Supplemental Figures:

Supplemental Figure 1. Growth of other cdc mutants. (A) cdc28-13 (A17896) cells were treated as described in Figure 1 and cell volume was determined at the indicated time points. Mode cell volume for each time point was plotted. (B) Expression of stable Glb2 causes cells to grow more than APC mutants even at 30ºC. GAL-CLB2dbD (A17873) and MET-CDC20 (A7334) were grown overnight in media w/o methionine and supplemented with 2% rafinose. Next morning cells were diluted in YEP supplemented with 2% rafinose, 1% galactose and 8mM methionine to induce the expression of CLB2dbD or to repress the expression of CDC20 (Time 0h). Cells were grown at 30ºC and cell size was measured at indicated time-points using the Coulter counter.

Supplemental Figure 2: FUS3 is required for the growth inhibitory effects of pheromone and BAR1 sensitizes cells to pheromone.

(A) cdc28-4 (A17132) and cdc28-4 fus3D (A18156) cells were treated as in Figure 3A and cell volume distributions were determined at the indicated times. The histograms above each plot displays the cell volume distribution 6 hours after temperature shift.

(B) cdc28-as1 mutants carrying either BAR1 (A18376) or bar1::HisG (A4370) alleles were grown in parallel in YPE media supplemented with 2% glucose at 30ºC. At time 0 hours, CDK inhibitor 1-NM-PP1 was added to both strains to 5mM final concentration. Each strain was further split into two equal volumes and one part was treated with pheromone (red symbols) to 20g/ml final concentration and the other part was left untreated (black symbols).

Supplementary Figure 3. Genes in the pheromone response pathway affect cell size during exponential growth.

The degree of enrichment or depletion [log(Cy5/Cy3)] was plotted versus the average intensity [0.5*log(Cy5*Cy3)] for each barcode in the deletion pool. Elutriation experiments are shown for (A) glucose and (B) ethanol. The approximate signal significance threshold is indicated by the dashed red line. Deletion of mating pathway genes confers a carbon source dependent large cell size. The barcodes for Ste4, Ste5, Ste7, and Ste11 are shown. In contrast, most DNA replication and cytokinesis gene deletions, for example, display a large size phenotype in both glucose and ethanol. Only barcodes for those DNA replication and cytokinesis gene deletions with an average large cell size in glucose (for at least one barcode) are shown. Similarly, deletion of cytosolic or mitochondrial ribosomal protein (RP) genes imparts a small cell size irrespective of carbon source. Mitochondrial RP gene deletions additionally display a loss of viability in ethanol and reduced abundance in the deletion pool, indicated by an overall decrease in barcode signal intensity.

Supplemental Figure 4: ste12D does not alleviate the growth defects of SCF and MEN mutants and SPA2 is required for full effects of pheromone. (A) Exponentially growing cdc34-2 (A1467) single and cdc34-2 ste12D (A21694) double mutants were shifted to 37ºC in YPE media supplemented with 2% glucose at time 0 h. Cell volume distribution was determined at the indicated times and the mode cell volume was plotted. The cell volume distribution for the 8 h time point is displayed next to the graph.

(B) The cell volume distributions of the MEN mutants cdc15-2 (A2596) and cdc15-2 ste12D (A21693) were determined as in (A).

(C) Exponentially growing cdc28-4 single and cdc28-4 spa2D (A23020) double mutants were shifted to 34ºC in YPE media supplemented with 2% glucose at time 0 h. At this time half of each culture was treated with pheromone (+af curves) and pheromone was re-added every 2h. Cell volume distribution was determined at the indicated times and the mode cell volume was plotted. The cell volume distribution for the 8 h time point is displayed next to the graph.

Supplemental Figure 5: Growth rates change during the cell cycle.

Multiple repetitions of the experiment described in Figure 7. Elutriation experiments (A) and cdc28-as1 releases (B) were performed as described in Figure 7 with the exception of panel B, where WT (A11311) cells were grown in 2% raffinose overnight and were incubated in YEP without sugar during elutriation.

Supplemental Figure 6: Measurement of cell volume increases of single cells. The measurements for the 20 individual cells used to construct the data in Figure 7C are presented. Cells were grown and analyzed as described in Figure 7C-D. Bud were detected at time 0min, and cell volume is plotted with respect to that time. Cells in panels 1 and 5 are the ones presented in Figure 7D.

Supplemental Figure 7: Statistical descriptors of populations of cells.

Cell volume distribution of cdc28-4 cells (A17321) at the permissive temperature (A) or after 6 h at 37ºC (B). (C) Cell volume distribution of cdc28-as1 cdc34-2 after 6 h at 37ºC. The mean, median, and mode for each population of cells are indicated in black. The percentage of cells falling within the “growing” population is indicated in red and the boundaries of the “growing” population are indicated by vertical lines. The mean, median, and mode specific to the “growing” population are indicated in red.

Supplemental Table 1: Raw Data from the Elutriation Size Screen.

Strains from the yeast deletion collection were manipulated as described in Figure 4 and Materials and Methods. Microarray signal intensity was calculated from the comparison of the samples collected before and after elutriation in the given experiments.

Conditions for each experiment are described in Materials and methods. Glucose-1

and Glucose-2 are different fractions obtained during elutriation, with glucose-1 cells having a median cell size of 29 fL and glucose-2 cells of 25 fL contain data from independent repetitions of the experiment. Negative values indicate large cells or cells sensitive to pheromone. Positive values indicate small cells or cells resistant to pheromone. Within the functional categories columns, "1" indicate that a gene is assigned to the given category, and "0" that it is not assigned to that category.

Supplemental Table 2. Saccharomyces cerevisiae strains used in this study*

W303 / ade2-1, leu2-3, ura3, trp1-1, his3-11,15, can1-100, GAL, psi+
A344 / MATa, cdc7-1
A785 / MATalpha, cdc23-1
A937 / MATalpha, cdc20-1
A1467 / MATa, cdc34-2
A1469 / MATa, cdc53-1
A2587 / MATa
A2589 / MATa, bar1::HisG
A2594 / MATa, cdc28-4, MET-
A2596 / MATa, cdc15-2
A2599 / MATalpha, cdc9-1
A2624 / MATa, cln1::hisG, cln2::del, cln3::LEU2, MET-CLN2::TRP1
A4370 / MATa, bar1::HisG, cdc28-as1
A5321 / MATa, cdc14-3
A7334 /

MATa, MET-CDC20::URA3

A11311 /

MATa, ade1::HIS3, lys2::KanMX

A17132 / MATa, cdc28-4, MET+
A17188 / MATa, cdc28-as1, cdc34-2
A17050 / MATalpha, cdc15-2,::cdc28-as1
A17051 / MATalpha, cdc20-1, cdc28-as1
A17872 / MATa, cdc15-as1::URA3,
A17873 / MATa, GAL-CLB2dbD:URA3,
A17874 / Mata, cdc15-as1::URA3, GAL-CLB2dbD:URA3
A17896 / MATa, cdc28-13
A17898 / MATa, cdc28-1
A18156 / MATa, cdc28-4, fus3D::KanMX
A18204 / MATa, cdc28-4, far1D::KanMX
A18205 / MATa, cdc28-4, ste11D::KanMX
A18221 / MATa, cdc42-6::URA3
A18238 / MATa, cdc28-4, bni1D::URA3
A18316 / MATalpha, cdc15-as1::URA3, cln1::hisG, cln2::del, cln3::LEU2, MET-CLN2::TRP1
A18367 / MATa, BAR1, cdc28-as1
A18434 / MATa, cdc28-4, ste12D::KanMX
A18982 / MATalpha, cdc15-as1::URA3, CLN2-3xHA::LEU2
A18986 / MATalpha, cdc15-as1::URA3, GAL-CLB2dbD::URA3, CLN2-3xHA::LEU2
A19911 / MATa, cdc28-4, bnr1D::KanMX
A20752 /

MATa, cdc25-1, ADE2

A21093 /

MATa, cdc25-1, cdc28-as1

A21693 /

MATa, cdc15-2, ste12D::KanMX

A21694 /

MATa, cdc34-2, ste12D::KanMX

A21947 /

MATa, cdc28-4, kss1D::KanMX

A22928 /

MATa, cdc28-4, bar1D::HisG

A23020 /

MATa, cdc28-4, spa2D::HIS3

MT1567 /

MATa ura3 leu2 his3::hisG trp1::hisG

*All strains except MT1567 are in the W303 strain background