In this study, ILM1, a gene of unknown function, was investigated in the yeast Saccharomyces cerevisiae. We investigated the sensitivity of an ILM1 deletion strain to a variety of stress conditions and compared the budding indices of wild-type and ilm1Δ strains. Our results showed that deletion of ILM1 increases sensitivity to caffeine and leads to a decrease in cell budding. These findings are consistent with a new role for ILM1 in regulating passage through the G1/S transition.
","acknowledgements":"The authors thank Heatherlynn Barrett, Indiana University East laboratory manager, for her help throughout the project, the Yeast ORFan Gene Project (www.yeastorfanproject.com) for protocols, resources, and inspiration, and the Saccharomyces Genome Database which is essential to work on yeast genes.
","authors":[{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["dataCuration","formalAnalysis","investigation","visualization","writing_reviewEditing"],"email":"dewittda@iu.edu","firstName":"Drew ","lastName":"Dewitt","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"aamyx@iu.edu","firstName":"Alyssa ","lastName":"Amyx","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"kmholder@iu.edu","firstName":"Kaitlynn ","lastName":"Holderman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"carmmaxw@iu.edu","firstName":"Carmen","lastName":"Maxwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"idball@iu.edu","firstName":"Ian ","lastName":"Ball","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dykbenni@iu.edu","firstName":"Dylan","lastName":"Bennington","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rijterry@iu.edu","firstName":"Rilee","lastName":"Terry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dafruizg@iu.edu","firstName":"Dafne","lastName":"Ruiz Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rbbranst@iu.edu","firstName":"Rudy ","lastName":"Branstetter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rowboyd@iu.edu","firstName":"Wesley","lastName":"Boyd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["conceptualization","formalAnalysis","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"jillschw@iu.edu","firstName":"Jill Kuglin","lastName":"Schweitzer","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded by the School of Natural Science and Mathematics at Indiana University East and by an Affinity Grant from the Women’s Philanthropy Leadership Council (IU Foundation).
","image":{"url":"https://portal.micropublication.org/uploads/efddbabccc88f7876bdac48705ee832a.jpg"},"imageCaption":"A, Tenfold serial dilution spot assays of paired wild-type and ilm1Δ, spotted on YPD media and 10mM caffeine incubated at 30°C for 2 days. Images show that ilm1Δ exhibit sensitivity to caffeine as compared to wild type strain. B, Independent spot assays of wild-type and ilm1Δ strains on YPG or YPD with 100mM CaCl2 or 5% ethanol. Cells were incubated at 30°C for 2-3 days. The growth of wild-type (top three rows) and ilm1Δ (bottom three rows) was similar under these stress conditions. C, Cell morphology analysis of fixed log-phase cells of both wild-type (left) and ilm1Δ (right). Micrographs show an increase in unbudded cells in the ilm1Δ strain. Scale bar = 1µm. D, Bar graph showing percent unbudded cells of both wild-type and ilm1Δ in log-phase cultures (average of three independent trials; 100 cells of each strain counted per trial). The ilm1Δ strain has a significantly higher percentage of unbudded cells than wild-type. Wild type: 53.3% ±8.14%; ilm1Δ: 74% ±8.54% (p=0.005).
","imageTitle":"Deletion of ILM1 sensitizes yeast to caffeine and decreases budding
","methods":"The methods for this work were adapted from procedures made available through the Yeast ORFan Project (Miller et al., 2024).
Yeast culture conditions
Yeast cells were grown at 30ºC in prepared YPD medium (1% yeast extract, 2% peptone, 2% dextrose) in liquid cultures or on solid medium with 2% agar. After yeast transformation, cells were incubated on SD-Ura agar plates for 4-5 days to select for successful transformants. Selection media (SD-Ura) contained 0.67% yeast nitrogen base without amino acids and 2% dextrose, supplemented with 20mg/L l-histidine, 20mg/L l-methionine, and 60mg/L l-leucine. Components for yeast media were from Dot Scientific, Inc., Burton, MI.
ILM1 knockout
An ILM1 knockout strain (ilm1Δ) was created in the BY4741 Saccharomyces cerevisiae background using a PCR-based strategy. First, plasmid DNA containing wild-type URA3 (pRS406) was isolated from E. coli using Qiagen mini-prep kit and quantitated using a NanoDrop 2000 spectrophotometer (ThermoScientific). Next, amplification of URA3 was accomplished using a forward primer to recognize twenty bases at the beginning of URA3 with an additional 60 bases of genomic DNA upstream of ILM1 and a reverse primer to recognize twenty bases at the end of URA3 plus an additional 60 bases of genomic DNA downstream of ILM1. See Table 2 for primer sequences. PCR reactions contained 0.5mM each primer, 0.2 mM dNTPs, 1X PCR buffer (GenScript), 0.5ml GenScript Green Taq DNA polymerase, and approximately 10 ng of plasmid template. Finally, the URA3 PCR product was introduced into yeast using yeast transformation (Gietz & Schiestl, 2007). Successful deletion strains were confirmed by isolating DNA from transformant colonies (Lõoke et al., 2011) and amplifying a confirmation PCR product—forward primer annealed to the genome upstream of ILM1 and the reverse primer annealed to URA3. All PCR products were analyzed using 0.8% agarose gel electrophoresis.
Spot assay
Liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1 and then serially diluted 1:10 to make five cell suspensions ranging from 1:100 to 1:104. Each strain was diluted in triplicate per experiment. 2ml of each cell suspension was spotted onto YPD agar plates and YPD containing 10mM caffeine, 100mM CaCl2, or 5% ethanol (Sigma Aldrich). Cell suspensions were also plated on media containing glycerol instead of dextrose (YPG). Plates were imaged after 2 or 3 days of incubation at 30 ºC. Each spot assay was replicated independently three times and representative images are shown.
Morphological Analysis
To prepare log-phase cells, liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1, and then incubated in fresh YPD for 4-5 hours. Cells were washed with phosphate-buffered saline (1X PBS), fixed with 3.7% formaldehyde for 5 min and sonicated for 20s to reduce clumping using a Fisher Scientific FB505 (amplitude—20%; pulse 1s on/1s off). Sonicated cells were mounted on slides and imaged using brightfield microscopy (Nikon Eclipse Ei with Digital Sight 1000 Camera). Micrographs were analyzed using Image J to measure the area of one hundred cells of each strain per experiment and determine the budding state. Morphological analysis was replicated independently three times. The unbudded percentage shown in Figure 1D is an average of three independent trials. A student t-test was used to compare the number of unbudded cells from wild-type and ilm1Δ to determine statistical significance.
","reagents":"Table 1. Yeast Strains
Name | Genotype | Source |
BY4741 (wild-type) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Horizon Discovery |
ilm1Δ | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ilm1::URA3 | This study |
Table 2. PCR Primers
Knockout of ILM1—Amplification of URA3 | |
Forward Primer: 5’ACAATTTACAACATAATAGAGTATCGCATTCAGCAAAAGTAAAGAATAAATTCTAAGAAAATGTCGAAAGCTACATATAAGG-3’ | |
Reverse Primer: 5’ATACATACACAGGTATCTACTATAAGAATAAAGGAAAGAAAAAATAAACGATTAAACATTTTAGTTTTGCTGGCCGCATC-3’ | |
Knockout of ILM1—Confirmation of ILM1 disruption with URA3 | |
Forward Primer 5’-GTTCAATAGCCTTTGTATTGCG-3’ | Reverse Primer 5’-AAACCGCTAACAATACCTGGG-3’ |
The function of the yeast gene ILM1 has not been well-characterized, but it may play a role in mitochondrial DNA maintenance (Entian et al., 1999) and cell wall integrity (Lockshon et al., 2007). The Ilm1p likely localizes to the endoplasmic reticulum (Huh et al., 2003) and only contains one functional protein domain, named Increased loss of mitochondrial DNA protein 1 (Engel et al., 2025). Based on analysis of ILM1 interacting genes using GeneMania and SPELL (Hibbs et al., 2007; Warde-Farley et al., 2010), a potential function for ILM1 in protein trafficking was identified. For example, GO terms related to endoplasmic reticulum-to-Golgi transport were enriched in the SPELL analysis of ILM1, and ILM1 interacts with genes involved in these processes, such as ERP2 and GET1.
To learn more about the potential function of ILM1 in yeast, a knock-out strain (ilm1Δ) was made using a PCR-based strategy that replaced ILM1 with a functional copy of URA3 in the BY4741 background strain of S. cerevisiae. Next, wild-type and ilm1Δ strains were grown using spot assays to test a variety of environmental stress conditions. Both wild-type and ilm1Δ produced the expected number of colonies on YPD media, but the ilm1Δ strain grew more slowly, taking longer to produce large colonies (Figure 1A). This finding is consistent with reduced colony size observed for tetrads produced by ilm1/ILM1 diploids (Entian et al., 1999). When incubated on media containing 5% ethanol or 100mM CaCl2, growth of ilm1Δ was like that of wild-type (Figure 1B). Also, ilm1Δ grew similarly to wild-type on YPG media (Figure 1B), suggesting that ILM1 is not required for mitochondrial function during respiratory growth and consistent with previous work (Entian et al., 1999). However, on YPD containing 10mM caffeine, the growth of the ilm1Δ strain was markedly inhibited compared to wild-type (Figure 1A). The ilm1Δ strain exhibited a hypersensitivity to caffeine.
Caffeine treatment has pleiotropic effects in yeast, including a disruption of the cell wall integrity (CWI) pathway and an inhibition of the TOR signaling pathway that regulates cell growth in response to environmental cues (Ruta & Farcasanu, 2020). Since ilm1Δ exhibited slowed growth on YPD and hypersensitivity to caffeine, its budding profile was investigated using morphological analysis. During log-phase growth in YPD, ilm1Δ accumulated more unbudded cells than wild-type (Figure 1C, D). The average percentage of unbudded cells increased from 53% in wild-type to 74% in ilm1Δ (p=0.005; t-test comparing results from three independent trials). This result shows that ilm1Δ cells appear to have a mild but measurable G1 delay, consistent with their slower growth observed on YPD media. Loss of ILM1 alone delays G1 progression and this is exacerbated in the presence of caffeine. Our findings suggest that the marked caffeine sensitivity of ilm1Δ cells may be due to its inhibition of TORC1.
The TORC1-Sch9 signaling pathway has been shown to regulate lifespan in S. cerevisiae and entry into S phase (Deprez et al., 2018; Moreno-Torres et al., 2015). In response to nutrient availability, TORC1 signaling promotes events that support cell division, including ribosome biogenesis and translation initiation (Wei & Zheng, 2011). Our findings show that loss of ILM1 delays G1 progression and sensitizes cells to TORC1 inhibition by caffeine. Since ILM1 is required for robust passage through G1/S, it may be acting in parallel with known TORC1 effectors to support efficient growth-dependent initiation of S phase.
","references":[{"reference":"Deprez MA, Eskes E, Winderickx J, Wilms T. 2018. The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 18 114.","pubmedId":"29788208","doi":"10.1093/femsyr/foy048"},{"reference":"Engel SR, Aleksander S, Nash RS, Wong ED, Weng S, Miyasato SR, Sherlock G, Cherry JM. 2025. Saccharomyces Genome Database: advances in genome annotation, expanded biochemical pathways, and other key enhancements. Genetics. 229 104.","pubmedId":"39530598","doi":"10.1093/genetics/iyae185"},{"reference":"Entian KD, Schuster T, Hegemann JH, Becher D, Feldmann H, Guldener U, et al., Et Al. 1999. Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol Gen Genet. 262: 683-702. 109.","pubmedId":"10628851","doi":"10.1007/pl00013817"},{"reference":"Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2: 31-4. 116.","pubmedId":"17401334","doi":"10.1038/nprot.2007.13"},{"reference":"Hibbs MA, Hess DC, Myers CL, Huttenhower C, Li K, Troyanskaya OG. 2007. Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 23: 2692-9. 106.","pubmedId":"17724061","doi":"10.1093/bioinformatics/btm403"},{"reference":"Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O Shea EK. 2003. Global analysis of protein localization in budding yeast. Nature. 425: 686-91. 108.","pubmedId":"14562095","doi":"10.1038/nature02026"},{"reference":"Lockshon D, Surface LE, Kerr EO, Kaeberlein M, Kennedy BK. 2007. The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function. Genetics. 175: 77-91. 107.","pubmedId":"17151231","doi":"10.1534/genetics.106.064428"},{"reference":"Looke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325-8. 111.","pubmedId":"21548894","doi":"10.2144/000113672"},{"reference":"Miller M, Tobin T, Aiello DP, Hanson P, Strome E, Johnston SD, et al., Keeney JB. 2024. CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence. J Microbiol Biol Educ. 25: e0016523. 110.","pubmedId":"38661403","doi":"10.1128/jmbe.00165-23"},{"reference":"Moreno Torres M, Jaquenoud M, De Virgilio C. 2015. TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway. Nat Commun. 6: 8256. 115.","pubmedId":"26356805","doi":"10.1038/ncomms9256"},{"reference":"Ruta LL, Farcasanu IC. 2020. Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell. Nutrients. 12 112.","pubmedId":"32823708","doi":"10.3390/nu12082440"},{"reference":"Warde Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al., Morris Q. 2010. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38: W214-20. 105.","pubmedId":"20576703","doi":"10.1093/nar/gkq537"},{"reference":"Wei Y, Zheng XF. 2011. Nutritional control of cell growth via TOR signaling in budding yeast. Methods Mol Biol. 759: 307-19. 113.","pubmedId":"21863495","doi":"10.1007/978-1-61779-173-4_18"}],"title":"Yeast Gene ILM1 Influences Passage Through G1/S.
","reviews":[{"reviewer":{"displayName":"Jill Keeney"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"2c93d12e-5116-4deb-81cd-8caf11ad2b9a","decision":"accept","abstract":"In this study, ILM1, a gene of unknown function, was investigated in the yeast Saccharomyces cerevisiae. We investigated the sensitivity of an ILM1 deletion strain to a variety of stress conditions and compared the budding indices of wild-type and ilm1Δ strains. Our results showed that deletion of ILM1 increases sensitivity to caffeine and leads to a decrease in cell budding. These findings are consistent with a new role for ILM1 in regulating passage through the G1/S transition.
","acknowledgements":"The authors thank Heatherlynn Barrett, Indiana University East laboratory manager, for her help throughout the project, the Yeast ORFan Gene Project (www.yeastorfanproject.com) for protocols, resources, and inspiration, and the Saccharomyces Genome Database which is essential to work on yeast genes.
","authors":[{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["dataCuration","formalAnalysis","investigation","visualization","writing_reviewEditing"],"email":"dewittda@iu.edu","firstName":"Drew ","lastName":"Dewitt","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"aamyx@iu.edu","firstName":"Alyssa ","lastName":"Amyx","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"kmholder@iu.edu","firstName":"Kaitlynn ","lastName":"Holderman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"carmmaxw@iu.edu","firstName":"Carmen","lastName":"Maxwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"idball@iu.edu","firstName":"Ian ","lastName":"Ball","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dykbenni@iu.edu","firstName":"Dylan","lastName":"Bennington","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rijterry@iu.edu","firstName":"Rilee","lastName":"Terry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dafruizg@iu.edu","firstName":"Dafne","lastName":"Ruiz Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rbbranst@iu.edu","firstName":"Rudy ","lastName":"Branstetter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rowboyd@iu.edu","firstName":"Wesley","lastName":"Boyd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East"],"departments":["Department of Natural Science"],"credit":["conceptualization","formalAnalysis","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"jillschw@iu.edu","firstName":"Jill Kuglin","lastName":"Schweitzer","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded by the School of Natural Science and Mathematics at Indiana University East and by an Affinity Grant from the Women’s Philanthropy Leadership Council (IU Foundation).
","image":{"url":"https://portal.micropublication.org/uploads/efddbabccc88f7876bdac48705ee832a.jpg"},"imageCaption":"A, Tenfold serial dilution spot assays of paired wild-type and ilm1Δ, spotted on YPD media and 10mM caffeine incubated at 30°C for 2 days. Images show that ilm1Δ exhibit sensitivity to caffeine as compared to wild type strain. B, Independent spot assays of wild-type and ilm1Δ strains on YPG or YPD with 100mM CaCl2 or 5% ethanol. Cells were incubated at 30°C for 2-3 days. The growth of wild-type (top three rows) and ilm1Δ (bottom three rows) was similar under these stress conditions. C, Cell morphology analysis of fixed log-phase cells of both wild-type (left) and ilm1Δ (right). Micrographs show an increase in unbudded cells in the ilm1Δ strain. Scale bar = 1µm. D, Bar graph showing percent unbudded cells of both wild-type and ilm1Δ in log-phase cultures (average of three independent trials; 100 cells of each strain counted per trial). The ilm1Δ strain has a significantly higher percentage of unbudded cells than wild-type. Wild type: 53% ±8.1%; ilm1Δ: 74% ±8.5% (p=0.005).
","imageTitle":"Deletion of ILM1 sensitizes yeast to caffeine and decreases budding
","methods":"The methods for this work were adapted from procedures made available through the Yeast ORFan Project (Miller et al., 2024).
Yeast culture conditions
Yeast cells were grown at 30ºC in prepared YPD medium (1% yeast extract, 2% peptone, 2% dextrose) in liquid cultures or on solid medium with 2% agar. After yeast transformation, cells were incubated on SD-Ura agar plates for 4-5 days to select for successful transformants. Selection media (SD-Ura) contained 0.67% yeast nitrogen base without amino acids and 2% dextrose, supplemented with 20mg/L l-histidine, 20mg/L l-methionine, and 60mg/L l-leucine. Components for yeast media were from Dot Scientific, Inc., Burton, MI.
ILM1 knockout
An ILM1 knockout strain (ilm1Δ) was created in the BY4741 Saccharomyces cerevisiae background using a PCR-based strategy. First, plasmid DNA containing wild-type URA3 (pRS406) was isolated from E. coli using Qiagen mini-prep kit and quantitated using a NanoDrop 2000 spectrophotometer (ThermoScientific). Next, amplification of URA3 was accomplished using a forward primer to recognize twenty bases at the beginning of URA3 with an additional 60 bases of genomic DNA upstream of ILM1 and a reverse primer to recognize twenty bases at the end of URA3 plus an additional 60 bases of genomic DNA downstream of ILM1. See Table 2 for primer sequences. PCR reactions contained 0.5mM each primer, 0.2 mM dNTPs, 1X PCR buffer (GenScript), 0.5ml GenScript Green Taq DNA polymerase, and approximately 10 ng of plasmid template. Finally, the URA3 PCR product was introduced into yeast using yeast transformation (Gietz & Schiestl, 2007). Successful deletion strains were confirmed by isolating DNA from transformant colonies (Lõoke et al., 2011) and amplifying a confirmation PCR product—forward primer annealed to the genome upstream of ILM1 and the reverse primer annealed to URA3. All PCR products were analyzed using 0.8% agarose gel electrophoresis.
Spot assay
Liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1 and then serially diluted 1:10 to make five cell suspensions ranging from 1:100 to 1:104. Each strain was diluted in triplicate per experiment. 2ml of each cell suspension was spotted onto YPD agar plates and YPD containing 10mM caffeine, 100mM CaCl2, or 5% ethanol (Sigma Aldrich). Cell suspensions were also plated on media containing glycerol instead of dextrose (YPG). Plates were imaged after 2 or 3 days of incubation at 30 ºC. Each spot assay was replicated independently three times and representative images are shown.
Morphological Analysis
To prepare log-phase cells, liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1, and then incubated in fresh YPD for 4-5 hours. Cells were washed with phosphate-buffered saline (1X PBS), fixed with 3.7% formaldehyde for 5 min and sonicated for 20s to reduce clumping using a Fisher Scientific FB505 (amplitude—20%; pulse 1s on/1s off). Sonicated cells were mounted on slides and imaged using brightfield microscopy (Nikon Eclipse Ei with Digital Sight 1000 Camera). Micrographs were analyzed using Image J to measure the area of one hundred cells of each strain per experiment and determine the budding state. Morphological analysis was replicated independently three times. The unbudded percentage shown in Figure 1D is an average of three independent trials. A student t-test was used to compare the number of unbudded cells from wild-type and ilm1Δ to determine statistical significance.
","reagents":"Table 1. Yeast Strains
Name | Genotype | Source |
BY4741 (wild-type) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Horizon Discovery |
ilm1Δ | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ilm1::URA3 | This study |
Table 2. PCR Primers
Knockout of ILM1—Amplification of URA3 | |
Forward Primer: 5’ACAATTTACAACATAATAGAGTATCGCATTCAGCAAAAGTAAAGAATAAATTCTAAGAAAATGTCGAAAGCTACATATAAGG-3’ | |
Reverse Primer: 5’ATACATACACAGGTATCTACTATAAGAATAAAGGAAAGAAAAAATAAACGATTAAACATTTTAGTTTTGCTGGCCGCATC-3’ | |
Knockout of ILM1—Confirmation of ILM1 disruption with URA3 | |
Forward Primer 5’-GTTCAATAGCCTTTGTATTGCG-3’ | Reverse Primer 5’-AAACCGCTAACAATACCTGGG-3’ |
The function of the yeast gene ILM1 has not been well-characterized, but it may play a role in mitochondrial DNA maintenance (Entian et al., 1999) and cell wall integrity (Lockshon et al., 2007). The Ilm1p likely localizes to the endoplasmic reticulum (Huh et al., 2003) and only contains one functional protein domain, named Increased loss of mitochondrial DNA protein 1 (Engel et al., 2025). Based on analysis of ILM1 interacting genes using GeneMania and SPELL (Hibbs et al., 2007; Warde-Farley et al., 2010), a potential function for ILM1 in protein trafficking was identified. For example, GO terms related to endoplasmic reticulum-to-Golgi transport were enriched in the SPELL analysis of ILM1, and ILM1 interacts with genes involved in these processes, such as ERP2 and GET1.
To learn more about the potential function of ILM1 in yeast, a knock-out strain (ilm1Δ) was made using a PCR-based strategy that replaced ILM1 with a functional copy of URA3 in the BY4741 background strain of S. cerevisiae. Next, wild-type and ilm1Δ strains were grown using spot assays to test a variety of environmental stress conditions. Both wild-type and ilm1Δ produced the expected number of colonies on YPD media, but the ilm1Δ strain grew more slowly, taking longer to produce large colonies (Figure 1A). This finding is consistent with reduced colony size observed for tetrads produced by ilm1/ILM1 diploids (Entian et al., 1999). When incubated on media containing 5% ethanol or 100mM CaCl2, growth of ilm1Δ was like that of wild-type (Figure 1B). Also, ilm1Δ grew similarly to wild-type on YPG media (Figure 1B), suggesting that ILM1 is not required for mitochondrial function during respiratory growth and consistent with previous work (Entian et al., 1999). However, on YPD containing 10mM caffeine, the growth of the ilm1Δ strain was markedly inhibited compared to wild-type (Figure 1A). The ilm1Δ strain exhibited a hypersensitivity to caffeine.
Caffeine treatment has pleiotropic effects in yeast, including a disruption of the cell wall integrity (CWI) pathway and an inhibition of the TOR signaling pathway that regulates cell growth in response to environmental cues (Ruta & Farcasanu, 2020). Since ilm1Δ exhibited slowed growth on YPD and hypersensitivity to caffeine, its budding profile was investigated using morphological analysis. During log-phase growth in YPD, ilm1Δ accumulated more unbudded cells than wild-type (Figure 1C, D). The average percentage of unbudded cells increased from 53% in wild-type to 74% in ilm1Δ (p=0.005; t-test comparing results from three independent trials). This result shows that ilm1Δ cells appear to have a mild but measurable G1 delay, consistent with their slower growth observed on YPD media. Loss of ILM1 alone delays G1 progression and this is exacerbated in the presence of caffeine. Our findings suggest that the marked caffeine sensitivity of ilm1Δ cells may be due to its inhibition of TORC1.
The TORC1-Sch9 signaling pathway has been shown to regulate lifespan in S. cerevisiae and entry into S phase (Deprez et al., 2018; Moreno-Torres et al., 2015). In response to nutrient availability, TORC1 signaling promotes events that support cell division, including ribosome biogenesis and translation initiation (Wei & Zheng, 2011). Our findings show that loss of ILM1 delays G1 progression and sensitizes cells to TORC1 inhibition by caffeine. Since ILM1 is required for robust passage through G1/S, it may be acting in parallel with known TORC1 effectors to support efficient growth-dependent initiation of S phase.
","references":[{"reference":"Deprez MA, Eskes E, Winderickx J, Wilms T. 2018. The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 18 114.","pubmedId":"29788208","doi":"10.1093/femsyr/foy048"},{"reference":"Engel SR, Aleksander S, Nash RS, Wong ED, Weng S, Miyasato SR, Sherlock G, Cherry JM. 2025. Saccharomyces Genome Database: advances in genome annotation, expanded biochemical pathways, and other key enhancements. Genetics. 229 104.","pubmedId":"39530598","doi":"10.1093/genetics/iyae185"},{"reference":"Entian KD, Schuster T, Hegemann JH, Becher D, Feldmann H, Guldener U, et al., Et Al. 1999. Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol Gen Genet. 262: 683-702. 109.","pubmedId":"10628851","doi":"10.1007/pl00013817"},{"reference":"Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2: 31-4. 116.","pubmedId":"17401334","doi":"10.1038/nprot.2007.13"},{"reference":"Hibbs MA, Hess DC, Myers CL, Huttenhower C, Li K, Troyanskaya OG. 2007. Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 23: 2692-9. 106.","pubmedId":"17724061","doi":"10.1093/bioinformatics/btm403"},{"reference":"Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O Shea EK. 2003. Global analysis of protein localization in budding yeast. Nature. 425: 686-91. 108.","pubmedId":"14562095","doi":"10.1038/nature02026"},{"reference":"Lockshon D, Surface LE, Kerr EO, Kaeberlein M, Kennedy BK. 2007. The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function. Genetics. 175: 77-91. 107.","pubmedId":"17151231","doi":"10.1534/genetics.106.064428"},{"reference":"Looke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325-8. 111.","pubmedId":"21548894","doi":"10.2144/000113672"},{"reference":"Miller M, Tobin T, Aiello DP, Hanson P, Strome E, Johnston SD, et al., Keeney JB. 2024. CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence. J Microbiol Biol Educ. 25: e0016523. 110.","pubmedId":"38661403","doi":"10.1128/jmbe.00165-23"},{"reference":"Moreno Torres M, Jaquenoud M, De Virgilio C. 2015. TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway. Nat Commun. 6: 8256. 115.","pubmedId":"26356805","doi":"10.1038/ncomms9256"},{"reference":"Ruta LL, Farcasanu IC. 2020. Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell. Nutrients. 12 112.","pubmedId":"32823708","doi":"10.3390/nu12082440"},{"reference":"Warde Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al., Morris Q. 2010. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38: W214-20. 105.","pubmedId":"20576703","doi":"10.1093/nar/gkq537"},{"reference":"Wei Y, Zheng XF. 2011. Nutritional control of cell growth via TOR signaling in budding yeast. Methods Mol Biol. 759: 307-19. 113.","pubmedId":"21863495","doi":"10.1007/978-1-61779-173-4_18"}],"title":"Yeast Gene ILM1 Influences Passage Through G1/S.
","reviews":[],"curatorReviews":[]},{"id":"8752eb11-eae2-48ea-a9a4-19a86c39706b","decision":"edit","abstract":"In this study, ILM1, a gene of unknown function, was studied in the yeast Saccharomyces cerevisiae. We investigated the sensitivity of an ILM1 deletion strain to a variety of stress conditions and compared the budding indices of wild-type and ilm1Δ strains. Our results showed that deletion of ILM1 increases sensitivity to caffeine and leads to a decrease in cell budding. These findings are consistent with a new role for ILM1 in regulating passage through the G1/S transition.
","acknowledgements":"The authors thank Heatherlynn Barrett, Indiana University East laboratory manager, for her help throughout the project, the Yeast ORFan Gene Project (www.yeastorfanproject.com) for protocols, resources, and inspiration, and the Saccharomyces Genome Database which is essential to work on yeast genes.
","authors":[{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["dataCuration","formalAnalysis","investigation","visualization","writing_reviewEditing"],"email":"dewittda@iu.edu","firstName":"Drew ","lastName":"Dewitt","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"aamyx@iu.edu","firstName":"Alyssa ","lastName":"Amyx","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"kmholder@iu.edu","firstName":"Kaitlynn ","lastName":"Holderman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"carmmaxw@iu.edu","firstName":"Carmen","lastName":"Maxwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"idball@iu.edu","firstName":"Ian ","lastName":"Ball","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dykbenni@iu.edu","firstName":"Dylan","lastName":"Bennington","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rijterry@iu.edu","firstName":"Rilee","lastName":"Terry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dafruizg@iu.edu","firstName":"Dafne","lastName":"Ruiz Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rbbranst@iu.edu","firstName":"Rudy ","lastName":"Branstetter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rowboyd@iu.edu","firstName":"Wesley","lastName":"Boyd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["conceptualization","formalAnalysis","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"jillschw@iu.edu","firstName":"Jill Kuglin","lastName":"Schweitzer","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded by the School of Natural Science and Mathematics at Indiana University East and by an Affinity Grant from the Women’s Philanthropy Leadership Council (IU Foundation).
","image":{"url":"https://portal.micropublication.org/uploads/efddbabccc88f7876bdac48705ee832a.jpg"},"imageCaption":"A, Tenfold serial dilution spot assays of paired wild-type and ilm1Δ, spotted on YPD media and 10mM caffeine incubated at 30°C for 2 days. Images show that ilm1Δ exhibit sensitivity to caffeine as compared to wild type strain. B, Independent spot assays of wild-type and ilm1Δ strains on YPG or YPD with 100mM CaCl2 or 5% ethanol. Cells were incubated at 30°C for 2-3 days. The growth of wild-type (top three rows) and ilm1Δ (bottom three rows) was similar under these stress conditions. C, Cell morphology analysis of fixed log-phase cells of both wild-type (left) and ilm1Δ (right). Micrographs show an increase in unbudded cells in the ilm1Δ strain. Scale bar = 1µm. D, Bar graph showing percent unbudded cells of both wild-type and ilm1Δ in log-phase cultures (average of three independent trials; 100 cells of each strain counted per trial). The ilm1Δ strain has a significantly higher percentage of unbudded cells than wild-type. Wild type: 53% ±8.1%; ilm1Δ: 74% ±8.5% (p=0.005).
","imageTitle":"Deletion of ILM1 sensitizes yeast to caffeine and decreases budding
","methods":"The methods for this work were adapted from procedures made available through the Yeast ORFan Project (Miller et al., 2024).
Yeast culture conditions
Yeast cells were grown at 30ºC in prepared YPD medium (1% yeast extract, 2% peptone, 2% dextrose) in liquid cultures or on solid medium with 2% agar. After yeast transformation, cells were incubated on SD-Ura agar plates for 4-5 days to select for successful transformants. Selection media (SD-Ura) contained 0.67% yeast nitrogen base without amino acids and 2% dextrose, supplemented with 20mg/L l-histidine, 20mg/L l-methionine, and 60mg/L l-leucine. Components for yeast media were from Dot Scientific, Inc., Burton, MI.
ILM1 knockout
An ILM1 knockout strain (ilm1Δ) was created in the BY4741 Saccharomyces cerevisiae background using a PCR-based strategy. First, plasmid DNA containing wild-type URA3 (pRS406) was isolated from E. coli using Qiagen mini-prep kit and quantitated using a NanoDrop 2000 spectrophotometer (ThermoScientific). Next, amplification of URA3 was accomplished using a forward primer to recognize twenty bases at the beginning of URA3 with an additional 60 bases of genomic DNA upstream of ILM1 and a reverse primer to recognize twenty bases at the end of URA3 plus an additional 60 bases of genomic DNA downstream of ILM1. See Table 2 for primer sequences. PCR reactions contained 0.5mM each primer, 0.2 mM dNTPs, 1X PCR buffer (GenScript), 0.5ml GenScript Green Taq DNA polymerase, and approximately 10 ng of plasmid template. Finally, the URA3 PCR product was introduced into yeast using yeast transformation (Gietz & Schiestl, 2007). Successful deletion strains were confirmed by isolating DNA from transformant colonies (Lõoke et al., 2011) and amplifying a confirmation PCR product—forward primer annealed to the genome upstream of ILM1 and the reverse primer annealed to URA3. All PCR products were analyzed using 0.8% agarose gel electrophoresis.
Spot assay
Liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1 and then serially diluted 1:10 to make five cell suspensions ranging from 1:100 to 1:104. Each strain was diluted in triplicate per experiment. 2μl of each cell suspension was spotted onto YPD agar plates and YPD containing 10mM caffeine, 100mM CaCl2, or 5% ethanol (Sigma Aldrich). Cell suspensions were also plated on media containing glycerol instead of dextrose (YPG). Plates were imaged after 2 or 3 days of incubation at 30 ºC. Each spot assay was replicated independently three times and representative images are shown.
Morphological Analysis
To prepare log-phase cells, liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1, and then incubated in fresh YPD for 4-5 hours. Cells were washed with phosphate-buffered saline (1X PBS), fixed with 3.7% formaldehyde for 5 min and sonicated for 20s to reduce clumping using a Fisher Scientific FB505 (amplitude—20%; pulse 1s on/1s off). Sonicated cells were mounted on slides and imaged using brightfield microscopy (Nikon Eclipse Ei with Digital Sight 1000 Camera). Micrographs were analyzed using Image J to measure the area of one hundred cells of each strain per experiment and determine the budding state. Morphological analysis was replicated independently three times. The unbudded percentage shown in Figure 1D is an average of three independent trials. A student t-test was used to compare the number of unbudded cells from wild-type and ilm1Δ to determine statistical significance.
","reagents":"Table 1. Yeast Strains
Name | Genotype | Source |
BY4741 (wild-type) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Horizon Discovery |
ilm1Δ | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ilm1::URA3 | This study |
Table 2. PCR Primers
Knockout of ILM1—Amplification of URA3 | |
Forward Primer: 5’ACAATTTACAACATAATAGAGTATCGCATTCAGCAAAAGTAAAGAATAAATTCTAAGAAAATGTCGAAAGCTACATATAAGG-3’ | |
Reverse Primer: 5’ATACATACACAGGTATCTACTATAAGAATAAAGGAAAGAAAAAATAAACGATTAAACATTTTAGTTTTGCTGGCCGCATC-3’ | |
Knockout of ILM1—Confirmation of ILM1 disruption with URA3 | |
Forward Primer 5’-GTTCAATAGCCTTTGTATTGCG-3’ | Reverse Primer 5’-AAACCGCTAACAATACCTGGG-3’ |
The function of the yeast gene ILM1 has not been well-characterized, but it may play a role in mitochondrial DNA maintenance (Entian et al., 1999) and cell wall integrity (Lockshon et al., 2007). The Ilm1p likely localizes to the endoplasmic reticulum (Huh et al., 2003) and only contains one functional protein domain, named Increased loss of mitochondrial DNA protein 1 (Engel et al., 2025). Based on analysis of ILM1 interacting genes using GeneMania and SPELL (Hibbs et al., 2007; Warde-Farley et al., 2010), a potential function for ILM1 in protein trafficking was identified. For example, GO terms related to endoplasmic reticulum-to-Golgi transport were enriched in the SPELL analysis of ILM1, and ILM1 interacts with genes involved in these processes, such as ERP2 and GET1.
To learn more about the potential function of ILM1 in yeast, a knock-out strain (ilm1Δ) was made using a PCR-based strategy that replaced ILM1 with a functional copy of URA3 in the BY4741 background strain of S. cerevisiae. Next, wild-type and ilm1Δ strains were grown using spot assays to test a variety of environmental stress conditions. Both wild-type and ilm1Δ produced the expected number of colonies on YPD media, but the ilm1Δ strain grew more slowly, taking longer to produce large colonies (Figure 1A). This finding is consistent with reduced colony size observed for tetrads produced by ilm1/ILM1 diploids (Entian et al., 1999). When incubated on media containing 5% ethanol or 100mM CaCl2, growth of ilm1Δ was like that of wild-type (Figure 1B). Also, ilm1Δ grew similarly to wild-type on YPG media (Figure 1B), suggesting that ILM1 is not required for mitochondrial function during respiratory growth and consistent with previous work (Entian et al., 1999). However, on YPD containing 10mM caffeine, the growth of the ilm1Δ strain was markedly inhibited compared to wild-type (Figure 1A). The ilm1Δ strain exhibited a hypersensitivity to caffeine.
Caffeine treatment has pleiotropic effects in yeast, including a disruption of the cell wall integrity (CWI) pathway and an inhibition of the TOR signaling pathway that regulates cell growth in response to environmental cues (Ruta & Farcasanu, 2020). Since ilm1Δ exhibited slowed growth on YPD and hypersensitivity to caffeine, its budding profile was investigated using morphological analysis. During log-phase growth in YPD, ilm1Δ accumulated more unbudded cells than wild-type (Figure 1C, D). The average percentage of unbudded cells increased from 53% in wild-type to 74% in ilm1Δ (p=0.005; t-test comparing results from three independent trials). This result shows that ilm1Δ cells appear to have a mild but measurable G1 delay, consistent with their slower growth observed on YPD media. Loss of ILM1 alone delays G1 progression and this is exacerbated in the presence of caffeine. Our findings suggest that the marked caffeine sensitivity of ilm1Δ cells may be due to its inhibition of TORC1.
The TORC1-Sch9 signaling pathway has been shown to regulate lifespan in S. cerevisiae and entry into S phase (Deprez et al., 2018; Moreno-Torres et al., 2015). In response to nutrient availability, TORC1 signaling promotes events that support cell division, including ribosome biogenesis and translation initiation (Wei & Zheng, 2011). Our findings show that loss of ILM1 delays G1 progression and sensitizes cells to TORC1 inhibition by caffeine. Since ILM1 is required for robust passage through G1/S, it may be acting in parallel with known TORC1 effectors to support efficient growth-dependent initiation of S phase.
","references":[{"reference":"Deprez MA, Eskes E, Winderickx J, Wilms T. 2018. The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 18 114.","pubmedId":"29788208","doi":"10.1093/femsyr/foy048"},{"reference":"Engel SR, Aleksander S, Nash RS, Wong ED, Weng S, Miyasato SR, Sherlock G, Cherry JM. 2025. Saccharomyces Genome Database: advances in genome annotation, expanded biochemical pathways, and other key enhancements. Genetics. 229 104.","pubmedId":"39530598","doi":"10.1093/genetics/iyae185"},{"reference":"Entian KD, Schuster T, Hegemann JH, Becher D, Feldmann H, Guldener U, et al., Et Al. 1999. Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol Gen Genet. 262: 683-702. 109.","pubmedId":"10628851","doi":"10.1007/pl00013817"},{"reference":"Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2: 31-4. 116.","pubmedId":"17401334","doi":"10.1038/nprot.2007.13"},{"reference":"Hibbs MA, Hess DC, Myers CL, Huttenhower C, Li K, Troyanskaya OG. 2007. Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 23: 2692-9. 106.","pubmedId":"17724061","doi":"10.1093/bioinformatics/btm403"},{"reference":"Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O Shea EK. 2003. Global analysis of protein localization in budding yeast. Nature. 425: 686-91. 108.","pubmedId":"14562095","doi":"10.1038/nature02026"},{"reference":"Lockshon D, Surface LE, Kerr EO, Kaeberlein M, Kennedy BK. 2007. The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function. Genetics. 175: 77-91. 107.","pubmedId":"17151231","doi":"10.1534/genetics.106.064428"},{"reference":"Looke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325-8. 111.","pubmedId":"21548894","doi":"10.2144/000113672"},{"reference":"Miller M, Tobin T, Aiello DP, Hanson P, Strome E, Johnston SD, et al., Keeney JB. 2024. CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence. J Microbiol Biol Educ. 25: e0016523. 110.","pubmedId":"38661403","doi":"10.1128/jmbe.00165-23"},{"reference":"Moreno Torres M, Jaquenoud M, De Virgilio C. 2015. TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway. Nat Commun. 6: 8256. 115.","pubmedId":"26356805","doi":"10.1038/ncomms9256"},{"reference":"Ruta LL, Farcasanu IC. 2020. Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell. Nutrients. 12 112.","pubmedId":"32823708","doi":"10.3390/nu12082440"},{"reference":"Warde Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al., Morris Q. 2010. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38: W214-20. 105.","pubmedId":"20576703","doi":"10.1093/nar/gkq537"},{"reference":"Wei Y, Zheng XF. 2011. Nutritional control of cell growth via TOR signaling in budding yeast. Methods Mol Biol. 759: 307-19. 113.","pubmedId":"21863495","doi":"10.1007/978-1-61779-173-4_18"}],"title":"Yeast Gene ILM1 Influences Passage Through G1/S
","reviews":[],"curatorReviews":[]},{"id":"ebaf35cd-3c4f-46c6-bbf5-9a17308a2be4","decision":"publish","abstract":"In this study, ILM1, a gene of unknown function, was studied in the yeast Saccharomyces cerevisiae. We investigated the sensitivity of an ILM1 deletion strain to a variety of stress conditions and compared the budding indices of wild-type and ilm1Δ strains. Our results showed that deletion of ILM1 increases sensitivity to caffeine and leads to a decrease in cell budding. These findings are consistent with a new role for ILM1 in regulating passage through the G1/S transition.
","acknowledgements":"The authors thank Heatherlynn Barrett, Indiana University East laboratory manager, for her help throughout the project, the Yeast ORFan Gene Project (www.yeastorfanproject.com) for protocols, resources, and inspiration, and the Saccharomyces Genome Database which is essential to work on yeast genes.
","authors":[{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["dataCuration","formalAnalysis","investigation","visualization","writing_reviewEditing"],"email":"dewittda@iu.edu","firstName":"Drew ","lastName":"Dewitt","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"aamyx@iu.edu","firstName":"Alyssa ","lastName":"Amyx","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"kmholder@iu.edu","firstName":"Kaitlynn ","lastName":"Holderman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"carmmaxw@iu.edu","firstName":"Carmen","lastName":"Maxwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"idball@iu.edu","firstName":"Ian ","lastName":"Ball","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dykbenni@iu.edu","firstName":"Dylan","lastName":"Bennington","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rijterry@iu.edu","firstName":"Rilee","lastName":"Terry","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"dafruizg@iu.edu","firstName":"Dafne","lastName":"Ruiz Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rbbranst@iu.edu","firstName":"Rudy ","lastName":"Branstetter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["investigation"],"email":"rowboyd@iu.edu","firstName":"Wesley","lastName":"Boyd","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Indiana University East, Richmond, IN"],"departments":["Department of Natural Science"],"credit":["conceptualization","formalAnalysis","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"jillschw@iu.edu","firstName":"Jill Kuglin","lastName":"Schweitzer","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This project was funded by the School of Natural Science and Mathematics at Indiana University East and by an Affinity Grant from the Women’s Philanthropy Leadership Council (IU Foundation).
","image":{"url":"https://portal.micropublication.org/uploads/efddbabccc88f7876bdac48705ee832a.jpg"},"imageCaption":"A, Tenfold serial dilution spot assays of paired wild-type and ilm1Δ, spotted on YPD media and 10mM caffeine incubated at 30°C for 2 days. Images show that ilm1Δ exhibit sensitivity to caffeine as compared to wild type strain. B, Independent spot assays of wild-type and ilm1Δ strains on YPG or YPD with 100mM CaCl2 or 5% ethanol. Cells were incubated at 30°C for 2-3 days. The growth of wild-type (top three rows) and ilm1Δ (bottom three rows) was similar under these stress conditions. C, Cell morphology analysis of fixed log-phase cells of both wild-type (left) and ilm1Δ (right). Micrographs show an increase in unbudded cells in the ilm1Δ strain. Scale bar = 1µm. D, Bar graph showing percent unbudded cells of both wild-type and ilm1Δ in log-phase cultures (average of three independent trials; 100 cells of each strain counted per trial). The ilm1Δ strain has a significantly higher percentage of unbudded cells than wild-type. Wild type: 53% ±8.1%; ilm1Δ: 74% ±8.5% (p=0.005).
","imageTitle":"Deletion of ILM1 sensitizes yeast to caffeine and decreases budding
","methods":"The methods for this work were adapted from procedures made available through the Yeast ORFan Project (Miller et al., 2024).
Yeast culture conditions
Yeast cells were grown at 30ºC in prepared YPD medium (1% yeast extract, 2% peptone, 2% dextrose) in liquid cultures or on solid medium with 2% agar. After yeast transformation, cells were incubated on SD-Ura agar plates for 4-5 days to select for successful transformants. Selection media (SD-Ura) contained 0.67% yeast nitrogen base without amino acids and 2% dextrose, supplemented with 20mg/L l-histidine, 20mg/L l-methionine, and 60mg/L l-leucine. Components for yeast media were from Dot Scientific, Inc., Burton, MI.
ILM1 knockout
An ILM1 knockout strain (ilm1Δ) was created in the BY4741 Saccharomyces cerevisiae background using a PCR-based strategy. First, plasmid DNA containing wild-type URA3 (pRS406) was isolated from E. coli using Qiagen mini-prep kit and quantitated using a NanoDrop 2000 spectrophotometer (ThermoScientific). Next, amplification of URA3 was accomplished using a forward primer to recognize twenty bases at the beginning of URA3 with an additional 60 bases of genomic DNA upstream of ILM1 and a reverse primer to recognize twenty bases at the end of URA3 plus an additional 60 bases of genomic DNA downstream of ILM1. See Table 2 for primer sequences. PCR reactions contained 0.5mM each primer, 0.2 mM dNTPs, 1X PCR buffer (GenScript), 0.5ml GenScript Green Taq DNA polymerase, and approximately 10 ng of plasmid template. Finally, the URA3 PCR product was introduced into yeast using yeast transformation (Gietz & Schiestl, 2007). Successful deletion strains were confirmed by isolating DNA from transformant colonies (Lõoke et al., 2011) and amplifying a confirmation PCR product—forward primer annealed to the genome upstream of ILM1 and the reverse primer annealed to URA3. All PCR products were analyzed using 0.8% agarose gel electrophoresis.
Spot assay
Liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1 and then serially diluted 1:10 to make five cell suspensions ranging from 1:100 to 1:104. Each strain was diluted in triplicate per experiment. 2μl of each cell suspension was spotted onto YPD agar plates and YPD containing 10mM caffeine, 100mM CaCl2, or 5% ethanol (Sigma Aldrich). Cell suspensions were also plated on media containing glycerol instead of dextrose (YPG). Plates were imaged after 2 or 3 days of incubation at 30 ºC. Each spot assay was replicated independently three times and representative images are shown.
Morphological Analysis
To prepare log-phase cells, liquid cultures of wild type and deletion strains were grown overnight in YPD with shaking, diluted to OD600 of 0.1, and then incubated in fresh YPD for 4-5 hours. Cells were washed with phosphate-buffered saline (1X PBS), fixed with 3.7% formaldehyde for 5 min and sonicated for 20s to reduce clumping using a Fisher Scientific FB505 (amplitude—20%; pulse 1s on/1s off). Sonicated cells were mounted on slides and imaged using brightfield microscopy (Nikon Eclipse Ei with Digital Sight 1000 Camera). Micrographs were analyzed using Image J to measure the area of one hundred cells of each strain per experiment and determine the budding state. Morphological analysis was replicated independently three times. The unbudded percentage shown in Figure 1D is an average of three independent trials. A student t-test was used to compare the number of unbudded cells from wild-type and ilm1Δ to determine statistical significance.
","reagents":"Table 1. Yeast Strains
Name | Genotype | Source |
BY4741 (wild-type) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Horizon Discovery |
ilm1Δ | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ilm1::URA3 | This study |
Table 2. PCR Primers
Knockout of ILM1—Amplification of URA3 | |
Forward Primer: 5’ACAATTTACAACATAATAGAGTATCGCATTCAGCAAAAGTAAAGAATAAATTCTAAGAAAATGTCGAAAGCTACATATAAGG-3’ | |
Reverse Primer: 5’ATACATACACAGGTATCTACTATAAGAATAAAGGAAAGAAAAAATAAACGATTAAACATTTTAGTTTTGCTGGCCGCATC-3’ | |
Knockout of ILM1—Confirmation of ILM1 disruption with URA3 | |
Forward Primer 5’-GTTCAATAGCCTTTGTATTGCG-3’ | Reverse Primer 5’-AAACCGCTAACAATACCTGGG-3’ |
The function of the yeast gene ILM1 has not been well-characterized, but it may play a role in mitochondrial DNA maintenance (Entian et al., 1999) and cell wall integrity (Lockshon et al., 2007). The Ilm1p likely localizes to the endoplasmic reticulum (Huh et al., 2003) and only contains one functional protein domain, named Increased loss of mitochondrial DNA protein 1 (Engel et al., 2025). Based on analysis of ILM1 interacting genes using GeneMania and SPELL (Hibbs et al., 2007; Warde-Farley et al., 2010), a potential function for ILM1 in protein trafficking was identified. For example, GO terms related to endoplasmic reticulum-to-Golgi transport were enriched in the SPELL analysis of ILM1, and ILM1 interacts with genes involved in these processes, such as ERP2 and GET1.
To learn more about the potential function of ILM1 in yeast, a knock-out strain (ilm1Δ) was made using a PCR-based strategy that replaced ILM1 with a functional copy of URA3 in the BY4741 background strain of S. cerevisiae. Next, wild-type and ilm1Δ strains were grown using spot assays to test a variety of environmental stress conditions. Both wild-type and ilm1Δ produced the expected number of colonies on YPD media, but the ilm1Δ strain grew more slowly, taking longer to produce large colonies (Figure 1A). This finding is consistent with reduced colony size observed for tetrads produced by ilm1/ILM1 diploids (Entian et al., 1999). When incubated on media containing 5% ethanol or 100mM CaCl2, growth of ilm1Δ was like that of wild-type (Figure 1B). Also, ilm1Δ grew similarly to wild-type on YPG media (Figure 1B), suggesting that ILM1 is not required for mitochondrial function during respiratory growth and consistent with previous work (Entian et al., 1999). However, on YPD containing 10mM caffeine, the growth of the ilm1Δ strain was markedly inhibited compared to wild-type (Figure 1A). The ilm1Δ strain exhibited a hypersensitivity to caffeine.
Caffeine treatment has pleiotropic effects in yeast, including a disruption of the cell wall integrity (CWI) pathway and an inhibition of the TOR signaling pathway that regulates cell growth in response to environmental cues (Ruta & Farcasanu, 2020). Since ilm1Δ exhibited slowed growth on YPD and hypersensitivity to caffeine, its budding profile was investigated using morphological analysis. During log-phase growth in YPD, ilm1Δ accumulated more unbudded cells than wild-type (Figure 1C, D). The average percentage of unbudded cells increased from 53% in wild-type to 74% in ilm1Δ (p=0.005; t-test comparing results from three independent trials). This result shows that ilm1Δ cells appear to have a mild but measurable G1 delay, consistent with their slower growth observed on YPD media. Loss of ILM1 alone delays G1 progression and this is exacerbated in the presence of caffeine. Our findings suggest that the marked caffeine sensitivity of ilm1Δ cells may be due to its inhibition of TORC1.
The TORC1-Sch9 signaling pathway has been shown to regulate lifespan in S. cerevisiae and entry into S phase (Deprez et al., 2018; Moreno-Torres et al., 2015). In response to nutrient availability, TORC1 signaling promotes events that support cell division, including ribosome biogenesis and translation initiation (Wei & Zheng, 2011). Our findings show that loss of ILM1 delays G1 progression and sensitizes cells to TORC1 inhibition by caffeine. Since ILM1 is required for robust passage through G1/S, it may be acting in parallel with known TORC1 effectors to support efficient growth-dependent initiation of S phase.
","references":[{"reference":"Deprez MA, Eskes E, Winderickx J, Wilms T. 2018. The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 18 114.","pubmedId":"29788208","doi":"10.1093/femsyr/foy048"},{"reference":"Engel SR, Aleksander S, Nash RS, Wong ED, Weng S, Miyasato SR, Sherlock G, Cherry JM. 2025. Saccharomyces Genome Database: advances in genome annotation, expanded biochemical pathways, and other key enhancements. Genetics. 229 104.","pubmedId":"39530598","doi":"10.1093/genetics/iyae185"},{"reference":"Entian KD, Schuster T, Hegemann JH, Becher D, Feldmann H, Guldener U, et al., Et Al. 1999. Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol Gen Genet. 262: 683-702. 109.","pubmedId":"10628851","doi":"10.1007/pl00013817"},{"reference":"Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2: 31-4. 116.","pubmedId":"17401334","doi":"10.1038/nprot.2007.13"},{"reference":"Hibbs MA, Hess DC, Myers CL, Huttenhower C, Li K, Troyanskaya OG. 2007. Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 23: 2692-9. 106.","pubmedId":"17724061","doi":"10.1093/bioinformatics/btm403"},{"reference":"Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O Shea EK. 2003. Global analysis of protein localization in budding yeast. Nature. 425: 686-91. 108.","pubmedId":"14562095","doi":"10.1038/nature02026"},{"reference":"Lockshon D, Surface LE, Kerr EO, Kaeberlein M, Kennedy BK. 2007. The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function. Genetics. 175: 77-91. 107.","pubmedId":"17151231","doi":"10.1534/genetics.106.064428"},{"reference":"Looke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325-8. 111.","pubmedId":"21548894","doi":"10.2144/000113672"},{"reference":"Miller M, Tobin T, Aiello DP, Hanson P, Strome E, Johnston SD, et al., Keeney JB. 2024. CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence. J Microbiol Biol Educ. 25: e0016523. 110.","pubmedId":"38661403","doi":"10.1128/jmbe.00165-23"},{"reference":"Moreno Torres M, Jaquenoud M, De Virgilio C. 2015. TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway. Nat Commun. 6: 8256. 115.","pubmedId":"26356805","doi":"10.1038/ncomms9256"},{"reference":"Ruta LL, Farcasanu IC. 2020. Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell. Nutrients. 12 112.","pubmedId":"32823708","doi":"10.3390/nu12082440"},{"reference":"Warde Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al., Morris Q. 2010. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 38: W214-20. 105.","pubmedId":"20576703","doi":"10.1093/nar/gkq537"},{"reference":"Wei Y, Zheng XF. 2011. Nutritional control of cell growth via TOR signaling in budding yeast. Methods Mol Biol. 759: 307-19. 113.","pubmedId":"21863495","doi":"10.1007/978-1-61779-173-4_18"}],"title":"Yeast Gene ILM1 Influences Passage Through G1/S
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