodriguez, M. R. Chevaiiier and R. Jund
segment between the Bci\- and Sph\ sites (Fig. 1A) which was known from previous results to include the entire FCY2 coding region (Schmidt et ai., 1984). When a DNA segment in the 3'-region of the fragment carrying the FCY2 gene {see Fig. lA) was sequenced, another open reading frame was found. By comparison of the deduced protein sequence with the NBRF protein data bank, this fragment was identified as the coding sequence of the
HiS1 gene encoding ATP phosphoribosyltransferase (Hinnebusch and Fink, 1983). The DNA region sequenced by these authors extended up to a reading frame which turned out to be the 3'-end of the FCY2 gene (up to Acc\ site in Fig. IA). Our sequence data agree with their determination except for one base which changes tyrosine at position 459 into aspartic acid. These data indicated that the FCY2 gene is closely linked to the HiS1
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Fig. 1. A. Restriction map and sequencing strategy of the FCV^ gene. Start and terminating codonsare indicated. The DNA fragments which were sequenced after cloning in phages M13mp10or M13mp11 are indicated by arrows reading 5' to 3'. o indicates that a synthetic oligonucleotide other than universal primer was used. Restriction sites: H, HindWV. K, Kpni. The second Kpn\ site was used for the construction otthe Bgl\\'Kpn\ deletion. The region already sequenced by Hinnebusch and Fmk (1983) ts indicated by 1he dotted line. B. Nucleotide sequence of the Bcl\-Sph\ DNA fragment containing the FCY2 gene and the deduced amino acid sequence of the purine-cytosine permease protein. The two glycosylation sites are indicated by the B O O symbols. The putative TATA boxes are underlined with a continuous line, for the most likely one, and dotted lines. ' = nucleotide favourable for the initiation of translation (Hamilton et al.. 1987). These sequence data will appear in the EMBLyGenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number X51751.
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gene. This finding was confirmed by genetic analysis (see below). The largest open reading frame identified in the 2.1 kb DNA segment (see Fig. IB) is 1599bp long and is borne by the coding strand defined by mRNA hybridization (see below). The first ATG codon of this reading frame is included in a sequence context similar to that found in most S. cerev/s/aemRNAs (Hamilton efa/., 1987); in particular, an adenine residue is present at positions - 1 and - 3 and G residues are underrepresented in the region preceding the predicted start codon (see Fig. IB). These features suggest the methionine triplet marked as position 1 in Fig. IB as the initiating codon, as predicted by previous site-directed mutagenesis experiments (Schmidt efa/., 1984).
(ii) A large central region is made up of an alternation of hydrophobic and short hydrophilic segments. (iii) The C-terminus (about 50 residues) is less hydrophobic than the bulk of the sequence. Apart from this overall similarity no homology was found, either with other sequenced transporters or with any of the sequences of the EMBLVGenBank Data Libraries (using program TFASTA).
Primary structure of the predicted purine-cytosine permease
Foiding pattern of the purine-cytosine permease deduced from predictive data
The FCY2 coding sequence does not contain the TACTAAC heptanucleotide shown to be necessary for splicing in S. cerevisiae (Langford and Gallwitz, 1983; Pikielny et al.. 1983), there is therefore probably no intron in the FCY2 gene. Consequently, the amino acid sequence of the purine-cytosine permease was directly deduced from the major open reading frame of the FCY2 gene (Fig. IB). It consists of 533 residues with a molecular weight of 58 200. This value is in agreement with experimental results: the determination of molecular weight of the photoaffinitylabelled permease treated with glycosidases gave two peaks of 60000 and 73000 (Schmidt et ai., 1984). The latter value probably corresponds to partially glycosylated forms of the protein.
In a previous paper (Weber et ai, 1988), we described secondary structure predictions of the purine-cytosine permease obtained with two methods designed for membrane-associated protein sequences (Engelman ef ai, 1986; Eisenberg ef ai., 1984). The primary assumption underlying all the prediction methods for membrane proteins is that the transmembrane segments span the lipid bilayer as u-helices of about 20 amino acids. The method of Eisenberg etai. relies on the hydropathy plotted against the hydrophobic moment of selected transmembrane helices, using a slightly different hydrophobicity scale than that proposed by Kyte and Doolittie (1982), namely a normalized 'consensus scale' derived from several different proposed scales. The plot of hydropathy versus hydrophobic moment allows the classification of the putative a-helices into different groups: TM (Transmembrane Muitimeric a-helices), TM° (Transmembrane Monomeric), S (Surface Seeking) and G (Globular) i.e. the u-helices which fall in the part of the diagram corresponding to a-helices found in globular proteins. On the other hand, the method of Engelman etai uses a polarity scale which these authors developed, combining separate experimental values for the polar and non-polar groups in the amino acid side-chains as they occur in an a-helix (Engelman efa/., 1986). From this scale (GES scale), H,the free energy of transfer from lipid phase into aqueous phase of stretches of amino acids in a-helical conformation can be calculated. According to the authors, a free energy of 20 kcal mol" ^ for a 20-amino-acid helix is a feature of membrane-spanning helices. The authors tested the predictive value of the method on proteins of the photosynthetic centre of Rhodopseudomonas, the partial structure of which has been elucidated by X-ray crystallographic analysis (Deisenhofer ef ai, 1986). Good agreement was found between the predicted membranespanning helices and the membrane-spanning segments
The predicted purine-cytosine permease resembles other yeast transport proteins whose primary structure has been determined (Hoffmann, 1985; Ahmad and Bussey, 1986; Tanaka and Fink, 1985; Celenza et ai., 1988; Jund etai., 1988; Rai etai. 1988): it is a protein of low polarity (40% polar residues, 60% non-polar). Alanine and glycine are the most abundant residues (20% of the total amino acids of the protein). Aromatic amino acids are also abundant in the sequence of the protein, particularly phenylalanine (7.3%) and tryptophan (2.8%) when compared to the respective average values of 3.6 and 1.3% calculated by Dayhoff etai. (1978) in a pool of 314 proteins. An abundance of aromatic amino acids is found in most transport proteins whose primary structures are available. A special feature of the predicted purine-cytosine permease is the excess of 10 negative charges at neutral pH. Of 533 residues, 39 are acidic and 29 are basic. This contrasts with the lactose (Kaback, 1986) and the melibiose (Yazu et ai., 1984) carriers of Escherichia coii and other yeast permeases which are predicted to be basic proteins. Like other yeast permeases, the deduced amino acid
sequence of the purine-cytosine permease can be divided into three major regions, as follows. (i) The hydrophilic A/-terminus consists of about 100 amino acids without a signal peptide and with a high content of acidic amino acids. The net charge of this protein segment
is-12,
Primary structure of purine-cytosine permease of yeast as deduced from the crystal structure. This more refined procedure is especially valuable for revealing possible transmembrane elements with polar groups in the helices. Therefore, it was particularly useful for the purine-cytosine permease, whose hydropathy profile (according to Kyte and Doolittle), although globally hydrophobic, contains several peaks of intermediate hydrophobicity; seven or ten peaks can be considered as putative membrane-spanning segments depending on the adopted threshold value of hydrophobicity for membrane-spanning segments. With the GES scale, nine peaks are retained above the threshold value. These same nine peaks and three additional ones (corresponding to helices 5, 10 and 12 in Table 1) are also retained by the first screening of the Eisenberg procedure with the additional information that helix 3 is strongly amphipathic and that helices 8 and 9, like helices 5, 10 and 12 not selected by the prediction method of Engelman et ai, fall into the globular area of the plot. This is another way of highlighting their less-hydrophobic character (see Table 1). Glycoproteins belonging to three major categories have been analysed in yeast: cell vi/all proteins, secreted proteins and vacuolar proteins. The case of the plasma membrane intrinsic proteins to which the purine-cytosine permease belongs is not documented. For cell wall proteins, long carbohydrate chains of up to 100-200 mannan residues have been reported, vi/hereas for secretory proteins the mannan extensions are shorter (about 50 residues) and for the vacuolar proteins even shorter (Kukuruzinska ef ai, 1987). In the case of the purine-cytosine permease, we find an apparent contribution of about 60000 Daltons for the mannan chains and only two /V-linked glycosylation sites. One must assume
Table 1. Prediction of transmembrane a-helices in the deduced purine-cytosine permease protein.
that the two sites are glycosylated with roughly 150 mannan residues per extension, if one admits that the 0-glycosylation does not contribute to long carbohydrate chains. Consequently, these two sites are expected to lie at the cell surface. The first of these two sites is at position 160, close to the start of the predicted helix 3, whereas the second one at position 180 in the amino acid sequence is located at the end of the proposed membrane-spanning helix 3. Since helix 3 is classified as 'surface seeking' in the diagram of Eisenberg et at., this led us to propose that instead of spanning the membrane, this helix, lies at the cell surface and defines the outer side of the membrane in the folding pattern. Since the first two predicted helices are retained by the three prediction methods, we think that these two helices actually span the membrane. It follows that the W-terminal end of the protein is at the cell surface, despite the fact that this /V-terminal end has no typical leader peptide. We propose the model shown in Fig. 4 as the folding pattern of the protein; all the segments of 20 amino acids selected by the prediction method of Engelman et at. are inserted into the bilayer, except segment 165-185, which corresponds to helix 3. According to the method of Engelman ef ai, six helices are predicted thereafter and, provisionally, the C-terminal end also appears at the cell surface.
The biosynthesis of the purine-cytosine permease enters the secretory pathway Core glycosylation in yeast has been shown to occur in the endoplasmic reticulum, and outer chain extension in a Golgi-like organelle (Novick et ai, 1981; Schekman, 1985).
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OUT
Fig, 2. Proposed folding pattern o( the purinecytosine permease according to the predicted transmembrane u-helices. All the helices retained by the GES scale are drawn. Helix 3 corresponds to a surface-seeking helix according to Eisenberg ef al. (1984). The thick arrows indicate the positions of the two potential glycosylation sites proposed to be at the ceil surface (see text).
IN
Since it has been shown that the purine-cytosine permease is a glycoprotein (Schmidt ef ai., 1984) it is expected that its biosynthesis goes through the yeast secretory pathway. To determine whether the purine cytosine permease enters the secretory pathway, we cloned the complete coding region of the gene behind the inducible GAL10 promoter on a multicopy plasmid (see the Experimental procedures). The resultant plasmid, pCRF2 (Fig. 3), was introduced into strain NC308-6B carrying a seel8 mutation, a deletion in the FCV^gene and ieu2. This strain when grown in glucose or lactate does not express purine-cytosine permease activity. When galactose is added to lactate-grown cells, induction of activity increases rapidly. If galactose is added and the cells are
I
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shifted to 37°G, no induction occurs (see Fig. 4), although the yeast galactokinase is induced at normal levels after a temperature shift of a sec18-1 mutant strain (Jund ef a/., 1988). We therefore conclude that, as anticipated, the purine-cytosine permease enters the secretory pathway.
Effect of the mnn9 mutation on the purine-cytosine permease activity The mnn9 mutation was described as a mutation which completely suppresses the addition of mannan chains to the core glycosylated proteins (Ballou ef ai., 1980). As the purine-cytosine permease is glycosylated we investigated whether the suppression of outer chains would affect the permease activity. Permease activity measurements were carried out in mnnS ieu2 mutant strains carrying a multicopy plasmid with or without the cloned FCY2 gene. The activities were compared with activities found in our wild-type strain and in strain X2180-1A (the parental strain of the mnn9 mutant). The activities were measured at several cytosine concentrations and we compared I/max relative to that of the wild-type strains. Table 2 summarizes the results: the two wild-type strains have identical Wnax values, whereas mnn9 strains have a significantly
Table 2. Uptake of cytosine in strain mnn9. Strains
YEp52PS (6,6 kb) Fig. 3. Plasmid pCRF2. Sequences from pBR322 E, coli plasmid are indicated by a bold line. All other sequences are from S, cerevisiae. The arrows indicate direction of transcription from the GAL W promoter to transcription terminator sequences. These are followed by 2 n DNA sequences conferring autonomous replication on the plasmid.
FLIOO X2180-1A mnn9 mnn9{pFCy2) fcy2- /S(pFCY2)
relative to FL100 1 1 0.25 0.58 2.9
Strains mnnGand X2180-1A were grown in minimal medium supplemented with 1 M sorbitol. Initial velocities of uptake were determined with four cytosine concentrations and the K^ and l/ma» values were extrapolated from Lineweaver-Burk plots. The Km was found to be about 2,2 (LM for all the strains tested.
Primary structure of purine-cytosine permease of yeast q b
591
Thus, even with high gene dosage, mnn9 does not express a wild-type level of cytosine permease activity. Therefore we can conclude that deficiency in outer chain glycosylation interferes with permease activity.
o
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Purine-cytosine permease mRNA ieveis in the wild-type strain and in the strain carrying a gene on multicopy plasmids
60
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Time (min) Fig. 4. Induction of galactose controlled purine-cytosine permease in thermosensitive secretory mutants {se
The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product E. Weber,^ C. Rodriguez, M. R. Chevallier^ and R.Jund I.B.M.C. du C.N.R.S., 15 rue R. Descartes, 67084 Strasbourg Cedex, Erance. Summary A 2.1kb DNA segment carrying the purine-cytosine permease gene {FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HISI gene.
Introduction In the yeast Saccharomyces cerevisiae, membrane transport is well documented by both physiological and genetic studies. Many phenomenological descriptions of specific transport systems are available {reviewed by Cooper, 1982) showing that most of the transport proteins in yeast (permeases) ensure active transport and function as profon symporters using the proton gradient created by the proton-translocating ATPase of the plasma membrane (for reviews on the plasma membrane ATPase of lower eukaryofes, see Goffeau and Slayman, 1981; Serrano, 1988). However, little is known at the molecular level about the recognition of substrate and the transport process itself. This requires structural information on the proteins which mediate the transport, elusive at the level of molecular analysis until recently. Indeed, several yeast permeases have been cloned and their sequences reported: the arginine pernnease (Hoffmann, 1986; Ahmad and Bussey, 1987), the histidine permease (Tanaka and Fink, 1987), the allantoate permease (Rai ef ai., '1988). the glucose carrier (Celenza et al., 1988), the uracil permease (Jund etal., 1988).
Three distinct permeases involved in pyrimidine uptake by the yeast S. cerevisiae have been described: (i) :he uracil permease, a very specific transport system which does not recognize other pyrimidines such as cytosine or thymine {Lacroute and Slonimski, 1964; Grenson, 1969; Jund and Lacroute, 1970; Jund efa/., 1977); {ii) fhe uridine permease, a permease with low affinity for uridine {Grenson, 1969; Losson et ai., 1977); and {iii) the purine-cytosine permease, a system with broad specifity towards purines and which also transports cytosine and 5-methylcytosine but neither uracil nor thymine {Grenson, 1969; Jund and Lacroute, 1970; Grenson and Polak, 1973; Reichert and Winter, 1974; Chevallier ef al., 1975). The broad specificity of this latter permease allowed the selection of mutants altered in affinity for one or several of its substrates as reported by Ghevallier et al. {1975). The uracil permease gene was cloned and sequenced (Chevallier, 1982; Jund ef al., 1988). The purine-cytosine permease gene (FCY2) was cloned and characterized, Northern blot analysis showed an mRNA band of 1.9kb which could encode a protein of up to 630 amino acids. By photoaffinity labelling of the permease with 8-azidoadenine in cells carrying a multicopy plasmid of FCY2, it was shown that this protein is extensively glyoosylated with an apparent molecular weight of about 120000 (Schmidt efa/.. 1984). Here we report the sequence of a DNA segment which encodes the FCY2 protein and the deduced primary structure of the protein (533 amino acids) whose calculated molecular weight is 58000 and which contains only two potential A/-glycosylation sites. Programs for secondary structure prediction designed for membrane proteins were applied to this sequence and lead us to propose a folding pattern in the membrane. Using the thermosensitive mutant seel8-1 blocked in the ER-Golgi transit (Schekman, 1985) we show that the expression of this protein requires a functional secretory pathway. Moreover, permease activity is significantly reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The ECY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene. Results
Received 20 September, 1989; revised 2 January, 1990, ''Present address: E.M.B.L., Meyerhofstrasse 1, 6900 Heidelberg 1, FRG. 'For correspondence. Tel. (6221) 88417039; Fax (6221) 88610680.
Nucleotide sequence analysis of the FCY2 region We have sequenced a 2.1 kb S. cerevisiae genomic DNA
586
E. Weber, C. Rodriguez, M. R. Chevaiiier and R. Jund
segment between the Bci\- and Sph\ sites (Fig. 1A) which was known from previous results to include the entire FCY2 coding region (Schmidt et ai., 1984). When a DNA segment in the 3'-region of the fragment carrying the FCY2 gene {see Fig. lA) was sequenced, another open reading frame was found. By comparison of the deduced protein sequence with the NBRF protein data bank, this fragment was identified as the coding sequence of the
HiS1 gene encoding ATP phosphoribosyltransferase (Hinnebusch and Fink, 1983). The DNA region sequenced by these authors extended up to a reading frame which turned out to be the 3'-end of the FCY2 gene (up to Acc\ site in Fig. IA). Our sequence data agree with their determination except for one base which changes tyrosine at position 459 into aspartic acid. These data indicated that the FCY2 gene is closely linked to the HiS1
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Fig. 1. A. Restriction map and sequencing strategy of the FCV^ gene. Start and terminating codonsare indicated. The DNA fragments which were sequenced after cloning in phages M13mp10or M13mp11 are indicated by arrows reading 5' to 3'. o indicates that a synthetic oligonucleotide other than universal primer was used. Restriction sites: H, HindWV. K, Kpni. The second Kpn\ site was used for the construction otthe Bgl\\'Kpn\ deletion. The region already sequenced by Hinnebusch and Fmk (1983) ts indicated by 1he dotted line. B. Nucleotide sequence of the Bcl\-Sph\ DNA fragment containing the FCY2 gene and the deduced amino acid sequence of the purine-cytosine permease protein. The two glycosylation sites are indicated by the B O O symbols. The putative TATA boxes are underlined with a continuous line, for the most likely one, and dotted lines. ' = nucleotide favourable for the initiation of translation (Hamilton et al.. 1987). These sequence data will appear in the EMBLyGenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number X51751.
588
£ Weber, C. Rodriguez, M. R. Chevaiiier and R. Jund
gene. This finding was confirmed by genetic analysis (see below). The largest open reading frame identified in the 2.1 kb DNA segment (see Fig. IB) is 1599bp long and is borne by the coding strand defined by mRNA hybridization (see below). The first ATG codon of this reading frame is included in a sequence context similar to that found in most S. cerev/s/aemRNAs (Hamilton efa/., 1987); in particular, an adenine residue is present at positions - 1 and - 3 and G residues are underrepresented in the region preceding the predicted start codon (see Fig. IB). These features suggest the methionine triplet marked as position 1 in Fig. IB as the initiating codon, as predicted by previous site-directed mutagenesis experiments (Schmidt efa/., 1984).
(ii) A large central region is made up of an alternation of hydrophobic and short hydrophilic segments. (iii) The C-terminus (about 50 residues) is less hydrophobic than the bulk of the sequence. Apart from this overall similarity no homology was found, either with other sequenced transporters or with any of the sequences of the EMBLVGenBank Data Libraries (using program TFASTA).
Primary structure of the predicted purine-cytosine permease
Foiding pattern of the purine-cytosine permease deduced from predictive data
The FCY2 coding sequence does not contain the TACTAAC heptanucleotide shown to be necessary for splicing in S. cerevisiae (Langford and Gallwitz, 1983; Pikielny et al.. 1983), there is therefore probably no intron in the FCY2 gene. Consequently, the amino acid sequence of the purine-cytosine permease was directly deduced from the major open reading frame of the FCY2 gene (Fig. IB). It consists of 533 residues with a molecular weight of 58 200. This value is in agreement with experimental results: the determination of molecular weight of the photoaffinitylabelled permease treated with glycosidases gave two peaks of 60000 and 73000 (Schmidt et ai., 1984). The latter value probably corresponds to partially glycosylated forms of the protein.
In a previous paper (Weber et ai, 1988), we described secondary structure predictions of the purine-cytosine permease obtained with two methods designed for membrane-associated protein sequences (Engelman ef ai, 1986; Eisenberg ef ai., 1984). The primary assumption underlying all the prediction methods for membrane proteins is that the transmembrane segments span the lipid bilayer as u-helices of about 20 amino acids. The method of Eisenberg etai. relies on the hydropathy plotted against the hydrophobic moment of selected transmembrane helices, using a slightly different hydrophobicity scale than that proposed by Kyte and Doolittie (1982), namely a normalized 'consensus scale' derived from several different proposed scales. The plot of hydropathy versus hydrophobic moment allows the classification of the putative a-helices into different groups: TM (Transmembrane Muitimeric a-helices), TM° (Transmembrane Monomeric), S (Surface Seeking) and G (Globular) i.e. the u-helices which fall in the part of the diagram corresponding to a-helices found in globular proteins. On the other hand, the method of Engelman etai uses a polarity scale which these authors developed, combining separate experimental values for the polar and non-polar groups in the amino acid side-chains as they occur in an a-helix (Engelman efa/., 1986). From this scale (GES scale), H,the free energy of transfer from lipid phase into aqueous phase of stretches of amino acids in a-helical conformation can be calculated. According to the authors, a free energy of 20 kcal mol" ^ for a 20-amino-acid helix is a feature of membrane-spanning helices. The authors tested the predictive value of the method on proteins of the photosynthetic centre of Rhodopseudomonas, the partial structure of which has been elucidated by X-ray crystallographic analysis (Deisenhofer ef ai, 1986). Good agreement was found between the predicted membranespanning helices and the membrane-spanning segments
The predicted purine-cytosine permease resembles other yeast transport proteins whose primary structure has been determined (Hoffmann, 1985; Ahmad and Bussey, 1986; Tanaka and Fink, 1985; Celenza et ai., 1988; Jund etai., 1988; Rai etai. 1988): it is a protein of low polarity (40% polar residues, 60% non-polar). Alanine and glycine are the most abundant residues (20% of the total amino acids of the protein). Aromatic amino acids are also abundant in the sequence of the protein, particularly phenylalanine (7.3%) and tryptophan (2.8%) when compared to the respective average values of 3.6 and 1.3% calculated by Dayhoff etai. (1978) in a pool of 314 proteins. An abundance of aromatic amino acids is found in most transport proteins whose primary structures are available. A special feature of the predicted purine-cytosine permease is the excess of 10 negative charges at neutral pH. Of 533 residues, 39 are acidic and 29 are basic. This contrasts with the lactose (Kaback, 1986) and the melibiose (Yazu et ai., 1984) carriers of Escherichia coii and other yeast permeases which are predicted to be basic proteins. Like other yeast permeases, the deduced amino acid
sequence of the purine-cytosine permease can be divided into three major regions, as follows. (i) The hydrophilic A/-terminus consists of about 100 amino acids without a signal peptide and with a high content of acidic amino acids. The net charge of this protein segment
is-12,
Primary structure of purine-cytosine permease of yeast as deduced from the crystal structure. This more refined procedure is especially valuable for revealing possible transmembrane elements with polar groups in the helices. Therefore, it was particularly useful for the purine-cytosine permease, whose hydropathy profile (according to Kyte and Doolittle), although globally hydrophobic, contains several peaks of intermediate hydrophobicity; seven or ten peaks can be considered as putative membrane-spanning segments depending on the adopted threshold value of hydrophobicity for membrane-spanning segments. With the GES scale, nine peaks are retained above the threshold value. These same nine peaks and three additional ones (corresponding to helices 5, 10 and 12 in Table 1) are also retained by the first screening of the Eisenberg procedure with the additional information that helix 3 is strongly amphipathic and that helices 8 and 9, like helices 5, 10 and 12 not selected by the prediction method of Engelman et ai, fall into the globular area of the plot. This is another way of highlighting their less-hydrophobic character (see Table 1). Glycoproteins belonging to three major categories have been analysed in yeast: cell vi/all proteins, secreted proteins and vacuolar proteins. The case of the plasma membrane intrinsic proteins to which the purine-cytosine permease belongs is not documented. For cell wall proteins, long carbohydrate chains of up to 100-200 mannan residues have been reported, vi/hereas for secretory proteins the mannan extensions are shorter (about 50 residues) and for the vacuolar proteins even shorter (Kukuruzinska ef ai, 1987). In the case of the purine-cytosine permease, we find an apparent contribution of about 60000 Daltons for the mannan chains and only two /V-linked glycosylation sites. One must assume
Table 1. Prediction of transmembrane a-helices in the deduced purine-cytosine permease protein.
that the two sites are glycosylated with roughly 150 mannan residues per extension, if one admits that the 0-glycosylation does not contribute to long carbohydrate chains. Consequently, these two sites are expected to lie at the cell surface. The first of these two sites is at position 160, close to the start of the predicted helix 3, whereas the second one at position 180 in the amino acid sequence is located at the end of the proposed membrane-spanning helix 3. Since helix 3 is classified as 'surface seeking' in the diagram of Eisenberg et at., this led us to propose that instead of spanning the membrane, this helix, lies at the cell surface and defines the outer side of the membrane in the folding pattern. Since the first two predicted helices are retained by the three prediction methods, we think that these two helices actually span the membrane. It follows that the W-terminal end of the protein is at the cell surface, despite the fact that this /V-terminal end has no typical leader peptide. We propose the model shown in Fig. 4 as the folding pattern of the protein; all the segments of 20 amino acids selected by the prediction method of Engelman et at. are inserted into the bilayer, except segment 165-185, which corresponds to helix 3. According to the method of Engelman ef ai, six helices are predicted thereafter and, provisionally, the C-terminal end also appears at the cell surface.
The biosynthesis of the purine-cytosine permease enters the secretory pathway Core glycosylation in yeast has been shown to occur in the endoplasmic reticulum, and outer chain extension in a Golgi-like organelle (Novick et ai, 1981; Schekman, 1985).
Engelman etai (1986)
Eisenberg e( a/. (1984) Amino acid number
H/21
99 122
0.65 0.95
16S 199 223 257 301 348 3%
0.60 0.80 0.55 0.56 0.83 0.55 0.50 0.50 0.77 0.57
421 466 503
589
Amino acid number
H/20
Helix number
TM TM
99 123
1.68 2,49
1 2
S TM" G TM TM G G G TM G
165 200 258 301 349 398 468 -
1.48 2.16 1.76 2.43 1.20 1.32 2.00 -
3 4 5 6 7 8 9 10 11 12
H: hydropathy of the «-helices. According to the authors, the mean hydropathy is calculated for stretches of 21 or 20 amino acids, respectively. In the GES scale, this value is expressed in kcal mol"' amino acid. The threshold value for membrane spanning segments is 1 kcal mol ' amino acid. The indicated amino acid numbers correspond to the starts of the predicted «-helices. The n-helices selected according to Eisenberg ef al. (1984) were classified according lo the authors as TM (transmembrane multimeric), G (globular), S (surface seeking) and TM° (transmembrane monomeric).
590
£ Weber, C. Rodriguez, M. R. Chevaiiier and R, Jund
OUT
Fig, 2. Proposed folding pattern o( the purinecytosine permease according to the predicted transmembrane u-helices. All the helices retained by the GES scale are drawn. Helix 3 corresponds to a surface-seeking helix according to Eisenberg ef al. (1984). The thick arrows indicate the positions of the two potential glycosylation sites proposed to be at the ceil surface (see text).
IN
Since it has been shown that the purine-cytosine permease is a glycoprotein (Schmidt ef ai., 1984) it is expected that its biosynthesis goes through the yeast secretory pathway. To determine whether the purine cytosine permease enters the secretory pathway, we cloned the complete coding region of the gene behind the inducible GAL10 promoter on a multicopy plasmid (see the Experimental procedures). The resultant plasmid, pCRF2 (Fig. 3), was introduced into strain NC308-6B carrying a seel8 mutation, a deletion in the FCV^gene and ieu2. This strain when grown in glucose or lactate does not express purine-cytosine permease activity. When galactose is added to lactate-grown cells, induction of activity increases rapidly. If galactose is added and the cells are
I
BamHI
FCY2 (1,9 hb)
shifted to 37°G, no induction occurs (see Fig. 4), although the yeast galactokinase is induced at normal levels after a temperature shift of a sec18-1 mutant strain (Jund ef a/., 1988). We therefore conclude that, as anticipated, the purine-cytosine permease enters the secretory pathway.
Effect of the mnn9 mutation on the purine-cytosine permease activity The mnn9 mutation was described as a mutation which completely suppresses the addition of mannan chains to the core glycosylated proteins (Ballou ef ai., 1980). As the purine-cytosine permease is glycosylated we investigated whether the suppression of outer chains would affect the permease activity. Permease activity measurements were carried out in mnnS ieu2 mutant strains carrying a multicopy plasmid with or without the cloned FCY2 gene. The activities were compared with activities found in our wild-type strain and in strain X2180-1A (the parental strain of the mnn9 mutant). The activities were measured at several cytosine concentrations and we compared I/max relative to that of the wild-type strains. Table 2 summarizes the results: the two wild-type strains have identical Wnax values, whereas mnn9 strains have a significantly
Table 2. Uptake of cytosine in strain mnn9. Strains
YEp52PS (6,6 kb) Fig. 3. Plasmid pCRF2. Sequences from pBR322 E, coli plasmid are indicated by a bold line. All other sequences are from S, cerevisiae. The arrows indicate direction of transcription from the GAL W promoter to transcription terminator sequences. These are followed by 2 n DNA sequences conferring autonomous replication on the plasmid.
FLIOO X2180-1A mnn9 mnn9{pFCy2) fcy2- /S(pFCY2)
relative to FL100 1 1 0.25 0.58 2.9
Strains mnnGand X2180-1A were grown in minimal medium supplemented with 1 M sorbitol. Initial velocities of uptake were determined with four cytosine concentrations and the K^ and l/ma» values were extrapolated from Lineweaver-Burk plots. The Km was found to be about 2,2 (LM for all the strains tested.
Primary structure of purine-cytosine permease of yeast q b
591
Thus, even with high gene dosage, mnn9 does not express a wild-type level of cytosine permease activity. Therefore we can conclude that deficiency in outer chain glycosylation interferes with permease activity.
o
ro
Purine-cytosine permease mRNA ieveis in the wild-type strain and in the strain carrying a gene on multicopy plasmids
60
90
120
Time (min) Fig. 4. Induction of galactose controlled purine-cytosine permease in thermosensitive secretory mutants {se
