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The Whi3 protein is associated with the endoplasmic reticulum, interacts with Cdc28, the budding-yeast Cdk, binds the mRNA of cyclin CLN3 and prevents accumulation of the Cdc28-Cln3 in the nucleus until late G(1). Besides its function as a cell size regulator, Whi3 is strictly required for filamentous growth. Here we show that emerging buds in Whi3-deficient cells are considerably rounder than in wild-type cells, indicating that Whi3 is required to maintain apical growth during S phase. This defect was not suppressed by deletion of CLB2, which is involved in switching from polar to isotropic bud growth, indicating that the observed phenotype is not the result of Whi3 acting solely as a negative regulator of cyclin Clb2. However, Cdc28 did not properly accumulate at the bud tip during S phase in whi3Delta cells, and their elongation defects were suppressed by CLN2 overexpression, suggesting a positive function for Whi3 in a Cdk-cyclin-dependent step required for apical growth. Additionally, the actin cytoskeleton was perturbed in Whi3-deficient cells, and WHI3 showed genetic interactions with actin patch components. Our results point to Whi3 as a key modulator of apical growth effectors to coordinate cell cycle events and morphogenesis. We propose that Whi3 is required for the apical localization of Cdc28-Cln1,2 complexes during bud growth and thereby, to promote the activation of Cdc42 and its effectors in the bud apex. Show less

Whi3 is an RNA-binding protein associated with the endoplasmic reticulum (ER) that binds the CLN3 mRNA and plays a key role in the efficient retention of cyclin Cln3 at the ER. In the present work, we have identified new Whi3-associated mRNAs by a genomic approach. A large and significant number of these Whi3 targets encode for membrane and exocytic proteins involved in processes such as transport and cell wall biogenesis. Consistent with the genomic data, we have observed that cell wall integrity is compromised in Whi3-deficient cells and found strong genetic interactions between WHI3 and the cell integrity pathway. Whi3-associated mRNAs are enriched in clusters of the tetranucleotide GCAU, and mutation of the GCAU clusters in the CLN3 mRNA caused a reduction in its association to Whi3, suggesting that these sequences may act as cis-determinants for binding. Our data suggest that Whi3 is involved in the regulation and/or localization of a large subset of mRNAs functionally related to the ER and, since it is important for different molecular processes such as cytoplasmic retention or exocytic traffic of proteins, we propose that Whi3 is a general modulator of protein fate in budding yeast. Show less

Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G(1) and released by specific chaperones in late G(1) to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start. Show less

G1 cyclin Cln3 plays a key role in linking cell growth and proliferation in budding yeast. It is generally assumed that Cln3, which is present throughout G1, accumulates passively in the nucleus until a threshold is reached to trigger cell cycle entry. We show here that Cln3 is retained bound to the ER in early G1 cells. ER retention requires binding of Cln3 to the cyclin-dependent kinase Cdc28, a fraction of which also associates to the ER. Cln3 contains a chaperone-regulatory Ji domain that counteracts Ydj1, a J chaperone essential for ER release and nuclear accumulation of Cln3 in late G1. Finally, Ydj1 is limiting for release of Cln3 and timely entry into the cell cycle. As protein synthesis and ribosome assembly rates compromise chaperone availability, we hypothesize that Ydj1 transmits growth capacity information to the cell cycle for setting efficient size/ploidy ratios. Show less

The G1 cyclin Cln3 is a key activator of cell-cycle entry in budding yeast. Here we show that Whi3, a negative G1 regulator of Cln3, interacts in vivo with the cyclin-dependent kinase Cdc28 and regulates its localization in the cell. Efficient interaction with Cdc28 depends on an N-terminal domain of Whi3 that is also required for cytoplasmic localization of Cdc28, and for proper regulation of G1 length and filamentous growth. On the other hand, nuclear accumulation of Cdc28 requires the nuclear localization signal of Cln3, which is also found in Whi3 complexes. Both Cln3 and Cdc28 are mainly cytoplasmic during early G1, and become nuclear in late G1. However, Whi3-deficient cells show a distinct nuclear accumulation of Cln3 and Cdc28 already in early G1. We propose that Whi3 constitutes a cytoplasmic retention device for Cln3-Cdc28 complexes, thus defining a key G1 event in yeast cells. Show less

Eukaryotic cells commit in G1 to a new mitotic cycle or to diverse differentiation processes. Here we show that Whi3 is a negative regulator of Cln3, a G1 cyclin that promotes transcription of many genes to trigger the G1/S transition in budding yeast. Whi3 contains an RNA-recognition motif that specifically binds the CLN3 mRNA, with no obvious effects on Cln3 levels, and localizes the CLN3 mRNA into discrete cytoplasmic foci. This is the first indication that G1 events may be regulated by locally restricting the synthesis of a cyclin. Moreover, Whi3 is also required for restraining Cln3 function in meiosis, filamentation, and mating, thus playing a key role in cell fate determination in budding yeast. Show less

Moderate hyperosmotic stress on Saccharomyces cerevisiae cells produces a temporary delay at the G1 stage of the cell cycle. This is accompanied by transitory downregulation of CLN1, CLN2 and CLB5 transcript levels, although not of CLN3, which codes for the most upstream activator of the G1/S transition. Osmotic shock to cells synchronized in early G1, when Cln3 is the only cyclin present, causes a delay in cell cycle resumption. This points to Cln3 as being a key cell cycle target for osmotic stress. We have observed that osmotic shock causes downregulation of the kinase activity of Cln3-Cdc28 complexes. This is concomitant with a temporary accumulation of Cln3 protein as a result of increased stability. The effects of the osmotic stress in G1 are not suppressed in CLN3-1 cells with increased kinase activity, as the Cln3-Cdc28 activity in this mutant is still affected by the shock. Although Hog1 is not required for the observed cell cycle arrest in hyperosmotic conditions, it is necessary to resume the cell cycle at KCl concentrations higher than 0.4 M. Show less

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition. Show less

Diploid yeast cells switch from mitosis to meiosis when starved of essential nutrients. While G1 cyclins play a key role in initiating the mitotic cell cycle, entry into meiosis depends on Ime1, a transcriptional activator regulated by both nutritional and cell-type signals. We show here that G1 cyclins downregulate IME1 transcription and prevent the accumulation of the Ime1 protein within the nucleus, which results in repression of early-meiotic gene expression. As G1-cyclin deficient cells do not require nutrient starvation to undergo meiosis, G1 cyclin would exert its role by transmitting essential nutritional signals to Ime1 function. The existence of a negative cross-talk mechanism between mitosis and meiosis may help explain why these two developmental options are incompatible in budding yeast. Show less

Nutrients are among the most important trophic factors in all organisms. When deprived of essential nutrients, yeast cells use accumulated reserves to complete the current cycle and arrest in the following G1 phase. We show here that the Cln3 cyclin, which has a key role in the timely activation of SBF (Swi4-Swi6)- and MBF (Mbp1-Swi6)-dependent promoters in late G1, is down-regulated rapidly at a post-transcriptional level in cells deprived of the nitrogen source. In addition to the fact that Cln3 is degraded faster by ubiquitin-dependent mechanisms, we have found that translation of the CLN3 mRNA is repressed approximately 8-fold under nitrogen deprivation conditions. As a consequence, both SBF- and MBF-dependent expression is strongly down-regulated. Mainly because of their transcriptional dependence on SBF, and perhaps with the contribution of similar post-transcriptional mechanisms to those found for Cln3, the G1 cyclins Cln1 and 2 become undetectable in starved cells. The complete loss of Cln cyclins and the sustained presence of the Clb-cyclin kinase inhibitor Sic1 in starved cells may provide the molecular basis for the G1 arrest caused by nitrogen deprivation. Show less