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Nucleotide sequence of the gene coding for SEC14p in Candida (torulopsis) glabrata
Gene 193 (1997) 115–118
Nucleotide sequence of the gene coding for SEC14p in Candida (torulopsis) glabrata William Dundon 1, Khalid Islam * Lepetit Research Center, Via R. Lepetit 34, 21040 Gerenzano, (Va), Italy Received 13 June 1996; received in revised form 30 October 1996; accepted 4 November 1996; Received by B. Dujon
Abstract A gene coding for SEC14p from Candida glabrata has been cloned and characterized. Nucleotide (nt) sequence analysis reveals an open reading frame of 909 bp and predicts the synthesis of a polypeptide of 302 amino acid (aa) residues. Comparison of nt and aa sequences shows that the gene exhibits a much higher homology to the Saccharomyces cerevisiae (72% and 87%, respectively) than to the Candida albicans (55% and 65%, respectively) SEC14 gene. © 1997 Elsevier Science B.V. Keywords: Recombinant DNA; Phospholipid transfer protein; Fungal gene; Candida albicans; Golgi
1. Introduction Eukaryotic cells contain an array of cytosolic factors, termed phospholipid transfer proteins (PLTPs), that catalyse the energy-independent transport of lipids between membrane bilayers in vitro ( Wirtz, 1974; Cleves et al., 1991). The product of the SEC14 gene in Saccharomyces cerevisiae is one such PLTP which has been shown to be essential for yeast growth (Bankaitis et al., 1989, 1990). It is an abundant cytosolic polypeptide that is specifically required for sustained transport of secretory glycoproteins from a late Golgi compartment. It has recently been suggested that SEC14p may act by inhibiting the CDP-choline pathway in vivo (Skinner et al., 1995). The primary function of SEC14p is therefore to maintain the phosphatidylinositol/ phosphatidylcholine (PI/PC ) ratio in Golgi membranes which is at least 3-fold higher when compared with bulk membranes. PI and PC play crucial roles in yeast cell
* Corresponding author. Address: Roussel Uclaf, 102 Route de Noisy, Baˆtiment Raulin-Sr 7205, Romainville Cedex, F-93235 France. Tel. +33 1 49913259; fax: +33 1 49915087. 1 Present address: Microbiology Dept., Trinity College, Dublin 2, Ireland. Abbreviations: aa, amino acid(s); bp, base pair(s); DTT, 1,4-dithiothreitol; kb, kilobase(s) or 1000 bp; PC, phosphatidylcholine; PI, phosphatidylinositol; PLTP, phospholipid transfer protein; SDS, sodium dodecyl sulfate; SEC14p, the SEC14 gene product. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 03 - 0
membrane functionality and structural integrity and if the PI/PC ratio decreases, Golgi dysfunction ensues, followed eventually by cell death. So far the SEC14 gene has been identified in S. cerevisiae (Bankaitis et al., 1989), Kluveromyces lactis (Salama et al., 1990) and more recently Yarrowia lipolytica (Lopez et al., 1994) and Candida albicans ( X81937; EMBL). In this paper we report the cloning and sequence of the SEC14 gene from C. glabrata NCYC 350.
2. Experimental and discussion 2.1. Cloning of the SEC14 gene In order to clone the SEC14 gene we designed two degenerate primers based on sequence homology between the SEC14 genes from S. cerevisiae and K. lactis. These oligonucleotides were used to prime a polymerase chain reaction (PCR) employing genomic DNA isolated from C. glabrata and resulted in the amplification of a 700 bp fragment which was cloned and sequenced (pCG10; 175–887; see Fig. 1). The sequence of the insert showed a 72% identity with the SEC14 gene from S. cerevisiae. C. glabrata genomic DNA, digested with a number of restriction enzymes, was also transferred to nylon membranes and hybridized under stringent conditions
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Fig. 1. The nt sequence and deduced aa sequence of SEC14 from C. glabrata NCYC350 ( EMBL X97320). The putative TATA box is underlined in bold and the putative TCAAT boxes are shown in bold italics. Methods: Two degenerate primers (N2B 5∞-CGGGATCCGAACGTCTTGAt/cGAc/tTCg/aACg/tTTG with a BamHI site at the 5∞ end and C1B 5∞-CCCAAGCTTt/cGGAGCTTCACCTTCc/ aGGTCC with a HindIII site at the 5∞ end ) were used to prime a PCR employing 50 pmol of each primer and 100 ng of genomic DNA isolated from C. glabrata NCYC350. The amplification programme consisted of 30 cycles: 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min. The resulting 700 bp fragment was excised from a 1% TAE agarose gel, purified and cloned into pUC18 giving the recombinant clone pCG10. The clone was sequenced (Sanger et al., 1977; ALF automatic Pharmacia DNA sequencer). 10 mg of genomic DNA were also digested with EcoRI-KpnI, loaded onto a 0.8% TAE low melting agarose gel ( FMC ) and the bands in the 2.1–3 kb and 1–2 kb regions were excised separately and purified using GELase ( Epicenter Technologies, Madison, WI ). The DNA was ligated to the appropriately digested pUC18, i.e., EcoRI-KpnI for the 2.1–3 kb and KpnI for the 1–2 kb bands and transformed into XL1-blue competent Escherichia coli (Stratagene). Dot-blot analysis was carried out on approx. 200 of the recombinants using both the HindIII-BamHI and the KpnI fragment of pCG10 as probe.
W. Dundon, K. Islam / Gene 193 (1997) 115–118
117
Fig. 2. Comparison of the deduced aa sequences of SEC14 genes from several yeast species. SEC14cg, C. glabrata NCYC350 (this paper); SEC14kl, K. lactis (Salama et al., 1990); SEC14sc, S. cerevisiae (Bankaitis et al., 1989); and SEC14yl, Y. lipolytica (first 350 aa; Lopez et al., 1994), SEC14ca, C. albicans ( X81937; EMBL). Appropriate gaps for alignment have been inserted (..) and identical aa are shown in bold.
using the HindIII-BamHI fragment of pCG10 as a probe. Two bands in the 1.5–2.5 kb were observed in the EcoRI-KpnI digestion, confirming the presence of a KpnI site in pCG10. The observed restriction profile suggested that the 2.5 kb fragment was derived by digestion with EcoRI+KpnI, while the 1.5 kb fragment was due to KpnI alone (data not shown). In this way two further clones were obtained, one containing the
first 252 bp at the 5∞ end of SEC14 (pCG1) and the other containing the remainder of the gene (pCG3). 2.2. Sequence analysis of the C. glabrata SEC14 The combined nt sequences from pCG1, pCG3 and pCG10 inserts revealed an open reading frame (ORF ) of 909 bp coding for a putative protein of 302 aa
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(Fig. 1). Comparison of the nt sequence with the SEC14 genes from S. cerevisiae, K. lactis, Y. lipolytica and C. albicans showed a 72%, 72.5%, 66% and 55% identity, respectively. Unlike the S. cerevisiae gene, C. glabrata SEC14 does not contain an intron. The sequence at the 5∞ end of SEC14 was analysed to locate a likely promoter region. In several yeast genes the promoter regions at position −9 are usually A+T rich containing TATA or TAAT sequences, proposed to substitute for TATA, and serve as messenger start signals ( Kurtz et al., 1990). Analysis of the 259 bp segment upstream from the ATG start codon revealed the presence of a TATA box at position −19. There are also two TCAAT consensus sequences at positions −50 bp and −39 bp, respectively, as found in several eukaryotic genes, around 80 bp upstream from the RNA synthesis start site (Myers et al., 1992). Several genes from C. albicans have been shown to contain long tracts of As (5 to 18) followed immediately by tracts of Ts (6 to 9) upstream from their start codon (Myers et al., 1992). These tracts were not observed upstream from C. glabrata SEC14. Likewise, the region downstream from the stop codon does not contain the TAG...TAGT...TTT sequence which is involved in transcription termination and found in several yeast genes ( Zaret and Sherman, 1982). The deduced aa sequence is highly related to the SEC14p from S. cerevisiae, showing 86.6% identity as compared to 79.9% with K. lactis and about 65% with C. albicans and Y. lipolytica ( Fig. 2). This suggests a high conservation of the SEC14p among fungi. A total of 605 bp were also sequenced downstream from SEC14 gene and this segment showed a 76% identity to the NAM7 gene from S. cerevisiae (Groundinsky et al., 1992), suggesting that SEC14 is contiguous to NAM7. This would also confirm the observation from the above authors that during the isolation of the S. cerevisiae NAM7 gene they had a clone overlapping SEC14. Genomic DNA from K. lactis, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and S. cerevisiae was probed using the HindIII-BamHI fragment of pCG10 which hybridized only with C. glabrata, S. cerevisiae, and K. lactis (data not shown), suggesting significant differences between SEC14 genes from C. glabrata and those from C. albicans, C. tropicalis and C. parapsilosis.
3. Conclusions (1) The SEC14 gene from C. glabrata has been isolated and the complete coding sequence determined. The nt and aa sequences show a high homology to the SEC14 genes from S. cerevisiae and K. lactis. The C. glabrata SEC14 gene, unlike the S. cerevisiae and Y. lipolytica genes, does not contain an intron. (2) The SEC14 gene from C. glabrata shows a much lower homology to the C. albicans gene, and proba-
bly with other Candida species, when compared with S. cerevisiae or K. lactis gene. To date very few genes from C. glabrata have been characterized but a preliminary study of URA3 from C. albicans, C. boidinii, and K. lactis similarly showed that at both the nt and aa levels the URA3 gene from C. glabrata exhibits a higher homology to the URA3 gene of K. lactis than to those from the other Candida species (Sakai et al., 1992).
Acknowledgement We would like to thank Ms. Franca Ripamonti and Dr. Anna Miele for providing us with the yeast strains and Dr. Stefano Donadio for critically reading the manuscript and for helpful suggestions.
References Bankaitis, V.A., Malehorn, D., Emr, S. and Greene, R., 1989. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108, 1271–1281. Bankaitis, V.A., Aitken, J., Cleves, A. and Dowhan, W., 1990. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347, 561–562. Cleves, A., McGee, T. and Bankaitis, V., 1991. Phospholipid transfer proteins: a biological debut – review. Trends Cell Biol. 1, 30–34. Groundinsky, O., Dujardin, G. and Slonimski, P., 1992. NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. J. Mol. Biol. 224, 575–587. Kurtz, M., Kelly, R. and Kirsch, D., 1990. Molecular genetics of Candida albicans. In: The Genetics of Candida. CRC Press, Boca Raton, FL, p. 21. Lopez, M., Nicaud, J., Skinner, H., Vergnolle, C., Kader, J., Bankaitis, V. and Gaillardin, C., 1994. A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form. J. Cell Biol. 125, 113–127. Myers, K., Fonzi, W. and Sypherd, P., 1992. Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans. Nucleic Acids Res. 20, 1705–1710. Sakai, Y., Kazarimoto, T. and Tani, Y., 1992. The orotidine-5∞phosphate decarboxylase gene (URA3) of a methylotrophic yeast, Candida boidinii: nucleotide sequence and its expression in Escherichia coli. J. Ferment. Bioeng. 73, 255–260. Salama, S., Cleaves, A., Malehorn, D., Whitters, E. and Bankaitis, V., 1990. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae. J. Bacteriol. 172, 4510–4521. Sanger, F., Nicklen, S. and Coulson, A., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Skinner, H.B., McGee, T.P., McMAster, C.R., Fry, M.R., Bell, R.M. and Bankaitis, V.A., 1995. The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity. Proc. Natl. Acad. Sci. USA 92, 112–116. Wirtz, K., 1974. Transfer of phospholipids between membranes – review. Biochim. Biophys. Acta 344, 95–117. Zaret, K. and Sherman, F., 1982. DNA sequence required for efficient transcription in yeast. Cell 28, 563–573.
Nucleotide sequence of the gene coding for SEC14p in Candida (torulopsis) glabrata William Dundon 1, Khalid Islam * Lepetit Research Center, Via R. Lepetit 34, 21040 Gerenzano, (Va), Italy Received 13 June 1996; received in revised form 30 October 1996; accepted 4 November 1996; Received by B. Dujon
Abstract A gene coding for SEC14p from Candida glabrata has been cloned and characterized. Nucleotide (nt) sequence analysis reveals an open reading frame of 909 bp and predicts the synthesis of a polypeptide of 302 amino acid (aa) residues. Comparison of nt and aa sequences shows that the gene exhibits a much higher homology to the Saccharomyces cerevisiae (72% and 87%, respectively) than to the Candida albicans (55% and 65%, respectively) SEC14 gene. © 1997 Elsevier Science B.V. Keywords: Recombinant DNA; Phospholipid transfer protein; Fungal gene; Candida albicans; Golgi
1. Introduction Eukaryotic cells contain an array of cytosolic factors, termed phospholipid transfer proteins (PLTPs), that catalyse the energy-independent transport of lipids between membrane bilayers in vitro ( Wirtz, 1974; Cleves et al., 1991). The product of the SEC14 gene in Saccharomyces cerevisiae is one such PLTP which has been shown to be essential for yeast growth (Bankaitis et al., 1989, 1990). It is an abundant cytosolic polypeptide that is specifically required for sustained transport of secretory glycoproteins from a late Golgi compartment. It has recently been suggested that SEC14p may act by inhibiting the CDP-choline pathway in vivo (Skinner et al., 1995). The primary function of SEC14p is therefore to maintain the phosphatidylinositol/ phosphatidylcholine (PI/PC ) ratio in Golgi membranes which is at least 3-fold higher when compared with bulk membranes. PI and PC play crucial roles in yeast cell
* Corresponding author. Address: Roussel Uclaf, 102 Route de Noisy, Baˆtiment Raulin-Sr 7205, Romainville Cedex, F-93235 France. Tel. +33 1 49913259; fax: +33 1 49915087. 1 Present address: Microbiology Dept., Trinity College, Dublin 2, Ireland. Abbreviations: aa, amino acid(s); bp, base pair(s); DTT, 1,4-dithiothreitol; kb, kilobase(s) or 1000 bp; PC, phosphatidylcholine; PI, phosphatidylinositol; PLTP, phospholipid transfer protein; SDS, sodium dodecyl sulfate; SEC14p, the SEC14 gene product. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 03 - 0
membrane functionality and structural integrity and if the PI/PC ratio decreases, Golgi dysfunction ensues, followed eventually by cell death. So far the SEC14 gene has been identified in S. cerevisiae (Bankaitis et al., 1989), Kluveromyces lactis (Salama et al., 1990) and more recently Yarrowia lipolytica (Lopez et al., 1994) and Candida albicans ( X81937; EMBL). In this paper we report the cloning and sequence of the SEC14 gene from C. glabrata NCYC 350.
2. Experimental and discussion 2.1. Cloning of the SEC14 gene In order to clone the SEC14 gene we designed two degenerate primers based on sequence homology between the SEC14 genes from S. cerevisiae and K. lactis. These oligonucleotides were used to prime a polymerase chain reaction (PCR) employing genomic DNA isolated from C. glabrata and resulted in the amplification of a 700 bp fragment which was cloned and sequenced (pCG10; 175–887; see Fig. 1). The sequence of the insert showed a 72% identity with the SEC14 gene from S. cerevisiae. C. glabrata genomic DNA, digested with a number of restriction enzymes, was also transferred to nylon membranes and hybridized under stringent conditions
116
W. Dundon, K. Islam / Gene 193 (1997) 115–118
Fig. 1. The nt sequence and deduced aa sequence of SEC14 from C. glabrata NCYC350 ( EMBL X97320). The putative TATA box is underlined in bold and the putative TCAAT boxes are shown in bold italics. Methods: Two degenerate primers (N2B 5∞-CGGGATCCGAACGTCTTGAt/cGAc/tTCg/aACg/tTTG with a BamHI site at the 5∞ end and C1B 5∞-CCCAAGCTTt/cGGAGCTTCACCTTCc/ aGGTCC with a HindIII site at the 5∞ end ) were used to prime a PCR employing 50 pmol of each primer and 100 ng of genomic DNA isolated from C. glabrata NCYC350. The amplification programme consisted of 30 cycles: 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min. The resulting 700 bp fragment was excised from a 1% TAE agarose gel, purified and cloned into pUC18 giving the recombinant clone pCG10. The clone was sequenced (Sanger et al., 1977; ALF automatic Pharmacia DNA sequencer). 10 mg of genomic DNA were also digested with EcoRI-KpnI, loaded onto a 0.8% TAE low melting agarose gel ( FMC ) and the bands in the 2.1–3 kb and 1–2 kb regions were excised separately and purified using GELase ( Epicenter Technologies, Madison, WI ). The DNA was ligated to the appropriately digested pUC18, i.e., EcoRI-KpnI for the 2.1–3 kb and KpnI for the 1–2 kb bands and transformed into XL1-blue competent Escherichia coli (Stratagene). Dot-blot analysis was carried out on approx. 200 of the recombinants using both the HindIII-BamHI and the KpnI fragment of pCG10 as probe.
W. Dundon, K. Islam / Gene 193 (1997) 115–118
117
Fig. 2. Comparison of the deduced aa sequences of SEC14 genes from several yeast species. SEC14cg, C. glabrata NCYC350 (this paper); SEC14kl, K. lactis (Salama et al., 1990); SEC14sc, S. cerevisiae (Bankaitis et al., 1989); and SEC14yl, Y. lipolytica (first 350 aa; Lopez et al., 1994), SEC14ca, C. albicans ( X81937; EMBL). Appropriate gaps for alignment have been inserted (..) and identical aa are shown in bold.
using the HindIII-BamHI fragment of pCG10 as a probe. Two bands in the 1.5–2.5 kb were observed in the EcoRI-KpnI digestion, confirming the presence of a KpnI site in pCG10. The observed restriction profile suggested that the 2.5 kb fragment was derived by digestion with EcoRI+KpnI, while the 1.5 kb fragment was due to KpnI alone (data not shown). In this way two further clones were obtained, one containing the
first 252 bp at the 5∞ end of SEC14 (pCG1) and the other containing the remainder of the gene (pCG3). 2.2. Sequence analysis of the C. glabrata SEC14 The combined nt sequences from pCG1, pCG3 and pCG10 inserts revealed an open reading frame (ORF ) of 909 bp coding for a putative protein of 302 aa
118
W. Dundon, K. Islam / Gene 193 (1997) 115–118
(Fig. 1). Comparison of the nt sequence with the SEC14 genes from S. cerevisiae, K. lactis, Y. lipolytica and C. albicans showed a 72%, 72.5%, 66% and 55% identity, respectively. Unlike the S. cerevisiae gene, C. glabrata SEC14 does not contain an intron. The sequence at the 5∞ end of SEC14 was analysed to locate a likely promoter region. In several yeast genes the promoter regions at position −9 are usually A+T rich containing TATA or TAAT sequences, proposed to substitute for TATA, and serve as messenger start signals ( Kurtz et al., 1990). Analysis of the 259 bp segment upstream from the ATG start codon revealed the presence of a TATA box at position −19. There are also two TCAAT consensus sequences at positions −50 bp and −39 bp, respectively, as found in several eukaryotic genes, around 80 bp upstream from the RNA synthesis start site (Myers et al., 1992). Several genes from C. albicans have been shown to contain long tracts of As (5 to 18) followed immediately by tracts of Ts (6 to 9) upstream from their start codon (Myers et al., 1992). These tracts were not observed upstream from C. glabrata SEC14. Likewise, the region downstream from the stop codon does not contain the TAG...TAGT...TTT sequence which is involved in transcription termination and found in several yeast genes ( Zaret and Sherman, 1982). The deduced aa sequence is highly related to the SEC14p from S. cerevisiae, showing 86.6% identity as compared to 79.9% with K. lactis and about 65% with C. albicans and Y. lipolytica ( Fig. 2). This suggests a high conservation of the SEC14p among fungi. A total of 605 bp were also sequenced downstream from SEC14 gene and this segment showed a 76% identity to the NAM7 gene from S. cerevisiae (Groundinsky et al., 1992), suggesting that SEC14 is contiguous to NAM7. This would also confirm the observation from the above authors that during the isolation of the S. cerevisiae NAM7 gene they had a clone overlapping SEC14. Genomic DNA from K. lactis, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and S. cerevisiae was probed using the HindIII-BamHI fragment of pCG10 which hybridized only with C. glabrata, S. cerevisiae, and K. lactis (data not shown), suggesting significant differences between SEC14 genes from C. glabrata and those from C. albicans, C. tropicalis and C. parapsilosis.
3. Conclusions (1) The SEC14 gene from C. glabrata has been isolated and the complete coding sequence determined. The nt and aa sequences show a high homology to the SEC14 genes from S. cerevisiae and K. lactis. The C. glabrata SEC14 gene, unlike the S. cerevisiae and Y. lipolytica genes, does not contain an intron. (2) The SEC14 gene from C. glabrata shows a much lower homology to the C. albicans gene, and proba-
bly with other Candida species, when compared with S. cerevisiae or K. lactis gene. To date very few genes from C. glabrata have been characterized but a preliminary study of URA3 from C. albicans, C. boidinii, and K. lactis similarly showed that at both the nt and aa levels the URA3 gene from C. glabrata exhibits a higher homology to the URA3 gene of K. lactis than to those from the other Candida species (Sakai et al., 1992).
Acknowledgement We would like to thank Ms. Franca Ripamonti and Dr. Anna Miele for providing us with the yeast strains and Dr. Stefano Donadio for critically reading the manuscript and for helpful suggestions.
References Bankaitis, V.A., Malehorn, D., Emr, S. and Greene, R., 1989. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108, 1271–1281. Bankaitis, V.A., Aitken, J., Cleves, A. and Dowhan, W., 1990. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347, 561–562. Cleves, A., McGee, T. and Bankaitis, V., 1991. Phospholipid transfer proteins: a biological debut – review. Trends Cell Biol. 1, 30–34. Groundinsky, O., Dujardin, G. and Slonimski, P., 1992. NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. J. Mol. Biol. 224, 575–587. Kurtz, M., Kelly, R. and Kirsch, D., 1990. Molecular genetics of Candida albicans. In: The Genetics of Candida. CRC Press, Boca Raton, FL, p. 21. Lopez, M., Nicaud, J., Skinner, H., Vergnolle, C., Kader, J., Bankaitis, V. and Gaillardin, C., 1994. A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form. J. Cell Biol. 125, 113–127. Myers, K., Fonzi, W. and Sypherd, P., 1992. Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans. Nucleic Acids Res. 20, 1705–1710. Sakai, Y., Kazarimoto, T. and Tani, Y., 1992. The orotidine-5∞phosphate decarboxylase gene (URA3) of a methylotrophic yeast, Candida boidinii: nucleotide sequence and its expression in Escherichia coli. J. Ferment. Bioeng. 73, 255–260. Salama, S., Cleaves, A., Malehorn, D., Whitters, E. and Bankaitis, V., 1990. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae. J. Bacteriol. 172, 4510–4521. Sanger, F., Nicklen, S. and Coulson, A., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Skinner, H.B., McGee, T.P., McMAster, C.R., Fry, M.R., Bell, R.M. and Bankaitis, V.A., 1995. The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity. Proc. Natl. Acad. Sci. USA 92, 112–116. Wirtz, K., 1974. Transfer of phospholipids between membranes – review. Biochim. Biophys. Acta 344, 95–117. Zaret, K. and Sherman, F., 1982. DNA sequence required for efficient transcription in yeast. Cell 28, 563–573.