Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis

Yeast Yeast 2003; 20: 881–892. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.994

Research Article

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis† Robin Wright1 *, Mark L. Parrish1,2 , Emily Cadera1‡ , Lynnelle Larson1§ , Clinton K. Matson1 , Philip Garrett-Engele2 , Chris Armour2 , Pek Yee Lum2 and Daniel D. Shoemaker2 1 University

of Minnesota, Department of Genetics, Cell Biology and Development, 321 Church Street, 6-160 Jackson Hall, Minneapolis, MN 55455, USA 2 Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, WA 98034, USA *Correspondence to: Robin Wright, University of Minnesota, Department of Genetics, Cell Biology and Development, 321 Church Street, 6-160 Jackson Hall, Minneapolis, MN 55455. E-mail: [email protected] † The first four authors contributed equally to this work. ‡ Current address: University of California, Department of Molecular and Cellular Biology, 401 Barker Hall 3202, University of California, Berkeley, CA 94720-202, USA. § Current address: Western Washington University, Woodring College of Education, MH 206E, 516 High Street, Bellingham, WA 98225-9090, USA.

Abstract Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely nonoverlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant’s sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright  2003 John Wiley & Sons, Ltd. Keywords: HMG-CoA reductase; endoplasmic reticulum; karmellae; DNA microarray; Saccharomyces cerevisiae

Received: 25 November 2002 Accepted: 28 January 2003

Introduction Increased levels of certain endoplasmic reticulum (ER) membrane proteins induce dramatic changes in the amount and organization of the ER. A well-characterized example of such proteininduced ER biogenesis occurs in cells expressing increased levels of HMG-CoA reductase, a key enzyme in sterol biosynthesis (Brown and Goldstein, 1980; Anderson et al., 1983; Wright et al., 1988, 1990; Goldstein and Brown, 1990), Copyright  2003 John Wiley & Sons, Ltd.

for example a 10-fold increase in the level of the HMG-CoA reductase isozymes, Hmg1p or Hmg2p, induces biogenesis of smooth ER membrane arrays in Saccharomyces cerevisiae that are morphologically and possibly functionally distinct (Lorenz and Parks, 1987; Koning et al., 1996). Hmg1p induces assembly of a nuclear-associated array of smooth ER membranes called karmellae (Wright et al., 1988; Lum and Wright, 1995; Parrish et al., 1995). Hmg2p induces assembly of shorter stacks of smooth ER membranes that may be located in

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the cytoplasm or associated with the nucleus or plasma membrane (Hampton et al., 1996; Koning et al., 1996). Analysis of HMG-CoA reductaseinduced membrane biogenesis provides opportunities to explore the regulation of ER biogenesis (Wright, 1993), for example an important component of the karmellae-inducing signal in Hmg1p is contained within a single loop of the membrane domain that is located within the ER lumen (Profant et al., 2000). Sequences on the cytoplasmic side of the ER are also important, suggesting that the process of ER biogenesis requires interactions or processes in both the cytoplasm and the ER lumen (Profant et al., 1999). To complement our studies of the karmellaeinducing signal in Hmg1p, we have conducted several genetic screens to identify trans-acting factors needed for karmellae assembly. A screen of 2500 randomly generated temperature-sensitive mutants for their ability to assemble karmellae identified vacuole mutants with karmellae-assembly defects, but did not identify mutants with karmellaedependent growth defects (Koning et al., 2002). It is likely that these vacuole biogenesis genes function indirectly in the cell’s ability to assemble karmellae. In another screen, potential karmellae assembly mutants were identified based on their inability to grow on galactose, which induced karmellae assembly via expression of HMG1 from the GAL1 promoter. However, subsequent analysis of these potential mutants revealed that the screen was efficiently identifying mutations in galactose metabolism, but not in karmellae assembly (unpublished observations). The recent availability of a complete set of oligonucleotide-tagged deletion mutants, produced by the Saccharomyces Genome Deletion Project, enables a systematic examination of non-essential genes involved in a process or pathway of interest (Winzeler and Davis, 1997; Giaever et al., 1999; Winzeler et al., 1999, 2000; Barth and Thumm, 2001; Bianchi et al., 2001; Giaever et al., 2002; Oshiro et al., 2002). We report that analysis of a population of tagged deletion mutants had a high success rate for identifying genes required for karmellae assembly and robust growth of cells expressing increased levels of Hmg1p, but was not effective for identifying mutations whose growth defects were suppressed by increased levels of Hmg1p. In addition, we found that this approach was sensitive to specific experimental parameters, Copyright  2003 John Wiley & Sons, Ltd.

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enabling subtle differences in experimental design to yield different classes of mutants.

Materials and methods Creation of deletion mutant population Each of 1557 different homozygous deletion mutants in the S288C derivative BY4743 (Mata/ αhis31/his31 leu20/leu20 ura30/ura30 met150/+ lys20/+) was independently patched onto solid YPD medium (2% each yeast extract, proteose peptone, dextrose and agar) containing 150 mg/l G418. The identity of all mutant strains in this population can be obtained by request. Equal amounts of each mutant were harvested and combined to generate a mixed mutant population in which each individual strain was present in approximately equal abundance. This mutant population was suspended in 15% glycerol and stored at 80 ◦ C. To transform the population, portions of the frozen culture were scraped into liquid YPD medium to a final concentration of 0.25 OD600 /ml. After the cells had resumed growth and completed one doubling time (about 8 h), the mutant population was harvested and made competent for transformation by washing three times in 0.1 M LiCl, followed by an overnight incubation in 0.1 M LiCl. Aliquots of the competent yeast were transformed with URA3-containing plasmids using minor modifications of the transformation protocol from the Saccharomyces Genome Deletion Project (Multiwell Transformation Protocol, http://wwwsequence.stanford.edu/group/yeast deletion project/deletions3.html). Transformants were selected on rich minimal medium lacking uracil (0.17% yeast nitrogen base, 0.5% ammonium sulphate, 2% casamino acids, 2% agar, with 30 µg/ml adenine, 20 µg/ml histidine, 40 µg/ml lysine, 40 µg/ml leucine, 20 µg/ml methionine, 30 µg/ml tryptophan and 20 µg/ml tyrosine). All of the resulting transformant colonies (approximately 30 000–140 000 independent transformants depending on the experiment) were pooled, rinsed once by centrifugation and resuspension in distilled water, resuspended in 15% glycerol at a concentration of 3 OD600 /ml, and frozen in 1 ml aliquots.

Competitive growth conditions An aliquot of the transformed mutant population containing 3 × 107 cells was thawed and used Yeast 2003; 20: 881–892.

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to inoculate 50 ml rich minimal medium broth. The initial inoculation was to a cell density of 0.05 OD600 /ml and cells were grown at 26 ◦ C, 28 ◦ C, or 30 ◦ C (depending on the experiment) in a shaking water bath until they reached a cell density of approximately 0.2 OD600 /ml. At this point, 25 ml of culture containing approximately 106 cells were removed and 25 ml of fresh medium were added to the remaining culture. The cycles of growth and dilution continued, maintaining cells in log-phase growth, until the culture had undergone 20 doublings (approximately 48 h for cultures grown with glucose as carbon source and 50 h for cultures grown with galactose as carbon source). Three different competition experiments were carried out (Table 1). The first experiment compared the composition of the same mutant population transformed with pAK266 (URA3, CEN6 with HMG1 under control of GAL1 promoter; Koning et al., 1996) following 20 generations of growth on 2% sucrose-containing medium (no karmellae induction) vs. 2% galactose plus 2% sucrose containing medium (karmellae induction). The second experiment, which was repeated three times, compared growth of the mutant population transformed with the vector control pBM258 (URA1, CEN4, GAL1/10 promoter sequences, equivalent to pBM150; Johnston and Davis, 1984) vs. the mutant population transformed

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with the karmellae-inducing plasmid pAK266; both populations were grown on rich minimal medium containing 2% galactose (karmellae-induction). The third experiment compared growth of the mutant population transformed with a control plasmid that expresses high levels of HMG-CoA reductase activity but no karmellae (pRH127-3, a 2µ URA3 plasmid containing the Hmg1p catalytic domain, Hmg1pcat , expressed from the constitutive TDH3 promoter; Donald et al., 1997, provided by Randy Hampton, UC, San Diego) vs. the mutant population transformed with a plasmid that expressed the entire Hmg1p, thus inducing high HMG-CoA reductase activity and also karmellae assembly (pLL716, a CEN6, URA3 plasmid containing HMG1 expressed from the constitutive TDH3 promoter; created by replacing the GAL1 promoter in pAK266 with the TDH3 promoter encoded on a PCR product using routine plasmid gap-repair strategy, as described by Orr-Weaver et al., 1988; Muhlrad et al., 1992). In this experiment, expression of Hmg1p and Hmg1pcat was constitutive, so that both populations were grown on rich minimal medium containing 2% glucose.

Oligonucleotide tag amplification and microarray analysis Genomic DNA was prepared from 3 OD600 units of each culture using a modification of the MasterPure

Table 1. Experimental growth conditions Experiment 1

2

3

Control culture Deletion mutant population containing the galactose-inducible HMG1 gene on pAK266; grown in sucrose to repress karmellae assembly Deletion mutant population transformed with pBM258 vector; grown in galactose

Deletion mutant population transformed with constitutively expressed HMG1 catalytic domain on pRH127-3; grown in glucose; expresses high levels of HMG-CoA reductase activity but does not assemble karmellae

Experimental culture Deletion mutant population transformed with the galactose-inducible HMG1 gene on pAK266; grown in galactose-sucrose to induce karmellae assembly1 Deletion mutant population transformed with the galactose-inducible HMG1 gene on pAK266; grown in galactose to induce karmellae assembly1 Deletion mutant population transformed with constitutively expressed HMG1 on pLL713; grown in glucose; expresses high levels of HMG-CoA reductase activity and assembles karmellae

Notes Growth in rich minimal medium at 26 ◦ C; 18 strains identified as potential karmellae-sensitive mutants2 Growth in rich minimal medium at 28 ◦ C; 60 strains identified as potential karmellae-sensitive mutants

Growth in rich minimal medium at 30 ◦ C; 61 strains identified as potential karmellae-sensitive mutants

1 This

strain also expressed increased HMG-CoA reductase activity under these growth conditions. is defined as a strain in which the hybridization signal from the experimental culture is at least five-fold lower than that from the control culture. 2 ‘Karmellae-sensitive’

Copyright  2003 John Wiley & Sons, Ltd.

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Yeast DNA protocol (Epicentre, Madison, WI) that included addition of 0.5 mm diameter glass beads to the meniscus of the resuspended cell sample and use of a Mini-Beadbeater (BioSpec Products, Bartlesville, OK) instead of vortexing. Samples that were examined included the pretransformation mutant population and the posttransformation mutant populations at time zero, and post-transformation mutant populations after 20 doublings under experimental or control conditions. The unique 20 bp oligonucleotide sequences (‘uptags’ and ‘downtags’) that mark each mutant strain were separately amplified from genomic DNA, using primers to the tag priming sites that are common to all deletions (B-U2-comp and U1 or B-D2-comp and D1). In the first experiment, both primer pairs were used. However, because no significant differences were observed in data obtained with different primer pairs, subsequent experiments used only probes prepared from the downtags. The PCR-amplified tags from control and experimental cultures were simultaneously hybridized, as previously described, to a DNA microarray containing oligonucleotides complementary to each individual mutant uptag or downtag sequence (Winzeler et al., 1999). The arrays were examined by laser scanning microscopy and Affymetrix GeneChip software, as previously described (Winzeler et al., 1999).

Results Three different competition experiments produced three different sets of potential karmellae-sensitive mutants To identify genes involved in karmellae assembly, we sought to identify deletion mutants in which karmellae assembly resulted in an observable change in cell division rate. To this end, we compared the composition of a population of 1557 different deletion mutants following growth for 20 generations under control vs. karmellaeinducing conditions. The deletion population contained 637 deletion strains corresponding to known genes, with the remainder corresponding to ORFs of unknown function (list available on request). Following the competitive growth period, genomic DNA was isolated from the two cultures and used as the template to amplify the unique oligonucleotide tags that mark each individual mutant strain. The primers for the PCR reaction were Copyright  2003 John Wiley & Sons, Ltd.

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labelled with either Cy-3 or Cy-5, allowing PCR products from experimental and control populations to be differentially labelled and simultaneously hybridized to a DNA oligonucleotide microarray representing every mutant in the population. If a particular mutant divides at the same rate in both conditions, equal PCR product will be present in both the experimental and control DNA samples, producing a hybridization signal ratio of 1. However, if a particular mutant divides at a decreased rate when karmellae are induced (i.e. is karmellae-sensitive), less PCR product representing that mutant will be present in the karmellaeinduced culture than in the control. Hybridization of these PCR products to the microarray produces a hybridization signal ratio of experimental vs. control DNA that is less than 1. If a particular mutant divides at a greater rate when karmellae are induced than in the control culture (i.e. is karmellae-suppressed), more PCR product representing that mutant will be present in the karmellaecontaining culture than in the control. Hybridization of these PCR products produces a hybridization signal ratio of experimental vs. control DNA that is greater than 1. Three separate competition experiments were carried out (Table 1). In the first experiment, the mutant population, transformed with a galactoseinducible HMG1 gene (pAK266), was grown under inducing (galactose/sucrose) or non-inducing (sucrose) conditions. This experiment was conducted at 26 ◦ C to allow survival of potential temperature-sensitive mutants that are defective for karmellae assembly, such as certain vacuole biogenesis mutants (Koning et al., 2002). The second experiment compared the growth of the mutant population transformed with a control plasmid (pBM258) to the mutant population transformed with a karmellae-inducing plasmid (pAK266). Both populations were grown at 28 ◦ C on galactose medium, minimizing differences in growth rate due to differences in carbon source. Finally, the third experiment compared growth of the mutant population transformed with a plasmid that produced high HMG-CoA reductase activity, but no karmellae (pRH127-3), to the mutant population transformed with a plasmid that produced both high HMG-CoA reductase activity and karmellae (pLL716). Because expression of the Hmg1p proteins from these plasmids was constitutive, both Yeast 2003; 20: 881–892.

Parallel analysis of tagged deletion mutants in ER biogenesis

cultures were grown on glucose-containing rich minimal medium. Among the three experiments, 120 potential karmellae-sensitive mutants were identified that displayed five-fold or greater decreases in the ratio of hybridization signal in the karmellae-induced culture vs. the control (Table 2). Based on the overall doubling rate of the mutant population, we estimate that this decreased signal ratio corresponds to an average increase in doubling time from 2.5 h to 2.7 h in galactose-containing medium or from 2.4 h to 2.6 h in glucose-containing medium. Of the 120 potential mutants, only two, yal011w and ubc7, were identified in all three experiments; 15 additional mutants were identified as karmellae-sensitive in two of the experiments. Thus, most of the potential mutants were identified in only one of the experiments, indicating that each experiment produced a largely unique set of mutants.

Most prospective mutants displayed an HMG-CoA reductase-dependent slow growth phenotype when grown clonally and assembled abnormal karmellae In theory, the competition experiment identified strains with only subtle growth differences under karmellae-inducing conditions (on average, a ∼12 min or greater increase in doubling time).

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However, this difference in growth rate occurred when the mutant was growing in liquid medium in competition with more than 1500 other strains. To test whether such subtle growth differences in a population would also be observable in the mutant strains growing clonally on solid medium, we examined the growth of all seventeen strains that were identified as karmellae-sensitive in two or more of the experiments. Each individual tagged deletion mutant strain was transformed independently with control or karmellae-inducing plasmids and the growth of serially diluted liquid cultures plated on solid rich minimal medium was examined at 16 ◦ C, 26 ◦ C, 30 ◦ C and 37 ◦ C (Figure 1). Eleven out of 17 multiply-hit strains showed observable decreases in growth on solid medium under karmellae-inducing vs. non-inducing conditions at one or more temperatures. Thus, for most potential karmellae-sensitive mutants, slow growth was not a consequence of competition with other mutants or of growth in liquid medium, but instead reflected a generalized slowing of growth rate upon karmellae induction. Expression of Hmg1p from the GAL1 promoter results in an approximately 10-fold elevation in Hmg1p. This increase has at least two consequences: induction of karmellae assembly and induction of a 10-fold increase in HMGCoA reductase activity. To determine whether or

Table 2. Genes predicted by microarray analysis to result in karmellae sensitivity1 when deleted Genes identified in

Genes

All experiments Experiments 1 and 2 Experiments 1 and 3 Experiments 2 and 3 Experiment 1 Experiment 2

UBC7 GOS1 CUE1; HTZ1; JNM1; YIM1 CKA2; DRS2; HIR2; HPC2; NUP53; RPS6B EFT2; GSG1; SIP1; STE20; VPS61 ARD1; ARG1; CCR4; CHA4; CIN1; CIN2; ECM18; GPD2; ILV1; NIP100; OPI1; RPL15B; RPL18B; RPS0A; RVS167; SAT4; SGS1; SHE4; SIC1; SNF2; SPT21; RPN10; SXM1; XDJ1

Experiment 3

ARP1; ARP6; CIN8; CKB2; COT1; CPR6; FOB1; GIM4; GRX3; HAC1; ICY1; MKK2; MLH1; OYE2; RGP1; RPL18A; RTG1; STA3; SWI5; THP2; UBP6; VIK1; VPS30; YEN1

ORFS YAL011W YML013W; YML035C-A YOL054W YMR153C-A YAL048C; YCL005W; YNL334C YCL060C; YDR359C; YDR386W; YEL007W; YEL031W; YER056C-A; YGR181W; YGR182C; YLR061W; YLR065C; YLR109W; YLR111W; YML010W-A; YML033W; YMR156C; YMR191W; YMR230W; YNL246W; YNL273W; YOR295W; YOR308C; YOR359W; YPL202C; YPL226W YDR049W; YDR120C; YDR383C; YEL001C; YER068C-A; YER079W; YFR007W; YGR164W; YHL023C; YHR115C; YHR126C; YHR133C; YHR156C; YLR190W; YLR217W; YMR044W; YMR162C; YMR166C; YMR299C; YOR051C; YOR066W; YOR314W; YOR342C

1 A potential karmellae-sensitive mutant is operationally defined as one that displays a five-fold or greater decrease in hybridization ratio of PCR-amplified DNA from that strain grown under karmellae-inducing vs. control conditions.

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Glucose (control)

Table 3. Growth and karmellae assembly phenotype of potential HMG-CoA reductase-sensitive mutants identified in multiple experiments1

Galactose (HMG1 induced)

pBM258 pAK266

gos1∆ 30°C

pDP304 pBM258 pAK266

yml 035c-a ∆ 37°C

Karmellae

Karmellae-sensitive

Abnormal

YML013W

HMG-CoA reductase-sensitive

Abnormal

CKA2 CUE1 DRS2 GOS1 HIR2 HTZ1 UBC7 YAL011W YMR153C-A HPC2 NUP53 RPS6B YOL054W

pDP304 pBM258 pAK266

ypl 120w ∆ 37°C

pDP304 pBM258 pAK266

Normal ylr 085c ∆ 26°C

pDP304

Normal

Figure 1. Serial dilution growth assay of potential HMG-CoA reductase-sensitive mutants. This figure shows examples of the growth assay used to test the validity of prospective mutants identified in the competition assays. Each mutant was transformed with a vector control (pBM258), a plasmid that induces both karmellae assembly and increased HMG-CoA reductase activity (pAK266), and a plasmid that induces karmellae but no increased HMG-CoA reductase activity (pDP304). The gos1 mutant transformed with all three plasmids grew similarly on galactose, indicating that it did not have a karmellae or HMG-CoA reductase-sensitive phenotype. The other three mutants displayed increasing sensitivity to HMG-CoA reductase activity (poor growth on galactose with pAK266). All mutants were assayed at three different temperatures; this figure shows only the single temperature at which the most severe phenotype was observed

not the slow-growth phenotype of the prospective karmellae-sensitive mutants was due to sensitivity of the mutant to karmellae, to increased HMGCoA reductase activity, or both, we compared the growth of strains expressing wild-type Hmg1p or catalytically-inactive Hmg1p in which the essential histidine at position 1020 has been replaced with glutamine (pDP304; Profant et al., 1999). This mutant protein induces normal levels of karmellae, but produces no measurable increase in HMG-CoA reductase catalytic activity. All but one (yl013w ) of the potential karmellae-sensitive mutants grew normally when they expressed catalytically inactive Hmg1p, indicating that, rather than being karmellae-sensitive, these strains were sensitive to elevated HMG-CoA reductase activity (Table 3). Copyright  2003 John Wiley & Sons, Ltd.

Gene or locus

Growth

Abnormal

JNM1 YIM1

1 Data represent clonal analysis of all mutants that were identified in two or more of the three experiments.

One possibility is that the sensitivity of the mutants to HMG-CoA reductase activity was coincident with abnormal karmellae assembly. If so, a cell’s poor growth in response to elevated HMG-CoA reductase activity might be due to its inability to properly organize karmellae membranes. To test this hypothesis, we used DiOC6 staining to examine the morphology of karmellae in the mutants. Eleven of the 17 mutants tested assembled karmellae membranes that were highly disorganized or less abundant than in wild-type controls, including four of the mutants that grew normally with elevated HMG-CoA reductase. Thus, the majority (11/16) of the putative ‘karmellaesensitive’ mutants identified in the screen were defective for karmellae assembly, whether or not they displayed a clonal slow-growth phenotype. This high success rate for identifying mutants with karmellae assembly defects might simply mean that many of the deletion mutants are karmellae-defective, whether or not they have a karmellae-dependent growth defect. To test this possibility, we randomly selected 10 mutants from the population that were not identified in any of the three experiments (arg4, asn2, cwh36, fre6, ntg2, pau2, pet100, rps7A, spa2, zrt2). Two of these mutants (cwh36 and pet100) did Yeast 2003; 20: 881–892.

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not grow on galactose and consequently could not be assayed for karmellae assembly using a galactose-inducible HMG1 gene. The remaining eight mutants assembled normal karmellae and grew normally with elevated HMG-CoA reductase. Thus, HMG-CoA reductase sensitivity and abnormal karmellae assembly appear to be rare among the mutant strains in this population and the microarray data effectively identified mutants with karmellae assembly defects.

No direct quantitative relationship was observed between the log(hybridization ratio) and measured growth rates of the mutants in clonal cultures To determine whether the differences in hybridization ratio observed via the microarray approach correlated closely with the actual differences in doubling times of the individual mutant strains, we compared the growth rates of all multiply-identified mutants in control and karmellae-inducing conditions (Table 4). Fourteen of the 17 strains examined showed the expected karmellae-dependent increase

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in doubling time, ranging from 4 to 99 min. However, no obvious direct correlation between the log(hybridization ratio) data obtained from the microarray and the measured doubling time was apparent, for example, the log(hybridization ratio) of the yol054w  mutant was −1.78, indicating a 60-fold decrease in the abundance of this mutant following competitive growth for 20 generations under karmellae-inducing conditions vs. the control. However, when cultured clonally, the yol054w  had a doubling time increase of only 26 min. In contrast, a 91 min increase in doubling time was observed for htz1  mutants, which had a log(hybridization ratio) in the competition experiment of −1.31, indicating a 20fold decrease in the abundance of this mutant. Finally, the doubling time of the rps6b mutant was not affected by karmellae assembly, in spite of the log(hybridization ratio) of −0.78. Thus, the hybridization ratio appeared to predict karmellae sensitivity with reasonable accuracy, but did not predict the degree of sensitivity of individual mutants in clonal growth. Thus, differences in the

Table 4. Hybridization ratio and doubling times Doubling time (min)

Mutation WT yol054w yml035c-a fun36 ubc7 htz1 yml013w drs2 cue1 jnm1 yim1 gos1 cka22 rps6b hpc22 ymr153c-a hir2 nup53 AVERAGE (for mutants)

Log (hybridization ratio, experimental/ control)1

Vector-transformed control

pAK266 transformed

Change

−1.78 −1.67 −1.49 −1.38 −1.31 −1.31 −1.22 −0.97 −0.97 −0.89 −0.87 −0.81 −0.78 −0.77 −0.76 −0.75 −0.66

214 139 256 170 140 138 274 196 133 216 230 219 147 474 154 155 216 287

210 164 320 173 225 230 281 236 158 315 278 244 177 470 196 146 315 365

−4 26 65 3 84 91 8 40 25 99 47 25 29 −4 42 −9 98 79

−1.08

208

250

44

1 Hybridization ratio is the log of the ratio of the signal produced by the PCR product representing the individual mutant strain in the experimental culture vs. the PCR product representing that gene in the control culture.

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log(hybridization signal) did not have predictive value for ordering the severity of mutant phenotype.

Table 5. Growth and karmellae assembly phenotype of potential HMG-CoA reductase-sensitive mutants identified in only one experiment1

Mutants identified in only one of the experiments were also enriched for strains with HMG-CoA reductase sensitivity and abnormal karmellae assembly

Growth

Karmellae

Karmellae and HMG-CoA reductase-sensitive

Abnormal

ARD1 SGS1

HMG-CoA reductase-sensitive

Abnormal

ARP6 GIM4 THP2 VIK1 YDR383C ARP1 CKB2 CPR6 SPT21 VPS30

To determine whether or not potential mutants identified in only one of the experiments were also of interest, we selected the 15 mutants from Experiment 3 that displayed the highest negative log(hybridization ratio). In addition, we randomly chose three mutants identified in only Experiment 1 and four mutants identified in only Experiment 2. Each of these strains was transformed independently with karmellae-inducing and control plasmids. The ability of the transformants to assemble karmellae and to grow on medium that induced elevated expression of Hmg1p was examined (Table 5). Most (14/19) of these potential mutants displayed HMG-CoA reductase-sensitive growth, abnormal karmellae assembly, or both. Thus, although the number of false positives was higher than that observed in potential mutants identified in multiple experiments, these data were also contained valid mutants of interest.

The microarray data were not useful for identifying karmellae- or HMG-CoA reductase-suppressed mutants The microarray data from Experiment 3 identified both potential HMG-CoA reductase-sensitive mutants, which grew more slowly with increased HMG-CoA reductase levels, and HMG-CoA reductase-suppressed mutants, which grew more rapidly with increased HMG-CoA reductase levels. We examined the growth and karmellae assembly phenotypes of 24 of the mutants that grew more rapidly with increased Hmg1p (Table 6). When grown clonally on solid medium, none of these strains grew better with increased levels of HMG-CoA reductase expression. In contrast, seven grew more slowly, indicating they were actually HMG-CoA reductase-sensitive. Indeed, two of these mutants (ard1 and sgs1) had been identified as potential HMG-CoA reductase sensitive mutants in Experiment 2. In addition, only six strains among the 24 had a karmellae-assembly defect. Thus, the Copyright  2003 John Wiley & Sons, Ltd.

Normal

Normal

Abnormal

Normal

Gene or locus

GRX3 YFR007W YOR051C EFT2 HAC1 YEN1

1 Data represent only the subset of mutants identified in only one of the experiments that were retested for clonal growth and karmellae assembly.

expected growth phenotype of the potential suppressed mutants was not reproduced in clonal growth. However, mutants of interest (HMGCoA reductase sensitive and/or karmellae assembly defective) were more common among these strains than in the general mutant population.

Discussion Several observations suggest that, in addition to increased expression of HMG1, karmellae assembly requires the action of other genes, for example, if only Hmg1p were needed for karmellae assembly, expression of Hmg1p in mammalian cells should also lead to assembly of karmellae. However, Hmg1p instead induces assembly of crystalloid ER rather than karmellae, suggesting that cellspecific factors are required (Wright et al., 1990). In addition, mutants that are defective in karmellae assembly can assemble normal Hmg2p-induced membranes, suggesting that the processes may be genetically distinguishable (Koning et al., 2002). With the knowledge that other genes were likely Yeast 2003; 20: 881–892.

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Table 6. Growth and karmellae assembly phenotype of prospective ‘HMG-CoA reductase-suppressed’ mutants1 Gene or locus

Growth

Karmellae

Karmellae and HMG-CoA reductase-sensitive

Abnormal

ARD12 SGS13

HMG-CoA reductase-sensitive

Abnormal Normal

BNI1 ESC1 ESC2 TYE7 YLR184W

Normal

Abnormal

KRH1 PHO8 YMR193C-A DPL1 EFT2 ECM14 GRE3 MSI1 RIT1 RPL36A SAL6 SPT213 VPS29 YPL102C YPL208W

Normal

Petite

Not tested

GLN3 MSS51 YOL027C

1 Defined as mutants that have a five-fold or greater increase in hybridization ratio in culture with high HMG-CoA reductase levels vs. control in Experiment 3. 2 Also identified and confirmed as potential HMG-CoA reductasesensitive mutant in Experiment 2. 3 Also identified as a potential karmellae-sensitive mutant in Experiment 1.

to be involved in karmellae assembly, we undertook a variety of genetic approaches over the past 13 years, each of which was at best marginally successful. The availability of the tagged deletion mutants provided an opportunity to approach the issue more systematically. The logic of the competitive growth screen described in this paper is based on the fact that karmellae do not confer a growth phenotype upon wild-type cells (Wright et al., 1988). We hypothesized that the ability of normal cells to grow robustly with karmellae required the cell to properly organize karmellae membranes, for example, in normal cells that contain karmellae, nuclear pores are no longer distributed uniformly but instead are concentrated in the region Copyright  2003 John Wiley & Sons, Ltd.

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of the nuclear envelope opposite from the nucleolus (Wright et al., 1988). If karmellae assembly were abnormal, nuclear pore clustering might be compromised, resulting in abnormal nuclear pore function and decreased growth rate or death. Consistent with this hypothesis, we were able to identify mutations that grew slowly when Hmg1p was expressed at increased levels and the majority of these mutants (7 of the 11 mutants identified in multiple experiments and 6 of 10 of the mutants identified in single experiments) also assembled abnormal karmellae. However, further experiments demonstrated that the slow growth phenotype was not directly due to abnormal karmellae but instead resulted from sensitivity of the mutants to increased levels of HMG-CoA reductase activity. Expression of very high levels of the Hmg1p catalytic domain leads to slow growth in wild-type cells, possibly due to the presence of excess squalene (Donald et al., 1997). Thus, these mutants may be particularly sensitive to even moderately increased levels of squalene that might accompany the, 10-fold elevation in Hmg1p levels. If the mutants identified in these screens were simply sensitive to increased HMG-CoA reductase activity, we would not expect to find such a high frequency of abnormal karmellae assembly among them, i.e. if the two processes were completely unlinked, we would expect most cells would be either karmellae-defective or HMG-CoA reductasesensitive. Nine of the 36 mutants that we studied in detail were sensitive to HMG-CoA reductase activity but assembled karmellae that appeared normal at the light microscope level. These observations are consistent with the hypothesis that karmellae assembly and HMG-CoA reductase activity are separate processes. However, the majority of the HMG-CoA reductase-sensitive mutants also were unable to assemble normal karmellae, suggesting that the two phenotypes may be linked. Indeed, it is possible that the ‘normal’ karmellae in the subset of HMG-CoA reductase-sensitive mutants may have defects that are not visible at the light microscope level. These observations lead us to hypothesize that proper organization of karmellae membranes may help to regulate HMG-CoA reductase activity, e.g. enclosure of the Hmg1p protein within the interior layers of karmellae may inhibit access of Yeast 2003; 20: 881–892.

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Table 7. Functional roles of genes identified as important for growth in the presence of elevated HMG-CoA reductase and/or normal karmellae assembly Gene

Function of gene product

Chromatin structure, transcription regulation Protein N-acetyltransferase required for transcriptional repression at telomeres and silent ARD11 MAT loci CKA21 Casein kinase II catalytic subunit required for normal transcription, ion homeostasis, cell cycle, polarity and cell size control Casein kinase II catalytic subunit required for normal transcription, ion homeostasis, cell CKB22 cycle, polarity and cell size control HIR21 Transcriptional repressor important for cell cycle regulation of histone gene transcription Required for cell cycle regulation of histone gene transcription HPC22 Histone-related protein involved in transcriptional silencing and regulation of Pol II HTZ11 transcription SGS11 DNA helicase involved in DNA repair, recombination and chromatin structure Regulation of Pol II transcription SPT212 Involved in transcriptional elongation and mitotic recombination THP21 Cytoskeleton structure and function Actin related protein of dynactin complex required for normal spindle and nucleus ARP12 positioning during mitosis ARP61 Actin related protein required for normal vacuole biogenesis and morphology Prefoldin subunit; promotes folding of tubulin and actin GIM41 Required for normal positioning of spindles and nuclear migration during mitosis JNM13 VIK11

Kinesin-related protein required for normal Kar3p localization

Secretory pathway, vacuole structure and function v-SNARE protein required for traffic through Golgi and vacuole biogenesis GOS13 Required for normal vacuole protein sorting and autophagy VPS302 Ca-ATPase required for normal ribosome assembly, Golgi function and DRS21 animophospholipid transport ER-associated protein degradation CUE11 ER membrane protein that recruits the soluble ubiquitin-conjugating enzyme Ubc7p Ubiquitin-conjugating enzyme UBC71

Location of gene product

Cytoplasm Nucleus Nucleus Nucleus Nucleus Nucleus, nucleolus Nucleus, nucleolus Nucleus Nucleus Nucleus, cytoplasm, spindle pole body Nucleus, cytoplasm Cytoplasm Nucleus, spindle pole body, cytoskeleton Spindle pole body, tubulin-cytoskeleton Membrane associated Endoplasmic reticulum (?) Golgi, plasma membrane

Endoplasmic reticulum Endoplasmic reticulum

Other CPR62 GRX33 NUP532 RPS6B2 YIM13

Cyclophilin that interacts with Rpd2p in protein folding Glutaredoxin Component of karyopherin complex of nuclear pore; required for nuclear traffic Ribosomal subunit Mitochondrial inner membrane protease

Cytoplasm, nucleus Cytoplasm

Unknown YAL011W 1 YDR049W YDR383C1 YFR007W 3 YML013W 2 YML035C-A2 YMR153C-A3 YOR051C3 YOL054W 2

Unknown, mutant is sensitive to rapamycin Unknown Unknown Unknown; mutant displays enhanced secretion Unknown Unknown Unknown Transcriptional elongation (Pol II)

Unknown Kinetochore Cytoplasm Unknown Unknown Unknown Nucleus Unknown

Cytoplasm, ribosome Cytoplasm, lipid particle, mitochondria

1 Slower

growth with elevated HMG-CoA reductase activity; abnormal karmellae assembly. growth with elevated HMG-CoA reductase activity; normal karmellae assembly. 3 Normal growth with elevated HMG-CoA reductase activity; abnormal karmellae assembly. Data about function and localization from Saccharomyces Genome Database, http://genome-www.stanford.edu/Saccharomyces/ 2 Slower

Copyright  2003 John Wiley & Sons, Ltd.

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Parallel analysis of tagged deletion mutants in ER biogenesis

the enzyme to its substrate, thus decreasing flux through the sterol biosynthetic pathway and minimizing squalene accumulation. If so, the primary defect in mutants that exhibit HMG-CoA reductase sensitivity may actually be karmellae assembly. Several specific results of this analysis help us understand the function of as yet unidentified ORFs, for example, the NUP53 gene overlaps at its 3 end with the 3 end of an open reading frame on the opposite strand, YMR053C-A. Both genes were identified in the screen as HMGCoA reductase-sensitive, but only ymr052c-a strains assembled abnormal karmellae. This result suggests that loss of NUP53 may cause HMG-CoA reductase-sensitivity because of the collateral loss of the carboxyl terminus of the YMR053C-A gene product. It also indicates that YMR053C-A probably encodes a valid gene. Genes that were not identified in the screens can also be informative, e.g. CKA2 and CKB2 were identified in the screens as HMG-CoA reductasesensitive. However, the related casein kinase gene, CKA1, was represented by a deletion mutant in the population but was not identified in any of the screens. Thus, the two kinases must have distinct roles with regard to resistance to elevated HMG-CoA reductase. An additional example involves ARP1. Loss of ARP1 makes cells sensitive to elevated levels of HMG-CoA reductase but arp1  mutants assemble karmellae that appear normal by light microscopy. Arp1p interacts physically with Arp10p, Jnm1p, Jsn1p, Nip100p, Nup1p, Srp1p, Ubc6p and YJR008Wp and genetically with BEM1, BIM1, BNI1, CIN8, KAR9 and STT4 (see the Saccharomyces Genome Database, http://biodata.mshri.on.ca/grid/servlet/Search Results?keywords=YHR129C). Of these interacting genes, the mutant population contained bem1, bim1, cin8 and jnm1. Among these mutants, bni1, jmn1 and cin8 were identified as HMGCoA reductase sensitive in the screens but bim1 was not, suggesting that these proteins may have different roles in the cell’s response to elevations in HMG-CoA reductase activity. The largest class of genes in which mutations produced both HMG-CoA reductase sensitivity and defective karmellae assembly were involved in chromatin structure and/or transcriptional regulation (Table 7). This observation may indicate that either changes in gene expression dependent on these genes (ARD1, CKA2, HIR1, HTZ1, SGS1 Copyright  2003 John Wiley & Sons, Ltd.

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and THP2) are important in karmellae assembly and/or HMG-CoA reductase resistance, or that the presence of karmellae alters the structure of the nucleus in a manner that necessitates the function of these previously non-essential genes, for example changes in the levels or function of histones may be important for maintaining proper chromatin function in nuclei with karmellae-induced changes in nuclear morphology or nuclear pore distribution. Alternatively, increased activity of HMG-CoA reductase may result in changes in ER membrane fluidity that disrupt normal associations between chromatin and the inner nuclear membrane, necessitating the function of chromatin remodelling proteins for the maintenance of cell growth. All three competition experiments were designed to identify karmellae-sensitive mutants and were expected to identify a largely overlapping set of mutants. Indeed, isolation of the same mutant in multiple experiments is frequently a good indication that the gene represented by the mutation has a valid role in the process under investigation. This expectation was not supported by the results. In fact, most genes were identified in only one of the experiments. Nevertheless, the non-overlapping sets of genes identified by each experiment were enriched for genes with roles in karmellae biogenesis and/or resistance to high levels of HMG-CoA reductase. Thus, in considering a comprehensive functional analysis of genes involved in a particular process using competition analysis, identification of all genes involved in a process may require completion of a variety of rather subtle experimental permutations and variations in cell growth conditions. These results underscore the fact that the role of a particular gene product in a cellular process may be greatly influenced by the physiological state of the cell.

Acknowledgement This work was supported by NSF Grant 0078287 to RW.

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