Nuclear Import of Ho Endonuclease Utilizes Two Nuclear Localization Signals and Four Importins of the Ribosomal Import System *

Activity of Ho, the yeast mating switch endonuclease, is restricted to a narrow time window of the cell cycle. Ho is unstable and despite being a nuclear protein is exported to the cytoplasm for proteasomal degradation. We report here the molecular basis for the highly efficient nuclear import of Ho and the relation between its short half-life and passage through the nucleus. The Ho nuclear import machinery is functionally redundant, being based on two bipartite nuclear localization signals, recognized by four importins of the ribosomal import system. Ho degradation is regulated by the DNA damage response and Ho retained in the cytoplasm is stabilized, implying that Ho acquires its crucial degradation signals in the nucleus. Ho arose by domestication of a fungal VMA1 intein. A comparison of the primary sequences of Ho and fungal VMA1 inteins shows that the Ho nuclear localization signals are highly conserved in all Ho proteins, but are absent from VMA1 inteins. Thus adoption of a highly efficient import strategy occurred very early in the evolution of Ho. This may have been a crucial factor in establishment of homothallism in yeast, and a key event in the rise of the Saccharomyces sensu stricto. Ho endonuclease initiates a mating type switch in Saccharomyces cerevisiae and related yeasts by making a site-specific double strand break in a 24-bp cognate site in the mating type gene, MAT. Repair of the double strand break is by gene conversion using one of the silent cassettes of mating type information (HML␣ or HMRa) as a template. Repair occurs before replication of the MAT locus and each daughter cell has the new mating type with a regenerated Ho cognate site (1). Ho activity is tightly regulated: HO is transcribed briefly at the end of G 1 , its transcription is restricted to haploid mother cells, i.e. cells that have divided at least once (2), and the protein is rapidly degraded by the ubiquitin-26 S proteasome system (3). Cells in which Ho is retained in the nucleus beyond its normal time window of activity show perturbation of the cell cycle (4). Ho is marked for degradation by functions of the DNA damage response (DDR), 7 specifically the MEC1, RAD9, and CHK1 pathway (5). Despite being a nuclear protein, Ho must exit the nucleus to be degraded in the proteasomes. The DDR functions are important for Ho phosphorylation: phosphorylation of threonine 225 is crucial for Ho nuclear export and additional phosphorylations are required for recruitment of Ho for ubiquitylation. Ho is ubiquitylated by the SCF (Skp1-Cdc53-F-box protein) E3 ubiquitin ligase complex, to which it is recruited by the F-box protein Ufo1 (6). In mec1 mutants Ho is stabilized and accumulates in the nucleus; conversely trapping Ho in the nucleus by deletion of its nuclear exportin, Msn5, leads to stabilization of the protein (4). Ddi1 binds ubiquitylated Ho and is required for interaction of Ho with the proteasome; in its absence Ho is stabilized. The finding that Ho is not degraded within the nucleus, but in the cytoplasm, is further strengthened by the direct demonstration of accumulation of ubiquitylated Ho in the cytoplasm of ⌬ddi1 mutants (7). Ho nuclear import is very rapid and efficient. Ectopic expression of HO leads to rapid cleavage of MAT (8), and to a mating type switch at any phase of the cell cycle in both mother and daughter cells. This indicates that there is no impediment to its nuclear import (9). Macromolecules are conveyed through nuclear pore complexes in the nuclear envelope by soluble karyopherins. Karyopherins comprise two structurally related families, ␣-and ␤-karyopherins. These recognize specific nuclear localization sequence (NLS) peptide motifs in the cargo molecule: NLSs may comprise a short stretch of basic residues (classical/ cNLS), or two basic clusters 10 -12 residues apart (bipartite NLS) (10). Cargoes may be recognized by an adaptor protein, ␣-karyopherin/Srp1, which mediates their binding to the transport receptor, ␤-karyopherin/ Kap95 (11). Additionally, a family of about 14 ␤-karyopherins bind an array of cargoes directly and also makes contacts with the nucleoporin subunits of the nuclear pore complexes. Directionality of transport is determined by interaction with the GTPase Ran/yeast Gsp1. RanGTP is at a high concentration in the nucleus due to the asymmetric distribution of the Ran regulators. The nuclear guanine nucleotide exchange factor, RanGEF/yeast Prp20, converts RanGDP to RanGTP, whereas the GTPase activating protein, RanGAP/yeast Rna1, is localized in the cytoplasm and catalyzes the hydrolysis of RanGTP. Importin-cargo complexes assemble in the cytoplasm and after translocation into the nucleus they dissociate upon binding of RanGTP to the importin (12). To investigate how the efficient nuclear import that supports the unique biological function of Ho is achieved we located and analyzed its nuclea

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

<i>ASH1</i> and <i>OXA1</i> mRNA granules interact with PBs.

<p>A. <i>ASH1-MS2L</i> cells at <i>A</i>₆₀₀ = 0.5 with p<i>CP-MS2L-GFPx3</i> and the PB marker, Edc3^mCherry, either untreated, treated with 1 mM arsenate or with 8.8 mM H₂O₂ for 30 minutes or transferred to SC without glucose for 30 minutes. Merge ×5 represents 5 times enlargement of selected granules indicated with white arrows in the whole cells. B. <i>OXA1-MS2L</i> cells treated as in A., and visualized by confocal microscopy. C. Histograms of <i>ASH1-MS2L</i> cells or D. <i>OXA1-MS2L</i> cells, showing percentages of overlapping, docked, or distinct granule types in a population of cells untreated, treated with 1 mM arsenate or with 8.8 mM H₂O₂ for 30 minutes or stressed in SC without glucose (n = >100 cells).</p

Transcription of <i>UFO1</i> in response to arsenate, H₂O₂, and UV.

<p>A. Wild type cells were grown in SC 2% glucose medium overnight, diluted to <i>A</i>₆₀₀ = 0.1, regrown to <i>A</i>₆₀₀ = 0.5 and treated with 1 mM arsenate, 8.8 mM H₂O₂, or irradiated with 40 mJ/cm² UV. Aliquots were collected at the indicated times and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells). B. p<i>GAL-GFP-UFO1</i> was expressed in <i>ufo1Δ</i> mutants by overnight induction with 2% galactose. Next morning cells were diluted to <i>A</i>₆₀₀ = 0.1, regrown to <i>A</i>₆₀₀ = 0.5 then untreated, or stressed with 1 mM arsenate or 8.8 mM H₂O₂ for 30 minutes, or irradiated with 40 mJ/cm² UV. The cells were washed and transferred to SC with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells). C. Western blot analysis of Ufo1^GFP protein produced from the tagged genomic <i>UFO1-GFP</i> gene. Anti-GFP antibodies were used to detect Ufo1^GFP and anti-α-tubulin antibodies to detect α-tubulin that serves as a loading control. D. <i>UFO1</i> mRNA levels in untreated wild type, <i>yap1Δ</i> or <i>pdr1Δ</i> mutants. mRNA levels were normalized to <i>ACT1</i> and to w.t. mRNA levels. E. <i>yap1Δ</i> or <i>pdr1Δ</i> mutants grown, treated and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002527#pgen-1002527-g001" target="_blank">Figure 1A</a>. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells).</p

<i>UFO1-MS2L</i> and <i>MFA2-U1A</i> mRNAs are sequestered in the same PBs after arsenate stress.

<p>A. Control wild type cells at <i>A</i>₆₀₀ = 0.5 producing U1A^GFP untreated or treated with 1 mM arsenate for 30 minutes. B. wild type <i>UFO1-MS2L</i> cells at <i>A</i>₆₀₀ = 0.5 expressing p<i>MFA2-U1A</i>, with their respective RNA-binding proteins, CP^mCherry and U1A^GFP, untreated or treated with 1 mM arsenate and stained with Hoechst 33342 at a final concentration of 2.5 µg/mL for 30 minutes.</p

Yeast strains.

<p>Yeast strains.</p

Stress-induced granules appear only in <i>UFO1-MS2L</i> stressed cells.

<p>A. Wild type and <i>UFO1-MS2L</i> cells transformed with p<i>CP-MS2L-GFPx3</i> at <i>A</i>₆₀₀ = 0.5, were transferred to SC 2% glucose without methionine for 1 hour to induce the CP^GFP. Cells were untreated or exposed to 1 mM arsenate, 8.8 mM H₂O₂, or UV-irradiated with 40 mJ/cm². Aliquots were collected 30 minutes after each treatment. Fluorescent granules appear only in cells with the tagged <i>UFO1-MS2L</i> gene. B. <i>UFO1-MS2L</i> cells expressing p<i>CP-MS2L-GFPx3</i> grown as above, treated with 1 mM arsenate for 30 minutes (stress), and transferred to fresh SC glucose medium. Stress recovery 30′ and 60′ indicate time after transfer to fresh SC. C. <i>UFO1-MS2L</i>, <i>yap1Δ</i> or <i>UFO1-MS2L</i>, <i>pdr1Δ</i> cells grown as above and untreated (Utrd), exposed to 1 mM arsenate, 8.8 mM H₂O₂, or UV-irradiated with 40 mJ/cm².</p

Pathways for mRNA under normal and stress conditions.

<p>Under normal growth conditions after transcription a mRNA could go directly to the polysomes for translation (a), or the mRNAs could be escorted by subunits of the RNA polymerase complex to the PBs and sorted either for the polysomes for translation (b), or the mRNA could undergo decay in the PBs (c). Under stress conditions pre-existing mRNAs could undergo enhanced translation as we propose for <i>HSP12</i> mRNA (α), or be retracted from the polysomes (β) for future sorting for storage or decay (γ or δ). Furthermore, after stress mRNAs can shuttle between PBs and SGs (γ) from where they can return to translation (ε). mRNA lifecycles under normal growth conditions have blue arrows, stress conditions are indicated with brown arrows. A more comprehensive description of the mRNA lifecycle, particularly of mRNA decay, can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002527#pgen.1002527-Harigaya1" target="_blank">[80]</a>.</p

Induction of Hsp12 protein and mRNA, and mRNA decay after stress.

<p>A. WB of protein produced from genomic <i>HSP12-GFP</i> in response to stress. B. <i>UFO1</i> and <i>HSP12</i> mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i>. C. Induction of <i>HSP12</i> mRNA by stress. Wild type cells at <i>A</i>₆₀₀ = 0.5, treated with 1 mM arsenate, 8.8 mM H₂O₂, irradiated with 40 mJ/cm² UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. <i>HSP12</i> mRNA decay. p<i>GAL-HSP12</i> was expressed in <i>hsp12Δ</i> mutants by overnight induction with 2% galactose. Next morning cells at <i>A</i>₆₀₀ = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H₂O₂ for 30 minutes, or irradiated with 40 mJ/cm² UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells).</p

Ectopic high level gene expression affects viability of mutants unable to form PBs and SGs.

<p>A. Visualization of wild type, <i>pat1Δ</i>, <i>edc3Δ</i>, <i>pat1Δ</i> or <i>edc3Δ</i>, <i>lsm4Δc</i> cells expressing the PB marker Dcp2^mCherry and the SG marker Pab1^GFP untreated or exposed for 30 minutes to 1 mM arsenate or 8.8 mM H₂O₂, irradiated with 40 mJ/cm² UV, or incubated in SC medium without glucose for 30 minutes. B. Viability of wild type, <i>pat1Δ; edc3Δ</i>, <i>pat1Δ</i> or <i>edc3Δ</i>, <i>lsm4Δc</i> cells untreated (Unt) or treated with 1 mM arsenate, 8.8 mM H₂O₂ or irradiated with 40 mJ/cm² UV analyzed by the spot test viability assay. C. Wild type, <i>pat1Δ; edc3Δ</i>, <i>pat1Δ</i> or <i>edc3Δ</i>, <i>lsm4Δc</i> cells expressing empty p<i>GAL</i>-vector (YCp), p<i>GAL-GFP-UFO1</i>, p<i>GAL-HSP12</i> or <i>MFA2</i>-U1A (pRP1193) were grown in SC medium with 2% glucose or induced in 2% galactose medium overnight, diluted and regrown in the same media to <i>A</i>₆₀₀ = 0.5 for spot test analysis on SC plates with 2% glucose or 2% galactose, respectively. D. WB analysis of w.t or <i>edc3Δ</i>, <i>pat1Δ</i> cells expressing <i>UFO1</i>, <i>HSP12</i> or <i>MFA2</i> from the <i>GAL</i> promoter. Glu (noninducing conditions) and Gal (inducing). The intensities of each protein band were normalized to the α-tubulin loading control using ImageJ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002527#pgen.1002527-Rasband1" target="_blank">[79]</a>. E. Comparison of <i>UFO1</i> and <i>HSP12</i> mRNA decay. p<i>GAL-GFP-UFO1</i> or p<i>GAL-HSP12</i> was expressed in w.t or <i>edc3Δ</i>, <i>pat1Δ</i> cells by overnight induction with 2% galactose. Next morning cells at <i>A</i>₆₀₀ = 0.5 were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to <i>ACT1</i> and to time 0 (untreated log cells).</p