The pH-sensing Rim101 pathway regulates cell size in budding yeast

Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry–based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wildtype cells. Furthermore, the size of wildtype cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wildtype cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101-induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101–V-ATPase axis under physiological conditions as well as in response to alkaline stresses.

Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genomewide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wildtype cells. Furthermore, the size of wildtype cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wildtype cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101-V-ATPase axis under physiological conditions as well as in response to alkaline stresses.
Although cell size varies among cell types, it is tightly controlled in each cell type (1). Many cellular processes, including the cell cycle (2), transcription (3,4), translation (5), and metabolism (3), are size dependent. Changes in cell size are observed in senescence and pathologies such as cancers (6).
Various intracellular and extracellular factors, including cell cycle regulators and nutrients, affect cell size. Previous studies have revealed that cell size homeostasis in dividing cells is controlled at the G1/S transition, called START, by preventing cell division until a certain size is reached (7,8). For example, bck2Δ, cln3Δ, swi4Δ, and swi6Δ cells are larger, while whi2Δ and whi3Δ cells are smaller than wildtype cells due to the dysregulation of G1 cyclins in Saccharomyces cerevisiae (9)(10)(11)(12)(13). Furthermore, cell size can change in response to extracellular conditions (14).
Several large-scale screens in yeast have revealed various cell size regulators, such as those involved in transcription, translation (including ribosome biogenesis), and cell cycle control (15)(16)(17)(18) (Table S1). In addition, a systematic analysis of the morphological traits of yeast deletion strains has provided cell size information (19). However, the correlations between these previous cell size screens are low (18), suggesting that additional cell size mutants were overlooked in previous screens.
Here, we conducted a genome-wide screen to identify additional cell size mutants using budding yeast. We identified the pH-sensing Rim101 pathway as a positive regulator of cell size under physiological and external alkaline conditions. Cell size increased by the activation of Rim101, and this increase was attributed to an increase in vacuolar and cytosolic volume and was mediated by the V-ATPase component Vph1. Collectively, we provide new mechanistic insight into the regulation of cell size in budding yeast.

Results
Identification of Rim21 as a positive regulator of cell size by genome-wide screening To identify genes that regulate cell size, we performed a genome-wide screen using a nonessential gene deletion array with haploid S. cerevisiae strains containing 4782 open reading frame deletions. The cell volume was determined for 4701 mutants (excluding 81 mutants that were not grown) based on the Coulter principle. We used cells in the stationary phase to avoid the effects of bud growth. As summarized in Figure 1 and Table S1, we identified cells ranked in the top or bottom 5% (236 mutants each) for normalized mean volume as cell size mutants. In total, 102 of 236 large mutants and 51 of 236 small mutants were identified in at least one previous screen (taking the top or bottom 5% are thresholds for screens that do not annotate cell size mutants) (15)(16)(17)(18)(19)(20) (Table S1).
The pH-sensing Rim101 pathway positively regulates cell size Among the cell size mutants identified through our screen, we focused on rim21Δ because Rim21 is a well-established component of the pH-sensing Rim101 pathway (21). We noticed that other deletion mutants of the Rim101 pathway, including rim21Δ, dfg16Δ, rim101Δ, rim8Δ, rim9Δ, rim13Δ, and rim20Δ, showed a reduction in cell size in the screen (Table S1). The Rim101 pathway is responsible for alkaline pH-responsive gene regulation and adaptation to alkaline conditions ( Fig. 2A) (22,23). The complex composed of Rim9, Rim21, Dfg16, and Rim8 senses environmental alkaline pH and activates the downstream signaling pathway in an endosomal sorting complex required for transport (ESCRT) protein-dependent manner, leading to the activation of the proteolytic complex containing Rim13, a calpain-like cysteine protease, and Rim20, an adaptor for substrate recognition. Rim13 cleaves and activates the transcriptional regulator Rim101, inducing the expression of alkaline-responsive genes ( Fig. 2A) (23). We confirmed that all Rim101 pathway mutants were significantly smaller than wildtype cells (Fig. 2B). In addition, we confirmed the cell size phenotype of rim21Δ in both the stationary and log phases (Fig. 2, C-E) and found that the expression of exogenous Rim21 restored the size of rim21Δ to that of wildtype cells (Fig. 2, C-E). Furthermore, the expression of the N-terminal fragment of Rim101 (1-532), the active form of Rim101 (24), increased the size of wildtype cells as well as rim21Δ (Fig. 2, A and F). These results suggest that the pH-sensing Rim101 pathway positively regulates cell size under physiological pH conditions.

Cell size increases in response to external alkalization
Because the Rim101 pathway is activated in response to external alkalization (23), we next evaluated whether external alkalization affects cell size. We found that wildtype cells were larger when they were cultured at pH 7.5 than at pH 5.5 or 3.5 (Fig. 3A). Note that pH 7.5 is regarded as an alkaline pH for yeast; the pH of yeast peptone dextrose (YPD), typically used to grow yeast, is approximately 6.8 (25) and becomes more acidic in the stationary phase (approximately 5.1-5.6).
We next examined whether the effect of external alkalization on cell size was dependent on the Rim101 pathway. The increase in the size of rim21Δ and rim101Δ cells in response to environmental alkalinization was largely suppressed (Fig. 3A). These data suggest that the Rim101 pathway positively regulates cell size under external alkaline conditions as well as under physiological pH conditions.

The Rim101 pathway regulates cell size via V-ATPase
To investigate the mechanism by which the Rim101 pathway regulates cell size, we evaluated RNA sequencing data for rim101Δ cells (26). Vacuolar-type ATPase (V-ATPase) components, such as Vph1, were downregulated in rim101Δ. In addition, the deletion of RIM101 causes the downregulation of the V-ATPase genes VMA2 and VMA4 (27). We thus hypothesized that the Rim101 pathway regulates cell size via V-ATPase expression.
We first measured the protein expression level of Vph1 in WT and rim101Δ cells. The amount of Vph1 was reduced in rim101Δ (Fig. 3B). These data confirmed that the Rim101 pathway positively regulates the expression of Vph1.
We found that vph1Δ cells were smaller than wildtype cells in both the stationary and log phases under physiological pH conditions (Fig. 3, C and D), although vph1Δ was not annotated as a cell size mutant in previous studies or ours (15)(16)(17)(18)(19)(20). The expression of the active form of Rim101 (1-532) failed to increase cell size in vph1Δ cells at physiological pH (Fig. 3E).  Figure 1. Genome-wide screening identifies Rim21 as a positive regulator of cell size. The cell volume of 4701 S. cerevisiae gene deletion mutants was measured. Each mutant is plotted as a dot, ranked left to right by mean volume normalized by mean volume of WT. Some known cell size mutants and rim21Δ are indicated. The red and blue plots show mutants ranked in the top 5% (red) and bottom 5% (blue) in size, respectively. See also Table S1.
To investigate whether Vph1 acts downstream of the Rim101 pathway, we compared the size of wildtype and rim101Δ cells with and without Vph1 overexpression. The overexpression of Vph1 increased the cell size of rim101Δ to the level of wildtype. This supports the connection between the Rim101 pathway and Vph1 (Fig. 3G).
Next, we investigated how V-ATPase increases cell size. One potential mediator is target of rapamycin complex 1 (TORC1) because V-ATPase is required for TORC1 activity in response to cytosolic pH in yeast (28). We checked TORC1 activity by quantifying the level of phosphorylated Sch9 (P-Sch9) (29). However, under all pH conditions, the level of P-Sch9 relative to total Sch9 did not decrease in rim101Δ cells compared with WT cells (Fig. 3H). This suggests that TORC1 does not mediate the cell size regulation by the Rim101 pathway.
Collectively, these results suggest that the Rim101 pathway regulates cell size via V-ATPase under physiological conditions as well as external alkaline conditions.
The vacuole and cytoplasm are enlarged by external alkalization via the Rim101-V-ATPase axis Next, we asked whether cell enlargement in response to alkalization via the Rim101-V-ATPase axis is associated with an increase in the volume of the cytoplasm (excluding the vacuole) or the vacuole. We found that vacuolar size (as determined by FM4-64 staining) was larger in wildtype cells at pH 7.5 than at pH 3.5, and this difference was abolished in rim21Δ cells (Fig. 4). In addition, an increase in the volume of the cytoplasm was also observed in wildtype cells at pH 7.5 but not in rim21Δ cells. These results suggest that both the vacuole and cytoplasm are enlarged by external alkalization via the Rim101-V-ATPase axis (Fig. 4).

Discussion
We identified Rim21, a component of the pH-sensing Rim101 pathway as a positive regulator of cell size by a genome-wide screen. Subsequently, we confirmed that other mutants defective in the Rim101 pathway were also smaller than wildtype cells. Consistent with the role of the Rim101 pathway in pH sensing, we found that cell size increases in response to external alkalinization in a manner dependent on the Rim101 pathway and V-ATPase. Based on these findings, we propose that the size of budding yeast cells is regulated by the Rim101-V-ATPase axis under both physiological and external alkaline conditions (Fig. 5).
Although the Rim101 pathway is activated in response to external alkalization, the Rim101 pathway mutants were also smaller around pH 5.5. This may be attributed to the basal activity of the Rim101 pathway; the processed active form of Rim101 can be detected in cells in the log phase cultured in YPD medium (30). The increase in the active form under alkaline conditions (23) is consistent with the increased effect of Rim101 deficiency on cell size under those conditions. We also found that the increase in cell size in response to alkaline conditions is not completely abolished in rim21Δ and rim101Δ cells (Fig. 3A), suggesting that other pH-sensing mechanisms contribute to this increase.
The mechanism by which V-ATPase increases cell size is unclear. A simple explanation is that V-ATPase-mediated proton translocation increases intravacuolar osmolarity and causes vacuolar swelling. This effect would be enhanced under  alkaline conditions to maintain acidity in vacuoles. In fact, V-ATPase activity in cells is higher at pH 7 than at pH 5 (28). Cell size regulation in response to the external pH by the Rim101 pathway is not likely to be conserved in mammals. In mammals, calpain-7 and ALIX are possible orthologs of yeast Rim13 and Rim20, respectively (23). These mammalian orthologs are not thought to function in ambient pH sensing and adaptation (23). However, it is possible that the cell size under alkaline conditions in mammals is regulated via V-ATPase, which regulates mTORC1 under some conditions in mammals (31).
In Cryptococcus neoformans, rim101Δ cells are smaller than wildtype cells in host mice (32). However, the size of rim101Δ cells does not differ from that of wildtype cells in vitro. The phenotype of rim101Δ in C. neoformans can likely be explained by a decrease in titan cells (characterized by enlarged size) and the small size of minimally encapsulated fungal cells. In Escherichia coli, environmental pH impacts the length at which cells divide and therefore cell size increases as the pH rises (33). Thus, the mechanism underlying cell size regulation in S. cerevisiae appears to differ from those of bacteria. Furthermore, the cell sizes of Staphylococcus aureus (33), Streptococcus pneumoniae (34), and Caulobacter crescentus (35) increase during growth in alkaline media. It is conceivable that cell size regulation in response to environmental pH has a common physiological role across taxa, despite differences in the underlying mechanisms.

Plasmids
The RIM21 gene (including the region 1 kb upstream of the coding sequence [CDS], CDS, and 0.3 kb downstream of the CDS) was amplified by PCR using BY4741 genomic DNA as a template and inserted into the pRS316 vector (38). The N-terminal region of RIM101 (encoding Met1-Gln532) was   ) and adjusted to pH 3.5 with HCl or to pH 5.5 or 7.5 with NaOH.

Yeast transformation
Yeast transformation was performed by a standard method as described (39).

Measurements of cell size
Volumes and diameters of approximately 10,000 cells were determined using an EC800 cell analyzer (Sony Japan). Data were processed using Kaluza (Beckman Coulter). The normalized mean volume, used as a proxy for cell size, was calculated by dividing the mean volume of each mutant by that of the wildtype.
Immunoblotting A volume of 3 ml of yeast cell cultures in the log phase grown in YPD medium or YPD medium buffered at pH 3.5, 5.5, and 7.5 was collected by centrifugation at 12,000g for 1 min at 4 C. The pellets were mixed with 140 μl alkaline solution (0.3 M NaOH, 100 mM dithiothreitol [Nacalai Tesque, Cat#: 14112-52]) containing protease inhibitor cocktail [Nacalai Tesque, Cat#: 03969-34]). The samples were then vortexed and incubated on ice for 5 min followed by the addition of 1 ml of ice-cold 15% trichloroacetic acid solution (Wako, Cat#: 200-08085). The samples were incubated on ice for 10 min and centrifuged at 15,000g for 5 min at 4 C. The pellets were washed in 600 μl of ice-cold acetone with sonication for 2 min and centrifuged at 15,000g for 5 min at 4 C.
The pellets were then mixed with sample buffer solution (Nacalai Tesque, Cat#: 09499-14) and heated at 55 C for 10 min. The samples were run on SDS-PAGE gel using Trisglycine-SDS buffer, transferred to Immobilon-P polyvinylidene difluoride membranes (IPVH00010; EMD Millipore) and subjected to immunoblotting using the antibodies described above. Immobilon Western Chemiluminescent HRP Substrate (P90715; EMD Millipore Corporation) or Super-Signal West Pico Chemiluminescent Substrate (1856135; Thermo Fisher Scientific) was used to visualize the signals. The signals were detected using a Fusion Solo 7S system (M&S Instruments Inc). Images were adjusted using Adobe Photoshop 2022 (Adobe). Quantitation of signal intensity was performed using Fiji (40).

Fluorescence imaging and quantification of the vacuolar size
Cells were incubated with 30 nM FM4-64FX (Thermo Fisher Scientific, Cat#: F34653) at 30 C for 20 min, washed, and resuspended in 5 ml of YPD. The cells were further incubated at 30 C for 90 min. Images were acquired on the FV3000 (Olympus) equipped with a UAPON 100XOTIRF lens (Olympus, Cat# N2709500, NA 1.49) and processed using Fiji software (40). The outline of each cell was determined based on phase-contrast observations. The major axes of the cells and their vacuoles were measured manually using Fiji software. The cube of the major axes was used as a proxy for the total cell and vacuolar volume. The volume of cytoplasm was estimated by subtracting the vacuolar volume from the total cell volume.

Statistical analysis
The Welch t test was used for two-group comparisons. Multiple comparisons were performed by one-way analysis of variance followed by Dunnett test, Sidak multiple comparison test, or Tukey multiple comparison test using GraphPad Prism 8 (GraphPad Software). Normal distributions were assumed but not formally tested.

Data availability
All data supporting the analyses described in the article are available from the corresponding author upon reasonable request.