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 CaCl ₂ , 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.

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 OD ₆₀₀ of 0.1 and then serially diluted 1:10 to make five cell suspensions ranging from 1:10 ⁰ to 1:10 ⁴ . 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 CaCl ₂ , 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 OD ₆₀₀ 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.

Table 1. Yeast Strains

Table 2. PCR Primers