{"paper_id":"16eb1696-1ff4-41ac-81d2-ca4c3bc69fa2","body_text":"=== R E V I E W   C O M M O N S   M A N U S C R I P T ===\nIMPORTANT:\nManuscripts subm itted to Review Com m ons are peer reviewed in a journal-agnostic way.\nUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor.\nThe identity of the referees will NOT be com m unicated to the authors unless the reviewers choose to sign their report.\nThe identity of the referee will be confidentially disclosed to any affiliate journals to which the m anuscript is transferred.\nGUIDELINES:\nFor reviewers: https://www.reviewcom m ons.org/reviewers\nFor authors: https://www.reviewcom m ons.org/authors\nCONTACT:\nThe Review Com m ons office can be contacted directly at: office@reviewcom m ons.org\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n1 \n \n \n \n \nTitle: Translational control of CAK and Cdk T-loop phosphorylation in response to \ngrowth in yeast \n \nAuthors: Heidi M. Blank1, Eun-Gyu No1, Ainsley E. Nelson1, Abigail Payne1, Sofia Lykidis1, \nMichael Polymenis1 \n \nAffiliation: 1Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen \nBlvd, College Station TX, 77843 \n \nContact: michael.polymenis@ag.tamu.edu \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n2 \nSHORT TITLE: Translational control of CAK in yeast \nKEYWORDS:  Cak1, Cdc28, uORF, stationary, chemostat \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n3 \nABSTRACT \nCyclin-dependent kinases (Cdks) require activating T-loop phosphorylation, a modification that \nis considered constitutive. Here, we examined the regulation of the Cdk-activating kinase, Cak1, \nin budding yeast. We measured Cak1 levels and the activating T169 phosphorylation of Cdc28 \n(the budding yeast Cdk) across various nutrient environments. We found that the abundance of \nCak1 and the T169 phosphorylation is significantly reduced in cells that are proliferating very \nslowly or have entered quiescence. A small upstream open reading frame (uORF) in the CAK1 \ntranscript represses Cak1 synthesis, especially in poor growth conditions. Eliminating the uORF \nincreased Cak1 levels but did not alter proliferation kinetics under most laboratory contexts. \nInstead, it reduced the viability of quiescent cells and the fitness of slowly proliferating \nchemostat cultures. In cells lacking several type 2C protein phosphatases, which remove the \nT169 phosphorylation, there was a pronounced acceleration of initiation of cell division in the \nabsence of the uORF in CAK1. Our results suggest an unexpected layer of control, impinging \non the activating phosphorylation of the Cdk. The uORF-mediated repression of Cak1 synthesis \ndirectly couples protein synthesis to the activity of the core cell cycle machinery.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n4 \nGRAPHICAL ABSTRACT \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n5 \nINTRODUCTION \nFrom late in the G1 phase of the cell cycle until anaphase, a rising Cdk activity drives cell cycle \nprogression (Polymenis, 2022; Morgan, 2007). Active Cdks are serine/threonine protein \nkinases. The Cdks are inactive as monomers and require binding to a regulatory cyclin subunit \nto form an active Cdk-cyclin heterodimer. When a cyclin binds to the Cdk, the conformation of \nthe Cdk's T-loop domain and a small nearby helix change, allowing protein substrates and ATP \nto access the active site of the kinase (Pavletich, 1999). In addition to cyclin binding, a \nconserved threonine residue in the T-loop must also be phosphorylated for Cdk activation. \n(Russo et al, 1996). T-loop phosphorylation is essential for Cdk function. Surprisingly, however, \na mutation mimicking constant T-loop phosphorylation at the same site is also lethal unless the \nCdk has more mutations elsewhere in the protein (Cross & Levine, 1998). Cells regulate the \nactivity of Cdk complexes in many ways, as one would expect for the 'master' regulator of cell \ndivision. For example, interactions with other proteins activate or inhibit the kinase, while \nperiodic phosphorylations at its N-terminus inhibit it (Polymenis, 2022; Morgan, 2007). Yet, cells \ndo not periodically phosphorylate and dephosphorylate the T-loop to adjust their Cdk activity in \nthe cell cycle (Kaldis, 1999; Cross & Levine, 1998). The seemingly constitutive nature of the \nactivating T-loop phosphorylation has raised long-standing questions about its physiological role \nin cell proliferation (Blank et al, 2025). \nCdk-activating kinases (CAKs) are the enzymes that phosphorylate Cdks at the T-loop \n(Kaldis, 1999; Blank et al, 2025). CAK themselves are part of the large Cdk family. In \nmetazoans, Cdks that do not have direct cell cycle roles but instead transcriptional ones serve \nas CAKs (Fisher & Morgan, 1994; Fisher, 2005; Fisher et al, 1995; Shiekhattar et al, 1995). In \nbudding yeast, Cak1 is the only CAK (Kaldis et al, 1996). Although Cak1 targets other proteins \nin meiosis (Whinston et al, 2013; Wagner et al, 1997), its single essential function is \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n6 \nphosphorylating the T169 residue of Cdc28, the main Cdk required for mitotic cell divisions in \nthis organism. There is no evidence that the abundance, activity, or localization of Cak1 is \nregulated in proliferating cells, nor do post-translational modifications alter it (Kaldis et al, 1998). \nLikewise, the levels and activity of metazoan CAKs do not change during somatic (Tassan et al, \n1994; Bartkova et al, 1996) or early embryonic cell divisions (Poon et al, 1994). The above \nobservations add to the puzzle of why an essential modification and the 'writer' enzymes of that \nmodification on the Cdk appear unregulated. \nContrary to expectations of constitutive control of Cak1 and T-169 phosphorylation, here \nwe show that Cak1 abundance and the activating T-loop phosphorylation of Cdc28 are \nregulated as cells transition from nutrient-rich to nutrient-poor environments or enter \nquiescence. A small upstream open reading frame (uORF) represses CAK1 mRNA translation. \nCells lacking the uORF lose viability when starved and entering quiescence and are less fit in \nslowly proliferating steady-state chemostat cultures. Furthermore, the effects of the \nuORF-mediated control of Cak1 synthesis become very prominent in the absence of the \ncounteracting phosphatases that remove the Cdk T169 phosphorylation. Our results suggest a \npreviously unappreciated layer of Cdk regulation in coupling cell growth with cell division in poor \nconditions. Lowering T-loop phosphorylation may safeguard against mitotic cycling in \nunfavorable contexts.   \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n7 \nRESULTS \nCak1 levels and the activating T169-Cdc28 phosphorylation vary in \ndifferent conditions \nWe decided to test if Cak1 abundance and the activating phosphorylation of Cdc28 at T169 vary \nin different media because past studies overwhelmingly focused on cells continuously dividing in \nstandard rich laboratory media. In addition, the only instance in the literature for any control over \nCak1 levels in S. cerevisiae vegetative cells was when they were in the stationary phase, at \nwhich point the protein's abundance dropped significantly (Kaldis et al, 1998). We isolated \nCdc28-TAP by immunoprecipitation from cells cultured in media with glucose, a preferred \ncarbon source, or glycerol (Figure 1A). The isolated protein was then examined by mass \nspectrometry, to see if there were any changes in the fraction of the Cdc28 protein \nphosphorylated at T169. Indeed, in the glycerol-grown cells the fraction of T169-phosphorylated \nCdc28 was reduced by about half (Figure 1B). To detect T169 phosphorylation in follow-up \nexperiments, we then used a custom anti-pT169 antibody (see Materials and Methods) to \nprovide independent validation of the mass spectrometry data and because it allows for faster, \nsimpler, cheaper, and more sensitive detection. The custom antibody detected a protein of the \nexpected size for Cdc28 (Figure 1C; compare the middle and right blots). The signal was also \nsensitive to phosphatase treatment (Figure 1C; middle blot, compare the left and right lanes) to \nthe same extent as the signal from a previously validated phospho-specific antibody against \nanother protein, Rps6 (Figure 1C; left blot, compare the left and right lanes). \n To monitor Cak1 levels, we used strains carrying an allele encoding Cak1 with a V5 \nepitope tag at its N-terminus (see Materials and Methods). We could then detect from the same \nsamples the abundance of Cak1 (with an anti-V5 antibody), Cdc28 and Pgk1 (with commercial \nantibodies), and phospho-T169-Cdc28 (with our custom antibody). For each experiment, the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n8 \nsignal for each quantified target was internally normalized by dividing its intensity by the \naverage intensity for that target across all conditions on the same gel, and then Log2 \ntransformed (Figure 1D legend). Our results suggest that the levels of Cak1 were the highest in \nexponentially proliferating cells in glucose-containing media, lower in glycerol-containing ones, \nand, as expected based on (Kaldis et al, 1998), even lower in stationary phase cells (Figure 1D, \nleft panel). In agreement with our mass spectrometry data (Figure 1B), T169 phosphorylation \nwas lower in cells proliferating with glycerol as the carbon source, compared to cells proliferating \nwith glucose, but undetectable in stationary phase cells (Figure 1D, second panel from the left). \nIn contrast, total Cdc28 (and Pgk1; a glycolytic enzyme used as reference) levels did not \nchange in all these conditions. We conclude that the levels of the activating Cdk phosphorylation \n(but not the levels of the Cdk itself) and the abundance of Cak1, the writer activating kinase, \nrespond to growth conditions. \nCAK1 expression is regulated translationally, in part through a uORF \nTo understand the regulation of Cak1 levels, we first quantified CAK1 mRNA abundance in the \nsame growth conditions described above (Figure 1D). We used digital droplet PCR (ddPCR) \nbecause it is a quantitative, highly sensitive method well-suited for detecting differences in \nmRNA abundance (Quan et al, 2018). We found that steady-state levels of CAK1 mRNA were \nnot significantly different among exponentially proliferating cells in glucose or glycerol-containing \nmedia, or in stationary phase cells (Figure EV1A, left). Hence, transcriptional control \nmechanisms are unlikely to explain the observed differences in Cak1 protein abundance. \nSince many cell cycle proteins are subject to regulated degradation, we next measured \nthe apparent half-life of Cak1 in glucose- and glycerol-containing media. To study Cak1 protein \nstability, we constructed a high-copy (2μ) plasmid enabling copper-inducible (CUP1 promoter) \nexpression of V5-CAK1 (Figure EV1B, top). As expected, V5-Cak1 synthesis was rapidly \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n9 \ninduced upon the addition of CuSO4 (0.5mM) to cells harboring this plasmid (Figure EV1B, \nbottom). In separate experiments, we examined otherwise wild-type cells (strain BY4742) \ncarrying this plasmid, growing with either glucose or glycerol as the carbon source. After a 20m \ninduction to drive V5-Cak1 synthesis, we added cycloheximide (CHX) to block new protein \nsynthesis. After normalizing the Cak1 signal (Figure EV1C, top) against the total protein signal \nat each time point (from the Ponceau-stained blots; Figure EV1C, bottom), there was little \ndegradation of Cak1 in either the glucose- or glycerol-containing media, with an estimated \nhalf-life of Cak1 >4h in both conditions. These results argue against Cak1 destabilization as the \nprimary mechanism explaining the observed differences in Cak1 protein abundance in glucose \nversus the poorer glycerol media. We note that the general notion that Cak1 is a very stable \nprotein agrees with prior studies (Christiano et al, 2014; Kaldis et al, 1998). \nWe then focused on potential mechanisms of translational control to explain the differing \nabundance of Cak1 under various growth conditions. We noticed that CAK1 has a uORF, \nencoding a 16-residue peptide, which initiates 37 nucleotides upstream of the CAK1 start codon \nand terminates downstream (marked as uORF-16 in Figure 2A, top). The uORF could explain \nthe differences in the levels of Cak1 we described (Figure 1D). Ribosomes initiating at the uORF \nwould bypass the start codon of CAK1, repressing Cak1 protein synthesis. The uORF-mediated \nrepression of Cak1 synthesis would be disproportionately greater in poor growth conditions, \nwhen the cell’s ribosome content is lower, according to the classic kinetic model of translation \n(Lodish, 1974). To test the uORF’s role, we generated strains carrying a mutation at its AUG \nstart codon to AAG (marked as uORF-0 in Figure 2A, second from bottom). Introducing the V5 \nepitope to monitor Cak1 abundance also extended the peptide produced by the uORF to 35 \nresidues (marked as uORF-35 in Figure 2A, second from top). Lastly, to test whether it is the \npresence of a uORF in general, or specifically the uORF-encoded peptide that impacts Cak1 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n10 \nsynthesis, we introduced a stop codon that terminates the uORF synthesis after only three \nresidues (marked as uORF-3 in Figure 2A, bottom). \nIn the uORF mutants, we monitored the Cak1, T169-phosphorylation, Cdc28, and Pgk1 \nlevels by immunoblotting (Figure 2B). When uORF-0 cells proliferated with glycerol as the \ncarbon source or were in stationary phase, the levels of Cak1 were derepressed, albeit not \ncompletely, compared to uORF-35 cells (compare the middle left panel to the bottom left panel). \nIn contrast, in uORF-3 cells, Cak1 levels remain repressed as in uORF-35 cells (compare the \ntop left panel to the bottom left panel). We conclude that the presence of the uORF represses \nCak1 synthesis in a growth-dependent manner. The uORF-encoded peptide is unlikely to be \nresponsible for this regulation. Instead, the repression likely arises from ribosomal re-initiation \nfailure, whereby ribosomes that successfully translate the uORF-3 are less likely to re-initiate \ntranslation at the downstream CAK1 start codon. We note that the uORF-mediated repression \nwas not the result of changes in the steady-state levels of the CAK1 transcript (Figure EV1A, \nright). Surprisingly, although Cak1 levels were derepressed in stationary phase uORF-0 cells, \nthe levels of T169 phosphorylation were not, and remained undetectable (Figure 2B, second \nfrom the left column of panels -compare the middle to the bottom panel). The levels of Cdc28 \nand Pgk1 remained largely unaffected in all conditions and mutants (Figure 2B, 3rd and 4th \ncolumn of panels from the left). These results suggest that the uORF regulates Cak1 levels in \ndifferent growth conditions, but the levels of the activating phosphorylation on the Cdk are \nrobust to those changes. \nTo provide independent evidence of uORF’s expression and its impact on Cak1 levels, \nwe generated plasmids encoding fluorescent proteins in their place (Figure 3A). In cells carrying \nthese plasmids, mTurquoise2-associated fluorescence reported on uORF expression, while \nmRuby2-associated fluorescence reported on Cak1 (Figure 3B). These plasmids were \nmaintained at a high copy number in cells, but the sequences driving expression of the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n11 \nfluorescent reporters were from the CAK1 locus (see Materials and Methods). Our results show \nthat mTurquoise2-associated fluorescence was readily detectable in all conditions we tested \n(Figure 3B,C), arguing that the start codon of the uORF is recognized by scanning ribosomes. \nFurthermore, the significant increase (~10-fold) in mRuby2-associated fluorescence when the \nuORF start codon was mutated (Figure 3C, compare the last two panels to the right) strongly \nsupports the notion that the uORF represses Cak1 synthesis. We note that the magnitude of the \nrepression appears greater with these reporters compared to our results from immunoblotting \n(Figure 2B, 2 to 3-fold), probably because these were high-copy plasmids. \nThe experiments we described thus far were in cells proliferating with glucose or glycerol \nas the carbon source in batch cultures, where the doubling time varies ~2-fold (90m vs. 160m), \nand one cannot distinguish nutrient-specific effects from growth rate-dependent ones. In \ncontrast, in chemostats, the growth rate is externally set by the rate of medium addition (D, \ndilution rate). It can be adjusted over a much greater range, independently of the limiting \nnutrient in the medium. For these reasons, we examined prototrophic uORF-35 and uORF-0 \ncells in chemostats under carbon or nitrogen limitation (Figure 4). Over a broad range of dilution \nrates, corresponding to a doubling time of 3.5h to 13.9h, Cak1 levels decreased linearly as the \ngrowth rate was lowered in uORF-35 cells, in both carbon- and nitrogen-limited cultures (Figure \n4A, bottom; and Figure 4B, bottom two panels in the left column). Hence, Cak1 levels respond \nto changes in growth rate, but not in a nutrient-specific manner. This regulation was not evident \nin uORF-0 cells, where Cak1 levels were not elevated in higher dilution rates (Figure 4A, top; \nand Figure 4B, top two panels in the left column). There were also no growth rate or \nnutrient-specific changes in the abundance of Cdc28 and Pgk1 in these strains and conditions \n(Figure 4B, second and third columns of panels). These results argue that the repressive \nfunction of the uORF in the CAK1 transcript mediates the growth-dependent changes in Cak1 \nabundance. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n12 \n In contrast to the steady decline of Cak1 levels as the dilution rate was lowered, the \nlevels of T169 phosphorylation displayed a different pattern: relatively constant until the cultures \nhad a doubling time of 6-7 hours (D = 0.10 h-1), but dropping precipitously in cells proliferating at \nslower rates (Figure 4C). The growth-dependent reduction in T169 phosphorylation was slightly \nmore acute in nitrogen-limited cells, occurring at doubling times of >5h (e.g., compare the two \nbottom panels in Figure 4C). In uORF-0 cells, the drop in T169 phosphorylation was still evident \nin low dilution rates, albeit it appears to have been pushed to even lower ones (e.g., in Figure \n4C, compare the second from the top to the bottom panels).  \nPhenotypes of cells lacking the CAK1 uORF \nThe data we described above suggest that a sizable change in T169 phosphorylation in uORF-0 \ncells manifests only in very slowly proliferating cells in chemostats (Figure 4C), despite an \nincrease in Cak1 levels. Consequently, we wondered if the 2-3 fold derepression of Cak1 \nsynthesis in uORF-0 cells (Figure 2B) would lead to any phenotypes associated with cell \nproliferation. We first examined cells proliferating in batch cultures with different carbon or \nnitrogen sources (Figure EV2), comparing wild-type cells expressing the 16 residue-long uORF \n(uORF-16) with those that did not (uORF-0). There was no significant change in the doubling \ntime (Td) between the two strains, or in budding and cell size (Figure EV2A), parameters often \nassociated with altered cell cycle kinetics (Polymenis, 2022). Next, we used centrifugal \nelutriation to obtain highly synchronous early G1 cells from the wild-type (uORF-16) and \nuORF-0 strains growing with glycerol as the carbon source, and monitored them as they \nprogressed through the cell cycle (Figure EV2B). In late G1, if budding yeast cells have grown \nenough in size, they commit to a new round of cell division at a point called START. Passage \nthrough START is followed by initiation of DNA replication and appearance of a bud on the cell \nsurface (Hartwell & Unger, 1977; Johnston et al, 1977), which will eventually become the new \ndaughter cell after cytokinesis. A parameter used to gauge START completion is the size at \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n13 \nwhich half of the cells are budded, known as the ‘critical size’ (Polymenis, 2022). We found that \nuORF-16 and uORF-0 cells had the same critical size (Figure EV2B), arguing that the kinetics of \nthe G1/S transition are unaffected in cells lacking the uORF in CAK1. Hence, although in media \nwith glycerol as the carbon source the uORF-0 cells have higher levels of Cak1 and T169 \nphosphorylation (Figure 2B), cell cycle progression was not measurably affected (Figure EV2). \nTo gain insight into phenotypes associated with Cak1 overexpression in general, we used the \ncopper-inducible plasmid driving ectopic CAK1 expression that we described earlier (Figure \nEV1B). Again, in proliferating cultures with either glucose or glycerol as the carbon source, there \nwas no difference in the doubling time or cell size when CAK1 was over-expressed (not shown). \nTaken together, our results suggest that under standard laboratory conditions, Cak1 is not \nlimiting and only a fraction of Cdc28 needs to be active for the timely completion of cell cycle \ntransitions. \nHowever, in stationary phase cells, where Cak1 levels are typically very low (Figure 1D), \nwe noticed that cells induced to overexpress CAK1 were smaller (Figure EV3A), albeit properly \narrested in G1, based on their DNA content (Figure EV3B). Moreover, CAK1 overexpression \nresulted in a 3- to 4-fold decrease in viability in stationary-phase cells (Figure EV3C). We note \nthat while some commonly used inducible systems (e.g., GAL-based) are sensitive to nutrients \nand the growth status of the cell, induction of CUP1-based expression is possible even in cells \nin the stationary phase (Hottiger et al, 1994). \nFollowing the above results, arguing that CAK1 overexpression may decrease the \nviability of quiescent cells, we next examined the viability of quiescent cells lacking the CAK1 \nuORF. Based on their ability to divide and form colonies on solid media, we found that uORF-0 \ncells lose viability at an accelerated rate compared to their uORF-16 counterparts (Figure 5A). \nNext, we measured the competitive fitness of uORF-0 cells over time in mixed populations \n(uORF-0 and uORF-16 started at equal proportions) in chemostat cultures, as there was a \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n14 \npronounced drop in T169 phosphorylation levels at low dilution rates (see Figure 4C), \nconcomitant with lower Cak1 levels (Figure 4A,B). Since uORF-0 cells carry the KanMX \nresistance marker, their proportion was scored based on their ability to grow on G418-containing \nsolid media. Note that the fitness of cells carrying this marker is unaffected in similar chemostat \nsettings (Baganz et al, 1997). Our results suggest that while at a higher dilution rate (D=0.13 h-1; \nFigure 5B, middle panel) the fitness of uORF-0 cells was unaffected, it progressively declined at \na lower dilution rate (D=0.05 h-1), in cultures limited for carbon (Figure 5B, left panel) or nitrogen \n(Figure 5B, right panel). We conclude that while cells lacking the CAK1 uORF are \nindistinguishable from their wild-type counterparts in most proliferative settings under standard \nlaboratory conditions, there are penalties in viability and fitness during quiescence and in \ncultures proliferating very slowly, respectively. \nProtein phosphatases counteract changes in cell cycle kinetics in cells \nlacking the CAK1 uORF \nOur data thus far suggest that eliminating the CAK1 uORF has limited phenotypic \nconsequences in standard laboratory settings, likely because T169 phosphorylation levels are \nrobust against a 2 to 3-fold change in Cak1 abundance. Since T169 phosphorylation is \nregulated by both 'writer' (Cak1) and 'eraser' (phosphatase) enzymes, we next examined the \nrole of protein phosphatases. Type 2C phosphatases dephosphorylate Cdc28 at T169 (Cheng \net al, 1999). The seven type 2C phosphatases in budding yeast (Ptc1-7) have overlapping roles \nwith each other and with other phosphatase classes. While cells lacking all seven PTC genes \nare viable in standard conditions, specific mutant combinations are sensitive to various stresses \n(Sharmin et al, 2014). To test for functional interactions, we first constructed uORF-0, ptc2,3Δ \ntriple mutants, because Ptc2 and Ptc3 were reported to dephosphorylate Cdc28 (Cheng et al, \n1999). The overall proliferation of triple uORF-0, ptc2,3Δ cells was indistinguishable from that of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n15 \nthe uORF-16, ptc2,3Δ control on solid media with glucose or glycerol as the carbon source \n(Figure EV4A) or in liquid glucose batch cultures (Figure EV4B). To generate a more sensitized \nbackground that enables the detection of functional interactions arising from the higher \nabundance of Cak1 in uORF-0 cells, we then constructed uORF-0, ptc1,2,3Δ quadruple \nmutants. We chose the additional Ptc1 deletion because Ptc1 is involved in more processes \nthan any other type 2C phosphatase in yeast (Ariño et al, 2019). Indeed, ptc1,2,3Δ mutants \n(with or without the CAK1 uORF) proliferated more slowly than wild type or ptc2,3Δ cells in \nglucose or glycerol-containing media (Figure EV4).  \nWe measured the DNA content of these cells proliferating exponentially in liquid batch \ncultures (Figure 6A). We noticed that a noticeable fraction of triple ptc1-3Δ cells (with or without \nthe uORF) had a higher than 2N DNA content in glucose and glycerol, consistent with mitotic \ndefects (Figure 6A, compare the right two panels to the others). There was a significant \ndecrease (from 39% to 23%, p<0.0001 based on robust bootstrap ANOVA) in the fraction of \ncells with G1 DNA content in uORF-0, ptc1,2,3Δ cells compared to uORF-16, ptc1,2,3Δ cells \n(Figure 6A, compare the two rightmost panels at the bottom). A similar effect was also evident in \nglycerol cultures (Figure 6A, compare the two rightmost panels at the top). These results \nsuggest that loss of the CAK1 uORF when ‘eraser’ phosphatases were also absent altered cell \ncycle kinetics significantly. \nWe then examined synchronous cultures obtained by elutriation (Figure 6B). We found \nthat ptc1,2,3Δ cells are difficult to elutriate because they proliferate slowly and the cultures \ncontain many dead cells. Even in glucose media, among the elutriated G1 fraction of cells, only \nabout two-thirds eventually budded, compared to >90% for the wild type (BY4742 strain \nbackground) cells (Figure 6B). Although uORF-16, ptc1,2,3Δ cells had the same critical size \n(the size at which half the cells were budded) as wild type cells (~37fL), there were nonetheless \nmore budded smaller cells (Figure 6B; compare the left and middle panels), arguing that loss of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n16 \ntype 2C PPases may promote initiation of cell division. The effect was greatly enhanced in \nuORF-0, ptc1,2,3Δ cells, which had a much lower critical size (~30fL), consistent with a \npronounced acceleration of START (Figure 6B, compare the right panel to the other two). Given \nthat their overall proliferation rate was similar to that of uORF-16, ptc1,2,3Δ cells (see Figure \nEV4), we conclude that uORF-0, ptc1,2,3Δ cells have accelerated entry into the S phase but \nalso delayed exit from mitosis, as expected for cells with higher Cdk activity. These results \nsupport a major role for phosphatases in counteracting the effect of higher Cak1 levels in \nuORF-0 cells.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n17 \nDiscussion \nThe results presented are significant for several reasons. Demonstrating growth-dependent \ncontrol of Cak1 and T169 phosphorylation provides an answer to a long-standing question \nabout the regulation of CAK activity. We describe the molecular mechanism underpinning this \ncontrol as a cis element (the uORF) in the CAK1 transcript, making Cak1 synthesis \ndisproportionately sensitive to the most central aspect of cell growth: the availability of \nribosomes. We had reported a similar mechanism in the control of the early G1 cyclin Cln3 \n(Polymenis & Schmidt, 1997; Blank et al, 2018). Hence, when yeast cells encounter different \nnutrient environments, they couple the levels of two direct activators of the Cdk, Cak1 and Cln3, \nto the capacity of their protein synthesis machinery (see Figure 7 for a schematic).  \nCells were impervious to changes in Cak1 levels in most laboratory contexts. This was \nthe result of counteracting type 2C protein phosphatases, which are apparently quite efficient in \nbuffering T169 phosphorylation against changes in Cak1 levels mediated by the uORF \nmechanism we described (Figure 6). Nonetheless, our data also suggest that translational \ncontrol of CAK1 may promote survival in the wild (Figures 4,5). In natural environments, where \nlimited and changing resources are the norm, the uORF in CAK1 would promote fitness by \nefficiently inhibiting Cak1 synthesis and cell division when nutrient availability drops significantly. \nFinally, our results contribute to a unifying view of CAK across all eukaryotes. \nObservations in animal cells, such as the higher threshold of T-loop Cdk phosphorylation \nrequired for the G1/S transition in response to extracellular signals and the heightened \ninvolvement of counteracting phosphatases at that proliferative transition (Schachter et al, 2013; \nBlank et al, 2025), point to a general and conserved role for CAK in safeguarding cells before \nthey commit to a new round of division.   \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n18 \nMATERIALS AND METHODS \nStrains and media \nAll S. cerevisiae strains used in this study are listed in the Key Resources Table. \nCulture Conditions \nUnless indicated otherwise, the cells were cultured on the standard, rich, YPD medium (1% w/v \nyeast extract, 2% w/v peptone, 2% w/v dextrose), at 30°C. Throughout the manuscript, glucose \nrefers to dextrose (D-glucose). In some experiments, as indicated, glycerol (2% w/v) replaced \ndextrose as the carbon source. Synthetic minimal media (SMM) contained 0.17% w/v yeast \nnitrogen base without amino acids and ammonium sulfate, 0.5% w/v nitrogen source (ammonium \nsulfate or proline, as indicated), 2% w/v dextrose, and were supplemented as needed for \nauxotrophies (Kaiser et al, 1994). For experiments involving the inducible expression of CAK1, \nthe media were standard synthetic complete, lacking uracil (SC-Ura) (Kaiser et al, 1994), with \n2% w/v dextrose or glycerol, as indicated, and the cells cultured at 30°C. For the experiments \nusing the reporter plasmids, the media were also SC, but lacking both uracil and leucine. For \nplasmid construction and propagation in E. coli bacteria, cells were cultured on the standard \nlysogeny broth media (0.5% w/v yeast extract, 1% w/v tryptone, 1% w/v sodium chloride), at 37°C. \nChemostat experiments \nChemostat experiments were conducted using a BioFlo 110 bioreactor system (New Brunswick \nScientific, Edison, NJ) with a working volume of 880 mL. To monitor the abundance of various \nproteins and T169 phosphorylation we used prototrophic strains with agitation set at 800 rpm for \nthese experiments, at room temperature (Blank et al, 2018; Henry et al, 2010; Guo et al, 2004). \nThe carbon-limited media were 0.17% w/v yeast nitrogen base without amino acids and \nammonium sulfate, 0.5% w/v ammonium sulfate, and 0.08% w/v dextrose. The nitrogen-limited \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n19 \nmedia were 0.17% w/v yeast nitrogen base without amino acids and ammonium sulfate, 0.002% \nw/v ammonium sulfate, and 2% w/v dextrose. The dilution rate was adjusted as indicated for each \nexperiment. The cell density remained >1E+07 cells/mL and did not vary more than threefold \nbetween the lowest and highest dilution rates. For experiments with auxotrophic strains (Figure \n5), the media were supplemented as needed. \nStrain construction \nTo monitor Cak1 protein abundance, we initially constructed strains carrying an allele encoding \na TAP-tag fused to CAK1 at the C-terminus. However, these strains lost viability after a few \ngenerations and the cells had elongated, sausage-like morphology, indicative of mitotic defects \n(not shown). Consequently, we then constructed strains carrying N-terminal V5-tagged CAK1 \nalleles, expressing CAK1 from the endogenous chromosomal locus. We first inserted a \nKanMX6 cassette upstream, at position ChrVI:79250, with PCR-mediated methodology \n(Longtine et al, 1998), using primers VI:79250-79325-F1 and VI:79159-79249-R1 (see Key \nResources Table), and plasmid pFA6a-KanMX6 as a template. We introduced the PCR product \ninto strain BY4742 to generate strain MSP256. The KanMX6 insertion was verified by PCR, \nusing primers VI:79600-FWD and VI:78815-REV. Then, we used genomic DNA from strain \nMSP256 as a template in a PCR reaction with primers V5-CAK1-REV and VI:79600-FWD. The \nPCR product was used to transform the prototrophic strain X2180-5A. The resulting strain \n(MSP258; denoted as uORF-35, V5-CAK1 throughout the manuscript) expressed a protein of \nthe expected size for V5-Cak1, based on immunoblots, and the genomic area was also \nsequenced to verify the absence of any introduced mutations. Note that the uORF normally \nencodes a 16-residue peptide (Figure 2, top; denoted as uORF-16). By inserting the V5 \nsequences at the CAK1 locus, the uORF was extended to 35 residues (Figure 2, second from \ntop; denoted as uORF-35). The doubling time and the percentage of budded cells between \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n20 \nstrains X2180-5A (uORF-16, CAK1) and MSP258 (ChrVI:79250::KanMX6, uORF-35, \nV5-CAK1, X2180-5A otherwise) were indistinguishable, in glucose and glycerol media.  \nTo mutate the start codon of the uORF (AUG → AAG substitution) upstream of the \nCAK1 ORF, we used primers VI:79250-79325-F1 and VI:79159-uORFm-R1 in a PCR reaction \nwith plasmid pFA6a-kanMX6 as a template. The PCR product was then used to transform \nstrain BY4742, generating strain MSP257 (ChrVI:79250::KanMX6, uORF-0, CAK1, BY4742 \notherwise). The KanMX6 insertion was verified by PCR, and the introduction of the mutation by \nsequencing. As above, we then used genomic DNA from strain MSP257 as a template in a \nPCR reaction with primers V5-CAK1-REV and VI:79600-FWD. The PCR product was used to \ntransform the prototrophic strain X2180-5A, generating strain MSP259 (Figure 2, second from \nbottom; denoted as uORF-0, V5-CAK1), which was verified as described above for strain \nMSP258 (uORF-35, V5-CAK1). We also generated a strain carrying a truncated uORF, \nencoding only its first 3 amino acids. We used primers VI:79600-FWD and uORFm1-3-REV in \na PCR reaction with genomic DNA from strain MSP256 as a template. The PCR product was \nused to transform strain X2180-5A, to generate strain MSP267. Introduction of the desired \nmutation was verified by sequencing as described above. To epitope-tag the CAK1 allele in this \nstrain background, we then used genomic DNA of strain MSP267 as a template in a PCR \nreaction with primers V5-CAK1-REV and VI:79600-FWD. The PCR product was used to \ntransform the prototrophic strain X2180-5A, generating strain MSP268 (uORF-3, V5-CAK1), \nverified by sequencing and immunoblotting to ensure that it expresses V5-Cak1 as expected. \nTo generate a strain lacking PTC2 (encoding a type 2C protein phosphatase), we \namplified the URA3 ORF using primers PTC2-URA3-FWD and PTC2-URA3-REV and genomic \nDNA from strain X2180-5A. We then used the resulting PCR product to transform strain \nBY4742, yielding a ptc2Δ::URA3 derivative (strain MSP269). We verified the correct \nreplacement of PTC2 with URA3 using primers V:337690-FWD and V:335594-REV and the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n21 \ngenomic DNA of each putative transformant as a template. A haploid strain (HB499) lacking \nPTC3 was obtained by sporulation of the homozygous diploid ptc3Δ::KanMX/ptc3Δ::KanMX \nstrain 33082. The double ptc2Δ::URA3 ptc3Δ::KanMX haploid strain (HB509) was a segregant \nof the cross between HB499 and MSP256. The ChrVI:79250::KanMX6, uORF-0, ptc2,3Δ \nhaploid strain (HB505) was a segregant of the cross between HB509 and MSP257. Lastly, we \ncrossed a ptc1Δ::HIS3 (BY4741 otherwise) strain described elsewhere (González-Rubio et al, \n2023) with strain HB509, or HB505. Segregants of these crosses were selected for being triple \nptc1-3Δ mutants without (HB531) or with the uORF-0 mutation (HB530), respectively, by \nco-segregation of the KanMXr, URA+, and HIS+ markers. Using genomic DNA from these strains \nin PCR reactions with primers VI:78815-REV and VI:79398-FWD, we validated the presence of \nthe uORF-0 mutation (strain HB531) or not (strain HB530) by sequencing the PCR product with \nthe VI:78815-REV primer. Since the PTC3 deletion was also marked with the KanMX marker, \nwe confirmed that these strains were indeed ptc3Δ::KanMX through PCR, with primers \nII:113444-FWD and KanMX-REV, and genomic DNA as template. We also confirmed that they \nhad no wild type PTC3 allele, in a PCR reaction with primers II:113444-FWD and \nII:114791-REV, and genomic DNA as template. \nPlasmids \nAll enzymes used in plasmid construction were from New England Biolabs. To generate the \nplasmid driving copper-dependent expression of V5-CAK1, we used the single-pot approach \nand the plasmids included in the MoClo kit (Lee et al, 2015), as detailed below. A synthetic \nDNA fragment was made (Integrated DNA Technologies, Coralville, IA) containing the \nV5-CAK1 allele flanked with adaptor sequences compatible with the MoClo multipart assembly. \nThe V5-CAK1 DNA fragment was digested with BsaI, electrophoresed through an agarose gel \n(1% w/v), and purified. It was then combined with plasmids YTK002, YTK067, YTK031, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n22 \nYTK063, YTK074, YTK082, and YTK083 in a ligase reaction containing T4 ligase together with \nthe BsaI restriction endonuclease. The reaction was carried out in a thermocycler at 37°C for \n20m; (37°C for 5m, 16°C for 5m) for 50 cycles; 60°C for 5m; 80°C for 10m. An aliquot was \nused to transform E. coli (high-efficiency DH5alpha cells; New England Biolabs). \nTransformants were selected and verified to contain the high-copy (2 micron; 2μ) \nYEp-pCUP1-V5-CAK1 plasmid, where the copper-inducible pCUP1 promoter drives V5-CAK1 \nexpression, the terminator is tADH1, the yeast selection marker is URA3, and AmpR-ColE1 are \nused for bacterial selection and propagation. The plasmid was sequenced with primers \npBR322ori-FWD and AmpR-FWD (see Key Resources Table). Expression of a protein of the \nexpected size upon induction with CuSO4 (0.5mM) was confirmed by immunoblotting for \nV5-Cak1 (see Figure EV1B). \n We also followed the same single-pot approach with reagents from the MoClo kit to \ngenerate several reporter constructs. Synthetic DNA inserts were made (Integrated DNA \nTechnologies, Coralville, IA) containing CAK1 upstream sequences (starting at position \nchrVI:79401) fused to sequences encoding fluorescent reporter proteins and flanked with \nadaptor sequences compatible with the MoClo multipart assembly. These inserts were: \n●  uORF-mTurquoise2: chrVI:79401-79196 fused to the mTurquoise2 ORF. \n● CAK1-mRuby2: chrVI:79401-79159 fused to the mRuby2 ORF. \n● uORFm-CAK1-mRuby2 (chrVI:79401-79159 fused to the mRuby2 ORF, but with the \nuORF start codon mutated to AAG). \nThe plasmids containing these synthetic insert DNA fragments were digested with BsaI, \nelectrophoresed through an agarose gel (1% w/v), and the inserts isolated. The inserts were \nthen combined with plasmids YTK002, YTK067, YTK031, YTK063, YTK074 (for the mRuby2 \nreporters) or YTK075 (for the mTurquoise2 reporter), YTK082, and YTK083 in a ligase reaction \ncontaining T4 ligase together with the BsaI restriction endonuclease. The reactions were \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n23 \nperformed as above and used to transform E. coli and isolate the relevant plasmids. The \nplasmids were verified by sequencing as above, and with additional oligonucleotides that \ncorrespond to their insert sequences (VI:79398-FWD, mTurquoise2-FWD, and mRuby2-FWD; \nsee Key Resources Table). The plasmids contained the tADH1 terminator, the 2 micron (2μ) \nelement for high-copy propagation in yeast cells, and the AmpR-ColE1 elements for bacterial \nselection and propagation. The yeast selection marker is URA3 for the mRuby2 reporters, and \nLEU2 for the mTurquoise2 one. \nCdc28-TAP immunoprecipitation and mass spectrometry for T169-Cdc28 \nphosphorylation \nTo detect the T169 phosphorylation by mass spectrometry (Figure 1), we used a strain (BY4741 \nbackground) carrying a CDC28-TAP allele expressed from its endogenous chromosomal \nlocation (Ghaemmaghami et al, 2003) (see Key Resources Table). \nOvernight cultures growing in rich undefined media with glucose (YPDextrose) or \nglycerol (YPGlycerol) as the carbon source were diluted 1:100 into fresh 1L cultures and \nincubated at 30°C for several hours until they reached a cell density of ~2-5E+07 cells/mL in \nYPD, or ~8E+06 to 1E+07 cells/mL in YPGlycerol. The cultures were quenched with sodium \nazide (at 0.1% final concentration) and cycloheximide (100μg/mL). The cells were collected by \ncentrifugation, and the pellets were stored at -80°C until needed. \nFor immunoprecipitation, the cell pellets were washed in ice-cold lysis/IP buffer (10mM \nTris-HCl, pH=8.0, 150mM NaCl), resuspended at ~1E+09 cells per reaction in 0.6mL of lysis \nbuffer, containing protease and phosphatase inhibitor cocktails, and kept on ice. Glass beads \n(~0.35mL) were then added. The cells were broken in 8 cycles of vortexing at the maximum \nspeed for 30s, followed by placement on ice for 30s. The samples were centrifuged at 5,000 g \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n24 \nfor 5m, and the supernatants were collected and placed in fresh tubes on ice. To each reaction \nwere added 50μL of IgG-Sepharose-6 fast-flow beads (pre-washed with lysis buffer), and \nincubated for 2h on a rotisserie mixer at 4°C. The beads were collected by centrifugation at 500 \ng for 1m, washed twice with lysis buffer and twice with 10mM ammonium bicarbonate buffer, \nprepared fresh on the day of the experiment. The beads were resuspended in 75μL of \nammonium bicarbonate buffer and shipped at 4°C to the UC Davis proteomics core facility. \nAt the core facility, samples underwent tryptic digestion and LC-MS/MS, and were \nprocessed according to their established protocols for phosphopeptide quantification. The signal \nin each sample for Cdc28 or pT169-Cdc28 was first normalized against the total ‘input’ signal in \neach case. The ratio of the two normalized values (i.e., pT169-Cdc28 : Cdc28) was used to \ncompare the different samples (Figure 1B). All the data from this mass spectrometry experiment \nare in Dataset EV1. \nProtein surveillance \nImmunoblotting \nProtein extracts were made as described (Wallace et al, 2022), and resolved on 12% \nTris‐Glycine SDS–PAGE gels, unless indicated otherwise. V5-Cak1 was detected using a \nmouse anti-V5 tag monoclonal antibody, conjugated with horseradish peroxidase (HRP), used at \na 1:5,000 dilution (ThermoFisher; Cat#: R961-25). Loading was measured with a mouse \nanti‐Pgk1 primary antibody, followed by a secondary antibody (at 1:5,000; ThermoFisher; Cat#: \n31431). To detect total amounts of Cdc28, we used a commercially available mouse monoclonal \nanti-Cdc28 antibody (at 1:1,000; Santa Cruz Biotechnologies #sc-515762). \nFor detecting the activating phosphorylation of Cdc28, we obtained a custom rabbit \npolyclonal antibody (Biomatic, Kitchener, Ontario, Canada) raised against a peptide \n(RAFGVPLRAY(pT)HEIVT) encompassing T169 in the yeast Cdc28 protein. The antibody \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n25 \n(α-pT169) recognized a protein of the same apparent mass as one recognized by the \nanti-Cdc28 antibody (Figure 1C, compare the middle and right panels on the top). The signal \nwas diminished (Figure 1C, middle panel on the top) when the samples were first treated for \n30m at 30°C with 400u of Lambda Protein Phosphatase (New England Biolabs #P0753). The \nreduction in signal intensity was comparable to the reduction observed for ribosomal protein S6 \nphosphorylation (Figure 1C, compare the left and middle panels on the top). The latter was \ndetected by a specific rabbit monoclonal antibody against Ser235/236 of the human protein (at \n1:5,000; Cell Signaling, Cat#: 4858), followed by a secondary antibody (at 1:5,000; \nThermoFisher; Cat#: 31466). \nV5-Cak1 Stability Assay \nTo monitor the stability of V5-Cak1 (Figure EV1C), we transformed BY4742 haploid cells with \nthe pCUP1-V5-CAK1 plasmid described above. The transformants were cultured in synthetic \ncomplete drop-out medium (SC-Ura), with glucose or glycerol as the carbon source. Cultures \nproliferating exponentially were induced with CuSO4 for 20m, and then treated with \ncycloheximide (CHX; 100μg/mL) to block protein synthesis. The levels of V5-Cak1 were then \nmonitored by immunoblotting as described above, from samples taken at various time points.   \nImage analysis \nImages were processed with the “Subtract Background” tool, and band intensities quantified \nusing the “Measure” tool to obtain a mean intensity for each band, using the ImageJ software \npackage. The area measured was kept constant for a particular sample series for each blot \nanalyzed. The source data for all the immunoblots are in the supporting files. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n26 \nTranscript abundance using digital droplet PCR (ddPCR) \nFor RNA surveillance, RNA extracts were made as described previously (Blank et al, 2024, \n2020).  Briefly, the ddPCR reaction mixture was prepared by following the manufacturer’s \nprotocol (One-Step RT-ddPCR Advanced Kit for Probes), using the TaqMan® hydrolysis probes \nlabeled with FAM-MBP for CAK1 and VIC-MBP for UBC6 reporter fluorophores. The abundance \nof the transcripts was obtained using the QuantaSoft™ Software. Transcript levels of CAK1 \nwere normalized against the corresponding transcript levels of UBC6 in each sample. \nCentrifugal elutriation and cell size measurements \nIn each experiment, a 250mL cell culture was grown to a density typical of mid-log cultures \n(~1-5E+07 cells/mL in YPD), then loaded onto a 40mL elutriator chamber at a flow rate of \n35mL/min and centrifuged at 3,200rpm. The cells were washed twice with 250mL of medium, \nfirst at 2,800rpm, then at 2,400rpm. Finally, cells were elutriated with 250mL of the same \nmedium at a pump speed of 38mL/min and centrifuge speed of 2,400rpm. This early G1 \ndaughter cell fraction was isolated and monitored regularly as it progressed through the cell \ncycle. \nFrom live, unfixed cells, the percentage of budded cells was determined via phase \nmicroscopy, and cell size was measured using a Beckman Z2 channelyzer (Hoose et al, 2012; \nSoma et al, 2014). The Beckman Accucomp software was used to generate size histograms, \nfrom which the geometric mean size was calculated. In addition, the ‘birth’ size of asynchronous \ncultures was determined from the size histograms using established methods (Maitra et al, \n2019; Truong et al, 2013). Briefly, the population to the left of the peak of the cell size histogram \nwas assumed to contain daughter cells, and the size corresponding to the 10% smallest of \nthese cells was taken as ‘birth size’. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n27 \nDoubling time measurements in batch cultures \nFor experiments shown in Figure EV2, cell numbers were determined by direct counting using a \nhemocytometer. Natural log-transformed cell numbers were plotted against time (h). The \nspecific growth rate (k, in h-1) was derived from the slope of the linear regression during the \nexponential phase. Doubling time (Td)  was calculated using the equation: Td=ln2/k \nFor experiments in Figure EV4, optical density at 600 nm was measured using an \nAgilent Synergy H1 plate reader as a proxy for cell density. Cultures (0.2mL per well) were \nincubated at 30°C with orbital agitation for 10s prior to hourly readings. Td estimates were \ncalculated as described above, using the linear portion of the growth curves between the 2h and \n10h timepoints. \nFlow cytometry and fluorescence microscopy \nThe percentage of cells in the G1 phase of the cell cycle (%G1) was quantified as detailed in \npreviously published protocols (Hoose et al, 2012, 2013). For the experiments in Figure 6A, we \nused a Cytek® Muse® Cell Analyzer. Because of S. cerevisiae's small genome size and the \nclose spacing of DNA peaks in the histograms, which often makes the application of software \ndeveloped for the analysis of DNA content in mammalian cells problematic, we used the \nfollowing approach: Briefly, the area under the left half of the G1 peak in the DNA content \nhistogram was measured, doubled to account for the entire G1 peak, and then divided by the \ntotal area under the histogram. This fraction, representing the proportion of cells with G1  DNA \ncontent (1N for haploid cells), was multiplied by 100 to express it as a percentage (%G1). For \nthe experiments in Figure EV3B, we used a BD Accuri C6 Flow Cytometer at the Flow \nCytometry Facility of the Texas A&M University College of Veterinary Medicine & Biomedical \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n28 \nSciences. The calculated %G1 fraction in this case was from FlowJoTM flow cytometry software \n(BD Biosciences, Franklin Lakes, NJ) \nFor the experiments in Figure 3, BY4743 cells carrying the mTurquoise2 and mRuby2 \nreporter plasmids were grown to mid-log phase at 30°C in synthetic complete (SC) medium \nlacking leucine and uracil, supplemented with either glucose or glycerol as the carbon source. \nTo prepare samples, exponentially proliferating cultures were briefly sonicated to separate cell \nclumps. Cells were then collected by centrifugation at 5,000g for 1m, and the pellets were \nresuspended in phosphate-buffered saline (PBS) containing 0.5% w/v bovine serum albumin \n(BSA). Samples were protected from light and processed immediately for analysis, using a BD \nAccuri C6 Flow Cytometer at the Flow Cytometry Facility of the Texas A&M University College of \nVeterinary Medicine & Biomedical Sciences. \nFor fluorescence microscopy, the samples were prepared as above and promptly \nimaged using a Nikon TS100 inverted microscope equipped with a Plan Apo 100X oil DIC \nobjective and a CoolSNAP DYNO camera. The filters used were an AT-DAPI filter set (excitation \nat λex ≅ 360-390nm) for imaging the mTurquoise2 reporter, and a DM575-TRITC filter set \n(excitation at λex ≅ 540-580nm) for imaging the mRuby2 reporter.  \nSample-size and replicates \nThe number of samples in each experiment was not determined based on a formal power \nanalysis. All replicates represented distinct biological samples from separate cultures. In \nexperiments with a minimum of three replicates, statistical comparisons between groups were \nperformed using a robust bootstrap ANOVA (t1waybt function, with the number of bootstrap \nsamples set to 599) followed by post hoc tests (mcppb20 function) from the WRS2 R package \n(Mair & Wilcox, 2020). For experiments with at least four replicates, nonparametric statistical \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n29 \nmethods were also employed, as indicated in each case. All collected data points, including \nany potential outliers, were included in the analyses.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n30 \nDATA AVAILABILITY \nStrains and plasmids are available upon request. The authors affirm that all data necessary for \nconfirming the conclusions of the article are present within the article, figures, and tables.  \n \nACKNOWLEDGEMENTS \nThis work was supported by the National Institutes of Health (NIH, grants R01 GM123139 and \nR35 GM161174 to M.P.). We thank Staci E. Hammer and Britt Faulk for technical assistance in \nsome experiments. We also thank Dr. Humberto Martin (Universidad Complutense de Madrid) \nfor his generous gift of the ptc1Δ::HIS3 strain. \n \nDISCLOSURE AND COMPETING INTERESTS STATEMENT \nThe authors have no conflicts of interest to declare that could be perceived to influence the \npresentation or interpretation of the data. \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n31 \nREFERENCES \nAriño J, Velázquez D & Casamayor A (2019) Ser/Thr protein phosphatases in fungi: structure, \nregulation and function. 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It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n35 \nFIGURES \n \nFIGURE 1. Cdc28 T169 phosphorylation and Cak1 protein levels vary with different \ngrowth conditions in batch cultures. (A) Schematic of the isolation of Cdc28-TAP  by \nimmunoprecipitation and processing for mass spectrometry to compare the extent of T169 \nphosphorylation in cells growing in media with glucose or glycerol as the carbon source. \nCreated with BioRender.com. (B) Plots showing the relative abundance of recovered Cdc28 \npeptides with the T169 phosphorylation over the abundance of all Cdc28 peptides (y-axis). The \nratio for cells growing with glucose was normalized to one, and the ratio for cells growing with \nglycerol is shown relative to this, from the indicated independent experiments (x-axis). The \nboxplot graphs were generated with R language functions. The central line inside the box \nrepresents the median of the data, while the box itself spans the interquartile range (IQR), and \nthe whiskers extend to a range of 1.5 times the IQR in either direction. The replicates were all \nbiological ones. The raw mass spectrometry data are in Dataset EV1. (C) An antibody against \nT169-Cdc28 phosphorylation. Protein extracts from exponentially proliferating cells (X2180-5A \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n36 \nbackground) in rich undefined medium (YPD) were split in half and either treated (+) or not (-) \nwith lambda phosphatase. The samples were then run on an SDS-PAGE gel and transferred to \na nitrocellulose blot. The blot was stained with Ponceau to indicate loading (bottom) and then \nprocessed with the antibodies indicated on top. The α-pT169 antibody was raised in rabbits, \nusing the peptide RAFGVPLRAY(pT)HEIVT as an antigen, corresponding to Cdc28 (see \nMaterials and Methods). (D) Representative immunoblots of V5-Cak1, pT169-Cdc28, Cdc28, \nand Pgk1 are shown at the top. The boxplot graphs below were drawn as in (B) and display the \nquantification of each target from four independent experiments. Band intensities were \nquantified using ImageJ software. For each experiment, the signal for each quantified target \nwas divided by the average intensity for that target in the series across all conditions on the \nsame gel. These ratios were then Log2 transformed. The Log2 transformed values are plotted \non the y-axis. The x-axis indicates the respective growth conditions. The extracts were prepared \nfrom cells growing exponentially in batch cultures with glucose or glycerol as the carbon source \nor until they reached the stationary phase after being cultured for 3 days in media with glucose \nas the carbon source. In all cases, the cells expressed CAK1 from its endogenous chromosomal \nlocation but from an N-terminal V5 epitope-tagged CAK1 allele. The indicated p-values were \ndetermined using robust bootstrap ANOVA (t1waybt function) followed by post hoc tests \n(mcppb20 function) from the WRS2 R package.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n37 \n \n \nFIGURE 2. A uORF represses Cak1 synthesis, yet T169 phosphorylation is robust to \nchanges in Cak1 levels. (A) A schematic of the CAK1 upstream genomic region. The wild-type \nuORF at position -37 encodes a 16-amino acid peptide. The engineered uORF changes are \nshown below, each with an N-terminal V5 epitope tag fused in-frame with the CAK1 ORF. (B) \nBoxplots showing the quantification of V5-Cak1, pT169-Cdc28, Cdc28, and Pgk1 in the \nindicated strains, drawn as in Figure 1. Data for the uORF-35 background is reproduced from \nFigure 1D for comparison. All experiments and analyses were performed as described in Figure \n1D. All the strains were in the prototrophic X2180-5A background.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n38 \n \nFIGURE 3. The CAK1 uORF is expressed and represses downstream translation. (A) \nSchematic of high-copy reporter plasmids. Reporter 1 (top) replaces the uORF coding sequence \nwith mTurquoise2 (uORF expression). Reporter 2 (middle) replaces the CAK1 open reading \nframe with mRuby2 and contains the uORF. Reporter 3 (bottom) is the same as Reporter 2 but \nwith a mutated uORF start codon (corresponding to the uORF-0 allele in Fig. 2A). (B) \nRepresentative images of cells carrying the indicated reporters. Top row: Cells with Reporter 1 \nonly (uORF-mTurquoise2, blue) show blue fluorescence, but not red. Middle/Bottom rows: Cells \nco-expressing Reporter 1 with either Reporter 2 (containing the uORF) or Reporter 3 (uORF-0). \nNote the marked increase in mRuby2 (red, CAK1 expression) in the Reporter 3 (uORF-0) \nco-transformants compared to Reporter 2, demonstrating uORF-mediated repression. The cells \nwere imaged live. The exposure was 500ms for the blue channel and 200ms for the red \nchannel. (C) Boxplots of single-cell mTurquoise2 (uORF) and mRuby2 (CAK1) fluorescence \nintensity from strain BY4743 co-transformed with Reporter 1 and either Reporter 2 (with uORF) \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n39 \nor Reporter 3 (uORF mutated). Cultures were grown in glucose or glycerol, as indicated. \nFluorescence per cell was quantified with flow cytometry. Boxplots were drawn as in Figure 1.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n40 \n  \nFIGURE 4. Cak1 levels and T169 phosphorylation respond to growth rate changes with a \ndifferent dynamic range. (A) Representative immunoblots of V5-Cak1, pT169-Cdc28, Cdc28, \nand Pgk1, with antibodies indicated, from chemostat cultures limited for nitrogen (N; ammonium \nsulfate) or carbon (C; glucose), for the strains shown. Samples were collected at the indicated \ndilution (D) rates (in h-1). (B,C) The graphs display the quantification of each indicated target \nfrom independent chemostat experiments under the limitations shown for each strain. Band \nintensities were quantified and their Log2-transformed expressed ratios were calculated as in \nFigure 1, for each series across the different dilution rates, and plotted on the y-axis; the x-axis \nindicates the respective dilution rates. Loess curves are shown in red. All the strains were in the \nprototrophic X2180-5A background.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n41 \n \n \nFIGURE 5. Cells lacking the CAK1 uORF exhibit reduced viability during quiescence and \nare outcompeted by wild-type cells in slow-proliferating chemostat cultures. (A) Barplot \nshowing the viability (y-axis) of quiescent cells over several days (x-axis), scored as described \nin the Materials and Methods. Cells without the CAK1 uORF (uORF-0, strain MSP257) had \nlower viability compared to cells with the uORF (uORF-16, strain MSP256). (B) Plots displaying \nthe percentage of uORF-0 cells (y-axis, strain MSP257) against wild-type uORF-16 cells (strain \nBY4742) in mixed chemostat cultures over several days (x-axis) across the indicated dilution \nrates (D) and nutrient limitations. Loess curves are shown in red.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n42 \n \nFIGURE 6. Type 2C protein phosphatases buffer changes in cell cycle kinetics in uORF-0 \ncells. (A) DNA content analysis of the indicated strains (all in the BY4742 background) in \nglucose (YPD) or glycerol (YPGlycerol) media growing exponentially in liquid cultures, \nmeasured by flow cytometry. Histograms show cell counts (y-axis) vs. fluorescence per cell \n(x-axis). The average percentage (± SD) of cells in the G1 phase from three independent \ncultures is indicated in select strains. (B) Cell cycle progression of elutriated synchronous early \nG1 cells in YP-glucose. The percentage of budded cells is plotted against cell size (fL) for the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n43 \nindicated strains. The plots show Loess curves with 95% confidence intervals. Red lines \nindicate the critical size for budding (the size at which 50% of cells have budded) for the wild \ntype cells (uORF-16, PTC+; strain BY4742), while green lines indicate the critical size of the \nuORF-0, ptc1-3Δ cells (strain HB530).  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n44 \n \n \nFIGURE 7. Schematic of our findings. The uORF-mediated translational control of the \ntranscripts encoding two core Cdk regulators, Cak1 and Cln3, couples cell division to ribosome \navailability. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n45 \nEXPANDED VIEW FIGURES \n \nFIGURE EV1. CAK1 expression is not regulated by transcription or proteolysis as a \nfunction of growth. (A) Plots of the relative steady-state levels of CAK1 mRNA from multiple \nindependent experiments (y-axis), in the respective growth conditions (x-axis). Cells and growth \nconditions were the same as in Figure 1C. The boxplots were drawn as in Figure 1. The mRNA \nlevels were measured by ddPCR, as described in the Materials and Methods. All values were \nnormalized to the mean for exponentially growing uORF-35, V5-CAK1 cells (first box). No \nsignificant differences were found between the strains and conditions (p > 0.05, Kruskal-Wallis \ntest). (B) A plasmid used for copper-inducible, ectopic V5-Cak1 synthesis is schematically \nshown on top and described in Materials and Methods. The immunoblot below is from cells \n(BY4742 background) carrying that plasmid showing the protein levels detected by the α-V5 \nantibody at the times indicated after induction with CuSO4. The band corresponds to a mass \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n46 \nconsistent with the V5-Cak1 protein. (C) The V5-Cak1 protein is highly stable in glucose and \nglycerol media. Exponentially proliferating cells (BY4742 background) carrying the \npCUP1-V5-CAK1 plasmid (described in A) were induced for 20 min with CuSO4 (added at 0.5 \nmM) and then treated with cycloheximide (CHX; 100 μg/mL) to block protein synthesis. The \ncultures were kept at 30°C, and the V5-Cak1 levels were monitored by immunoblotting (top) of \nsamples taken at the indicated time points. Loading is shown at the bottom from the same blots \nstained with Ponceau. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n47 \n \n \nFIGURE EV2. Eliminating the uORF of CAK1 does not affect cell cycle kinetics in batch \ncultures. (A) Cell cycle parameters for the uORF-16 CAK1 (MSP256) and uORF-0 CAK1 \n(MSP257) strains shown on the x-axis, grown in the indicated media. The boxplots, drawn as \ndescribed in Figure 1, show (from top to bottom): birth size, mean cell size, %budding, and \ndoubling time (Td), on the y-axis. Values were obtained from independent experiments. No \nsignificant differences were found between the strains (p > 0.05, Wilcoxon rank sum test). (B) \nCell cycle progression of elutriated synchronous early G1 cells in YP-glycerol. The percentage \nof budded cells is plotted against cell size (fL) for the same strains as in (A). The plots show \nLoess curves with 95% confidence intervals. Red lines indicate the critical size for budding (the \nsize at which 50% of cells have budded).  \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n48 \n \n \nFIGURE EV3. Overexpression of CAK1 in the stationary phase reduces cell size and \nviability. (A) Cell size distribution of stationary phase cells (BY4743) with (right panel) or without \n(left panel) the pCUP1-V5-CAK1 plasmid. Histograms show cell counts (y-axis) by size (x-axis) \nfrom 6-day cultures. Density lines represent cultures induced with 0.5 mM CuSO4 to express \nV5-Cak1 (yellow) versus mock-treated controls (blue). (B) DNA content analysis of the same \nsamples shown in (A), measured by flow cytometry. Histograms show cell counts (y-axis) vs. \nfluorescence per cell (x-axis). The average percentage (± SD) of cells in the G1 phase from \nthree independent cultures is indicated. (C) Representative cell viability plates for cultures \ncarrying the pCUP1-V5-CAK1 plasmid, as shown in (A, right panel). Cultures were either \nmock-treated (left plates) or induced with CuSO4 (right plates) before plating on media lacking \nCuSO4. Viability dropped from 67% in mock-treated cultures to 19% in CuSO₄-treated cultures \n(χ2= 8123.4, df = 1, p < 2.2e-16). \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint \n\n \n49 \n \nFIGURE EV4. Proliferation of type 2C protein phosphatase mutants with or without the \nuORF in CAK1. (A) Growth spot assays to assess the relative growth fitness of the indicated \nstrains (all in the BY4742 background) on glucose (YPD) or glycerol (YPGlycerol) media. \nOvernight cultures were normalized to an initial cell density and aliquots were spotted at 10-fold \nserial dilutions. The plates were imaged after 2 (glucose) or 3 (glycerol) days at 30°C. (B) \nGrowth curves of the strains shown in (A), cultured in YPD glucose medium. Ln-transformed \nabsorbance values (600nm) are plotted on the y-axis against time (x-axis). Measurements were \ntaken using a microplate reader and corrected for background absorbance of the sterile \nmedium. Data are presented as Loess regression curves; shaded regions represent 95% \nconfidence intervals (n=8 independent replicates per strain). \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.28.702203doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}