Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces cerm'siae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype. D ETERMINATION of the Saccharomyces cerevisiue genome sequence focuses attention on how to make effective use of this unique resource to provide a global description of eukaryotic cell function (GOFFEAU et al. 1996). Strategies to determine the role of each of the approximately 6000 yeast genes, especially the 2400 of unknown function, remain unclear (DUJON 1996). Two main strategies have been proposed (OLIVER 1994, 1996). The ease of gene disruption in yeast has led to efforts to undertake the task of sequentially disrupting every gene in the genome. Such a comprehensive collection of mutants would complement the sequence and aid the study of gene function. A "genome-wide" disruption series has been started by the international yeast community and should be completed in 2-3 years (OLIVER 1996). The collection will be distributed among researchers, who will apply their own specialized phenotypic tests to the mutants. The hierarchical classification of the many new and unknown yeast genes into families related by function constitutes a second approach (OLIVER 1994, 1996). A potential strength of this strategy is that classifying genes into functional subgroups avoids having to do detailed analysis on each and every gene in the genome. In the simplest case, only those genes within a subgroup Corresponding author: Howard Bussey, Department of Biology, McGill University, 1205 Dr. Penfield Ave., Montreal, Quebec, Canada H3A lB1. E-mail: hbussey@monod.biol.mcgill.ca Genetics 147: 435-450 (October, 1997) are further analyzed by more specific tests. Here we have made an initial attempt to identify a broad functional class of genes: those involved with the biology of the cell surface. The cell wall is composed of the major polymers, glucan, glucomannoproteins and mannoproteins and chitin, which are synthesized and elaborated into an extracellular matrix (FLEET 1991; BULAWA 1993; HERS COVICS and ORLEAN 1993; KLIS 1994; LEHLE and TANNER 1995; VAN DERVAART et al. 1995). This extracellular matrix constitutes an organelle that is dynamically engaged with the plasma membrane and the underlying secretory organelles (PRYER et al. 1992) along with cytoskeletal and cytoplasmic components to maintain cell integrity during growth and morphogenesis (MULHOL LAND et al. 1994; CID et al. 1995). The cell surface varies in shape and composition throughout the life of a fungal cell; in the budding of vegetative cells, in mating projection formation, in cell fusion in haploid cell conjugation, in spore wall formation following meiosis and in the specialized cell surfaces and morphogenesis seen in pseudohyphal growth (MADDEN et al. 1992; FLESCHER et al. 1993; KRON et al. 1994; MULHOLLAND et al. 1994; CHANT and PRINGLE 1995; CID et al. 1995). In view of the complexity of this organelle, the number of genes directly or indirectly involved in cell wall synthesis and elaboration is expected to be large. However, only a relatively small fraction of these genes have been identi436 M. Lussier et al. fied and functionally characterized (WIS 1994; RAM et al. 1994; CID et al. 1995). The aim of this study is to identify, phenotypically analyze and attempt to classify genes involved in these processes. MATERIALS AND METHODS Yeast strains, cultme conditions and methods: All yeast manipulations were done in the AWM3CA630 (MATa ci4 h 2 3,2-112 ura3-6?9 his3-11,3-15) (VERNET et al. 1987), PRY441 (MATa ci4 leu2-A1 ura3-52 his3-100 lys2-801a ade2-lo ga13) or PRY442 (MATa ci4 leu2-A1 ura?-52 his3-100 lys2-801" ade2-1" gal?) backgrounds. Yeast cells were grown under standard conditions, (YEPD, YNB and Halvorson medium) as previously described (BROWN et al. 199413). Calcofluor white solutions were either prepared fresh at 20 mg/ml and filter sterilized or were prepared at a stock concentration of 10 mg/ml in 50% ethanol and stored, in the dark, at -20" for a period of up to 1 mo. Calcofluor white containing plates were made as follows: calcofluor white solution was added to either pH 6.4 YNB agar (melted and kept at 70") containing glucose and required supplements or to YEPD agar (melted and kept at 55") containing glucose. Generation of transposon-mutagenized yeast library: Haploid strains AWM3CA630 and PRY441 were mutagenized using transposon Tn3::LEU2::lacZ according to BURNS et al. (1994). Briefly, a yeast genomic library was mutagenized in Escherichia coli to generate a large number of independent gene-containing transposon insertions (kindly provided by Dr. MICHAEL SNYDER). The mutated yeast DNA was then released from vector DNA by digestion with Not1 and was transformed into the appropriate strains using the LiAc/SSDNA/ PEG procedure (GIETZ et al. 1995) or the rapid transformation procedure of SONI et al. (1993). Yeast cells carrying the transposon as a recombinational replacement of the genomic copy with the transposon-mutagenized version were selected on synthetic minimal medium with auxotrophic supplements but lacking leucine. Southern a alysis of transposon i sertions: In the Tn3 :: lacZ::LEU2 transposon, the lacZ gene is flanked on its 3' side by an EcoRI site. Mutant yeast genomic DNA was consequently digested with EcoRI, separated through a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a "P-labeled-probe covering most of the lac2 sequence. The Tn lacZ-containing fragment detected after Southern analysis reflects a particular integration event since the other EcoRI site (5' from the Tn lac4 is within the flanking genomic sequence. Each band visualized after autoradiography corresponds to an individual integration event. Isolation of calcofluor white mutants: Mutagenized AWM3CA630 yeast cells were replica plated on YNB plates without leucine containing 20 pg/ml calcofluor white and all mutants that showed calcofluor white hypersensitivity were reverified in a plate assay according to RAM et al. (1994). Briefly, mutant AWM3CA630 cells were grown to an ODs00 value of 0.5 and lo-', lo-*, and lo-' cell dilutions were made. Three microliters of each dilution series were then spotted onto a series of YNB petri dishes containing varying amounts of calcofluor white up to 20 pg/ml. Final identification of mutants was made by scoring for growth after 48 hr at 30". Mutagenized PRY441 yeast cells were picked and resuspended in YEPD liquid broth in a 96well dish. Each 96well dish contained three wells into which the parent strain (PRY441) and two predetermined mutants (one resistant and one hypersensitive) had been inoculated. The transformants were then replica plated, using a pronged manifold, to YEPD solid medium (in rectangular Nunc plates) and allowed to grow for 48 hr. The transformants were then serially diluted using a pronged manifold into 2 X 100 p1 ddH20. Each of the dilutions was then plated, using a pronged manifold onto rectangular plates containing 5, 10 or 15 pg/ml calcofluor white. For reverification, PRY441 mutants were grown overnight at 30" and then diluted to concentrations of -1000, 100, 10, and 1 cell per pl. Five microliters of each dilution was then spotted onto plates containing 1-15 pg/ml calcofluor white. All mutants obtained showing hypersensitivity or resistance upon reverification were further analyzed. Mating To determine if the calcofluor white phenotype resulted from a transposon gene disruption, mutants obtained with strain PRY441 were crossed with PRY442 and the diploids were sporulated. All four spores were analyzed for calcofluor white resistance or hypersensitivity. All mutant phenotypes segregated with the transposon insertion. Identification of genes causing calcofluor white phenotypes: Transposondisrupted genes causing calcofluor white phenotypes were identified by plasmid rescue and DNA sequence analysis. Individual mutant yeast cells were transformed with 50-75 ng of URAibased HpaI-linearized pRSQl or PouI-linearized YIp5 plasmids using the lithium acetate procedure with sheared, denatured carrier DNA (GIETZ et al. 1995) or electroporation (SIMON 1993). Transformants were selected on YNB plates lacking both leucine and uracil. Yeast genomic DNA from each rescued mutant was prepared by the DTAB lysis method as previously described (GUSTINCICH et al. 1991; BURNS et al. 1994). The recovered genomic DNA was digested overnight by EcoRI (pRSQ1) or Nszl (YIp5) and afterwards ligated for 4 hr at 16". The ligation mixture was transformed in E. coli strain DHlOB and transformants were selected on ampicillin. Plasmid DNA was prepared from individual colonies and verified by restriction digesting with BamHI plus EcoRI (pRSQ1) or EcoRI alone (YIp5). Rescued vector pRQSl results in a 3-kilobase (kb) band with additional bands coming from genomic DNA. Correct rescue of mutant genes with vector YIp5 results after digestion in diagnostic bands of 1.0 and 1.3 kb. The identity of transposon-disrupted genes was made following the determination of the DNA sequence flanking the transposon insertion using an AB1 sequencer (Applied Biosystems Inc., model 373A) or manually using the di