The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle

Abstract

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.

FIG. 1

Overexpression of the Ser/Thr phosphatase Ppz1 produces a phenotype of slow growth that is rescued by overexpression of Hal3. (A) Wild-type strain JA-100 was transformed with plasmid YEplac195 (●), plasmid YEplac195-PPZ1 (▾), plasmid YEp-PPZ2 (■), or plasmids YEplac195-PPZ1 and YEp351-HAL3 (⧫). Positive clones were grown overnight in CM medium lacking uracil, diluted to an OD₆₆₀ of 0.15, and then grown for the indicated times in the same medium. Data are means ± SEM of three experiments. The inset shows the flow cytometry analysis of cells carrying the empty plasmid YEplac195 (upper profile) or plasmid YEplac195-PPZ1 (lower profile). Samples were taken at equivalent optical densities to avoid interferences due to the availability of nutrients. (B) Wild-type JA-100 cells were transformed with plasmid pYES2 (●), plasmid pYES2-PPZ1 (▾), or plasmid pYES2-PPZ1 plus plasmid YEp351-HAL3 (■). Cells were grown overnight on CM medium lacking uracil in the presence of 2% glucose; then an aliquot was inoculated (to achieve a starting optical density of 0.01) in minimal medium lacking uracil and containing 2% galactose. Growth was resumed, and samples were taken at the indicated times. Data are means ± SEM of four experiments.

FIG. 2

Overexpression of Ppz1 causes a delay in G₁/S transition and G₁ cyclin expression. (A) JA-100 cells carrying plasmid YEplac181 (●) or YEplac181-PPZ1 (▾) were arrested in G₁ phase by incubation with α-factor (20 μg/ml) for 3 h. Cells were collected, washed, and resuspended in fresh CM medium lacking leucine. Samples were taken at the indicated times, and the budding index of the culture was determined by microscopic observation. Data are means ± SEM of four experiments. (B) Control cells (with plasmid YEplac181; left) and cells overexpressing Ppz1 (with plasmid YEplac181-PPZ1; right) were grown as indicated, and samples were taken for DNA content determination by flow cytometry. (C) Cells were arrested as indicated. After release, samples of cells were taken at the indicated times and total RNA was isolated. Equivalent amounts (5 μg) of total RNA were run in formaldehyde-agarose gels, transferred to membranes, and hybridized with the indicated DNA probes. Hybridization with the ACT1 probe and the ethidium bromide staining of rRNA are shown for comparison.

FIG. 3

Deletion of PPZ1 mimics the effect of overexpression of HAL3 in sit4 cells. The following strains were streaked on CM plates lacking leucine: wild-type JA-100, JA-110 (PPZ1 sit4), JA-110 containing plasmid YEp351-HAL3, JA-112 (ppz1 sit4), JA-112 with plasmid YEp351-HAL3, and JA-112 with plasmid YCp111-PPZ1. Strains without an specific plasmid were transformed with YEplac181 to allow growth in the selective medium. Growth was scored after 3 days.

FIG. 4

Deletion of PPZ1 accelerates the entry into G₁/S transition of a sit4 mutant. (A) Strains JA-100 (wild type; ●), JA-110 (sit4; ▿), JA-110 containing plasmid YEp351-HAL3 (■), JA-112 (ppz1 sit4; ◊), JA-112 containing plasmid YEp351-HAL3 (▴), and JA-112 containing plasmid YCplac111-PPZ1 () were arrested in G₁ phase by incubation with α-factor and released from arrest as indicated. Cells were taken at the indicated times, and the budding index was determined by microscopic counting. Data are means ± SEM of six experiments. (B) Analysis of DNA content of wild-type (wt), sit4 (strain JA-110), and sit4 ppz1 (strain A-112) cells by flow cytometry. Cells were arrested as described above, and samples were taken at different times after release from α-factor arrest. (C) The indicated strains were released form α-factor arrest, and samples were taken at various times. Total RNA was prepared, electrophoresed, transferred to membranes, and hybridized with the indicated probes. Ethidium bromide staining of rRNAs is shown for comparison.

FIG. 5

Ppz1 function is not mediated by BCK2 or CLN3. (A) Strains JA-100 (wild type [wt]), CML211 (cln3Δ), and JA-170 (bck2Δ) were transformed with plasmid YEplac195 (upper half) or YEplac195-PPZ1 (lower half). Positive clones were streaked on CM plates lacking uracil and grown at 28°C for 2 days. (B) Strain JA-114 (MATα sit4Δ pz1Δ) was crossed with strain CML211 (MATa cln3Δ). The resulting diploid strain, JA-190, was sporulated, and ppz1 sit4 cln3 triple (but not sit4 cln3 double) mutants were recovered. Growth of the triple mutant was compared with that of cln3 cells (which grow nearly like a wild-type strain), sit4 cells, and sit ppz4 cells by streaking the cultures in YPD plates and incubating them at 28°C for 2 days.

FIG. 6

Genetic interactions between the sit4, bem2, and ppz1 mutations. (A) Strains JA-100 (wild type [wt]), JA-110 (sit4), JA-130 (bem2), JA-140 (sit4 bem2), JA-112 (sit4 ppz1), and JA-151 (sit4 ppz1 bem2) were grown at 28°C on YPD plates. Growth was scored after 2 days. (B) Strains JA-100 (wild type [wt]), JA-130 (bem2), JA-131 (ppz1 bem2), JA-150 (bem2 hal3), and JA-152 (ppz1 bem2 hal3) were streaked on YPD plates and grown at 37°C for 2 days.

FIG. 7

Schematic diagram of the role of the Ser/Thr phosphatase Ppz1 in cell cycle regulation. See Discussion for details.