Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae

Abstract

In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Delta mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Delta cells, as in mec1Delta cells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1Delta mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Delta mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

FIG. 1

Interaction between Mec1 and Pie1 in the two-hybrid assay. (A) Pie1 interacts with Mec1 in the two-hybrid assay. Strain PJ69-4A carrying pBD-MEC1(2–2368) was transformed with pAD-PIE1 or the control vector. Transformants were streaked on an SC-Ura-Leu-His plate containing 10 mM AT. (B) Identification of the Mec1 region required for its interaction with Pie1. Strain PJ69-4A carrying pAD-PIE1 was transformed with pBD-MEC1(2–2368), pBD-MEC1(2–1399), pBD-MEC1 (2–938), pBD-MEC1(2–500), pBD-MEC1(496–1590), or pBD-MEC1(1586–2368). The kinase domain and central homologous region of Mec1 are indicated as black and gray bars, respectively. Transformants were streaked on an SC-Ura-Leu-His plate containing 10 mM AT. Interaction with Pie1 was assessed by measuring the growth of transformants.

FIG. 2

Structure and alignment of Pie1, S. pombe Rad26, and A. nidulans UVSD. The Pie1, S. pombe Rad26, and A. nidulans UVSD proteins contain 747, 614, and 778 amino acids, respectively. Alignment of the conserved regions of these three proteins is shown below. Amino acids that are identical or conserved are indicated by black and gray boxes, respectively.

FIG. 3

Interaction between Mec1 and Pie1 in vivo. Extracts were prepared from MEC1-HA (KSC1212), PIE1-myc (KSC1213), and MEC1-HA PIE-myc (KSC1214) cells, and subjected to immunoprecipitation (IP) with anti-HA (left panel) or anti-myc (right panel) antibodies. The immunoprecipitates were separated by SDS-PAGE and subjected to immunoblot analysis with anti-HA or anti-myc antibodies.

FIG. 4

pie1Δ lethality is suppressed by the sml1 Δ mutation. The pie1Δ∷TRP1/+ sml1Δ∷LEU2/+ diploid was sporulated and dissected on a YEPD plate. Seven tetrads are displayed vertically. Sporulated tetrads grown up on a YEPD plate were replica-plated to a SC-Trp or SC-Leu plate. Cells proliferating on SC-Trp are all Leu⁺, indicating that pie1Δ∷TRP1 sml1Δ∷LEU2 double mutants are viable.

FIG. 5

Sensitivity of pie1Δ, mec1Δ, and mec1Δ pie1Δ mutants to HU, MMS, and UV. sml1Δ (KSC1178), pie1Δ sml1Δ (KSC1180), mec1Δ sml1Δ (KSC1186), and mec1Δ pie1Δ sml1Δ (KSC1196) cells were grown to log phase and treated with HU, MMS, or UV light. The viability of cells was estimated as described in Materials and Methods.

FIG. 6

G₂/M-phase DNA damage checkpoint in pie1Δ and mec1Δ mutants. sml1Δ (KSC1178), pie1Δ sml1Δ (KSC1180), and mec1Δ sml1Δ (KSC1186) cells were arrested with nocodazole and irradiated or not irradiated with UV. At the indicated times after release of UV-irradiated (+UV) and unirradiated (−UV) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining.

FIG. 7

HU- and MMS-induced Rad53 modification in pie1Δ and mec1Δ mutants. sml1Δ (KSC1178), pie1Δ sml1Δ (KSC1180), and mec1Δ sml1Δ (KSC1186) cells carrying YCp-RAD53-HA were left untreated or treated with 10 mg/of HU per ml for 120 min or 0.1% MMS for 30 min and then subjected to immunoblot analysis as described in Materials and Methods.

FIG. 8

Analysis of mutations of Pie1 at the conserved domain and the carboxyl terminus. (A) Mutations of Pie1 at the conserved and carboxyl-terminal regions. The structures and conserved regions of Pie1 and S. pombe Rad26 are shown. The pie1-KA mutation changes lysine to alanine at amino acid positions 177 and 178 (indicated by dots) within a conserved region. The pie1-ΔC gene product lacks the carboxyl-terminal 47 amino acids. An asterisk marks the location of the rad26.a14 mutation. (B) Sensitivity of the pie1-KA and pie1-ΔC mutants to HU, MMS, and UV. pie1Δ sml1Δ (KSC1234) cells were transformed with YCpT-PIE-myc, YCpT-PIE1-KA-myc, YCpT-PIE1-ΔC-myc, or the control vector. The transformants were grown to log phase and treated with HU, MMS, or UV light. The viability of cells was estimated as described in Materials and Methods. (C) Interaction of the Pie1-KA and Pie1-ΔC mutant proteins with Mec1. Extracts were prepared from MEC1-HA pie1Δ sml1Δ (KSC1286) cells carrying YCpT-PIE-myc (WT), YCpT-PIE1-KA-myc (KA), or YCpT-PIE1-ΔC-myc (ΔC) and subjected to immunoprecipitation (IP) with anti-HA antibodies. The extracts and immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-HA or anti-myc antibodies.

FIG. 9

Effect of the pie1-KA mutation on the intracellular distribution of Mec1 and Pie1. MEC1-HA pie1Δ sml1Δ (KSC1286) cells carrying YCpT-PIE-myc, YCpT-PIE1-KA-myc, or the control vector YCplac22 were harvested and spheroplasted. Spheroplasts were homogenized to prepare whole-cell extracts (W) and then separated into the cytoplasmic (C) and nuclear (N) fractions. Samples from each fraction were separated by SDS-PAGE and immunoblotted with anti-HA, anti-myc, anti-Ssb1, or anti-nuclear pore complex (NPC) antibodies.

FIG. 10

Protein kinase activity associated with Mec1. Cells grown to the mid-log phase were incubated with or without 0.1% MMS for 1 h and harvested for preparation of crude extracts. Extracts were subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitated HA-tagged Mec1 proteins were assayed for kinase activity using GST-Rad53 as a substrate, as described in Materials and Methods. In the top panel, ³²P incorporation into GST-Rad53 was detected by autoradiography. In the bottom panel, the amount of the Mec1 protein used for the kinase assay was examined by immunoblotting. (A) mec1-1 sml1-1 (KSC783) cells carrying YEp-MEC1-HA (WT), YEp-MEC1-KN-HA (KN), or the control vector pRS426 (−), (B) MMS-treated (+) or untreated (−) sml1Δ (KSC1178) and MEC1-HA sml1Δ (KSC1215) cells; (C) MEC1-HA sml1Δ (KSC1215) and MEC1-HA pie1Δ sml1Δ (KSC1286) cells.