An MFal-SUC2 (a-factor-invertase) gene fusion for study of protein localization and gene expression in yeast

The peptide mating pheromone a-factor and the hydrolytic enzyme invertase (f3D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and preproleader portion of the coding sequence of the a-factor gene (MFa1l) to a large fragment of the invertase gene (SUC2) lacking its 5'flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the a-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-a-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (secl8). Therefore, prior to its efficient proteolytic removal, the a-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MFal, the MFal-SUC2 fusion is expressed in a haploids at levels 65-75 times higher than in a haploids or in a/a diploids; also, high-level expression is eliminated in matal mutants but not in matat mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer a cell-specific expression to the hybrid gene. Fusions of bacterial genes to the lacZ gene of Escherichia coli have been extremely useful for determining the level of expression of gene products that otherwise would be difficult to detect because there are convenient methods for monitoring the activity of 3galactosidase (,B-D-galactoside galactohydrolase, EC 3.2.1.23) (1, 2). This approach has been extended to analyze the regulation of expression of several yeast genes (3-7). Studies in E. coli (8, 9) and more recently in yeast (unpublished data) demonstrate that ,3galactosidase is unable to be translocated through a membrane, even when coupled to secretory proteins. In E. coli, this property results in certain characteristic phenotypes that have been used to select mutations that have permitted the genetic dissection of the process of prokaryotic protein export (10, 11). In yeast, however, this feature of 83-galactosidase might limit its utility in the analysis of eukaryotic secretory transport (which includes protein translocation into the endoplasmic reticulum, delivery to the Golgi complex, protein sorting, packaging into vesicles, and final targeting to unique cellular destinations). To overcome this limitation, but to take advantage nonetheless of the utility of gene fusions for studying protein secretion and gene regulation in yeast cells, we sought a different and more appropriate indicator enzyme for use in constructing gene fusions. The SUC2 gene of yeast codes for the enzyme invertase (,3 D-fructofuranoside fructohydrolase, EC 3.2.1.26) (12), which is required for growth on sucrose as sole carbon source. This gene and its product have several biochemical and genetic advantages that should permit development of a generally applicable gene fusion system for yeast. The SUC2 gene has been cloned (13) and its entire nucleotide sequence has been determined (14). Invertase itself is a very stable dimeric protein that is secreted via the yeast secretory pathway (15, 16). The secreted molecule bears long, Nlinked, mannose-rich oligosaccharide chains (17) on at least 9 of its 13 potential glycosylation sites (14, 18, 19). The enzyme is too large to pass through the yeast cell wall and remains trapped in the so-called periplasmic space. Extracellular invertase is produced from a transcript that codes for a precursor containing a 19-residue hydrophobic signal sequence (20, 21). Synthesis of the external form is repressed by the presence of glucose because transcription of the gene is reduced and only a short transcript, which does not encode the signal sequence, is made (13). Thus, a low level of an internal form is made. Invertase activity present in whole cells, either intact or made permeable by various methods, can be determined through simple and sensitive colorimetric assays that measure either the glucose or the fructose released upon sucrose hydrolysis (22). Furthermore, these assays can be adapted to be performed directly on colonies on a solid medium or on filter paper replicas of such clones (ref. 23; unpublished results). Finally, growth on sucrose provides a direct genetic selection for functional invertase. We chose the structural gene MFal, which encodes the 165amino acid precursor of the yeast mating pheromone, a-factor (aF), to determine how successful gene fusions to SUC2 might be. aF is a peptide hormone-like effector that is secreted only by a haploids and is required (24) to prime haploid yeast cells of the opposite mating type, a cells, for eventual conjugation with a cell partners (for review, see ref. 25). The MFal gene has been cloned and its entire nucleotide sequence has been determined (26, 27). Because only MATa cells contain a mRNA complementary to MFal (27), expression of the gene is apparently under the direct transcriptional control of the mating type locus (28). aF is excised from its larger precursor, preproaF (27, 29), which contains four tandem copies of the pheromone (26, 27). The precursor enters the secretory system, is glycosylated with short core oligosaccharides at three sites (unAbbreviations: aF, yeast a-factor mating pheromone; bp, base pair(s). t Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125. § To whom reprint requests should be addressed. 7080 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S.C. §1734 solely to indicate this fact. Proc. Nati. Acad. Sci. USA 80 (1983) 7081 published data), and is processed at a late stage in the secretory pathway by both endoproteolytic and exoproteolytic cleavages (29) to yield the mature pheromone. Unlike processing of the secreted form of invertase, the amino-terminal hydrophobic leader of 20 or so residues is apparently not removed during translocation of prepro-aF into the lumen of the endoplasmic reticulum (unpublished data). In this report, we describe the construction of a MFal-SUC2 gene fusion, demonstrate the cell type-specific regulation of its expression, and characterize the processing and secretion of its hybrid protein product. MATERIALS AND METHODS Yeast and Bacterial Strains. Genetic crosses (30) were used to construct the following Saccharonyces cerevisiae strains, each of which contains a complete deletion (suc2-A9) of the chromosomal copy of SUC2 (C. Falco and M. Carlson, personal communication) and contains no other unlinked invertase structural gene (SUC1, SUC3-SUC7) (13): SEY2101, MATa ura3-52 leu2-3,-112 ade2-1; SEY2102, MATa ura3-52 leu2-3,-112 his4519; SEYD2112, MATa/MATa ura3-52/ura3-52 leu2-3,-112/ leu2-3,-112 +/ade2-1 +/his4-519; SEY5188, MATa secl8-1 ura3-52 leu2-3,-112; and XMF40-3a, matal-ochre ura3-52 leu2 trpl-289 Iysl-l. Strain XBH15a-34c, mata2 SUC2 ura3-52 leu23,-112 his4-580 trpl-289 (from L. C. Blair), was used in certain experiments. Bacterial transformations were performed with the following E. coli K-12 strains: JM83, Fara A(lac-pro) rpsL thi [080dlacZAM15] (from J. Messing); MC1061, FhsdRhsdM+ araDl39 A(araABOIC-leu)7679 A(lac)X74 galU galK rpsL; and MC1066, FhsdRhsdM+ pyrF: :Tn5 A(lac)X74 galU galK rpsL trpC9830 leuB600 (from M. Casadaban). Plasmid Vectors and Recombinant DNA Methodology. pBR322 (31), pUC8 (32), YEp24 (33), pAB101 (27), and pRB58 (13) have been described previously. pCGS139 was the gift of G. Vovis. Restriction endonuclease digestions and ligations with phage T4 DNA ligase were conducted as recommended by the suppliers. Plasmid purifications, agarose gel electrophoresis, DNA-mediated transformations of yeast and bacteria, and other manipulations of nucleic acids were performed by standard methods (30, 34, 35). Enzyme Assay. Invertase activity was measured at the glucose generated from sucrose hydrolysis by minor modifications (36) of the colorimetric procedure of Goldstein and Lampen (22). All values given represent the average of duplicate determinations and were the results of measurements performed under conditions in which the amount of glucose generated was linear with respect to both time and the number of cells added. In situ staining for invertase activity in nondenaturing polyacrylamide gels has been described (15, 37). Radiolabeling and Immunoprecipitation. Labeling of yeast cells with [IS]SO42 in low-sulfate medium in the presence and absence of tunicamycin (Sigma), subcellular fractionation of the radioactive cells and removal of the cell wall by lytic enzyme (38) digestion, immunoprecipitations with anti-invertase antibody (gift of I. Schauer), and electrophoresis of proteins in slabs of polyacrylamide gel in the presence of NaDodSO4 have all been described (15, 39). To remove N-linked glycosyl chains, protein samples were resuspended in a final volume of 50 ,ul of 50 mM 2-mercaptoethanol/0.5% NaDodSO4/5 mM NaN3/ 100 ,ug of bovine serum albumin per ml/0.27 M sodium citrate, pH 5.5, boiled for 3 min, cooled to ambient temperature, incubated with 12.5 ng of endoglycosidase H (40) (gift of P. Robbins) for 10-12 hr at 370C, subjected to a second boiling, and redigested with 6 ng of endoglycosidase H for 10-12 hr at 37TC. After this treatment, the reaction mixtures were dialyzed exhaustively against water, lyophilized, and resuspended in electrophoresis buffer (15, 39).