The Erf2–Erf4 palmitoyltransferase drives major changes in the meiotic palmitoylome during meiosis.

<p>(A–B) Alk-16-associated fluorescence of immunopurified Ras1 from vegetatively growing cells with the indicated palmitoyltransferase deletions (top panels). Western blots are probed for Ras1 (bottom panels). Wild-type (<i>DHHC</i>) or catalytically inactive (<i>DHHA</i>) Erf2 was expressed from a thiamine-repressible promoter in <i>erf2Δ</i> cells (B). (C) Fluorescence detection of palmitoylated substrates (top panel) and Ras1 palmitoylation (bottom panel) in homozygous diploid <i>pat1-114/pat1-114</i> cells with the indicated palmitoyltransferase deletions 8 h after meiotic induction. Ctrl, DMSO control; Veg, vegetative cells.</p

Palmitoylation-dependent Rho3 function is required for the Erf2-induced meiotic phenotype.

<p>(A) DAPI (top) and DIC (bottom) images of <i>erf2</i> and <i>erf4</i> co-overexpressing cells with indicated genotypes at 96 h postinduction. Vectors expressing internally tagged wild-type and mutant (Cys4→Ala) Rho3-HA₃ proteins from the <i>nmt41</i> promoter were integrated into the chromosome of <i>rho3Δ</i> cells. Scale bars, 10 µm. (B) Alk-16-associated fluorescence of Rho3 and palmitoylation-deficient Rho3(C4A) (top panel). Western blots were probed for HA (bottom panel).</p

Erf2 substrates are differentially modified in vegetative and meiotic cells.

<p>(A) Filter criteria for candidate substrates that are palmitoylated by Erf2 during meiosis. (B) Each of the 238 candidates obtained is represented as a data point reflecting its molecular weight and enrichment (net spectral counts) in alk-16 over DMSO labeled <i>erf2⁺</i> meiotic cells. Isp3 and Rho3 are the top two candidates with molecular weights ∼23 kDa (shaded). (C) Fluorescence profiles of meiotic cells with indicated gene deletions or expressing endogenous or tagged Isp3 (top panels). Western blot is probed for HA (bottom panels). Ctrl, DMSO control. (D) Isp3-HA₃ expression as determined by anti-HA blot of lysates from cells in distinct cellular states. Veg, vegetative cells. Mei, meiotic cells. (E) Rho3-HA₃ palmitoylation in meiotic <i>erf2⁺</i> and <i>erf2Δ</i> cells (top panel). Western blot is probed for HA (bottom panel). (F) Ras1 and Rho3 palmitoylation states in vegetative and meiotic cells (top panels). Western blots were probed for Ras1 and HA (bottom panels). Synchronous meiosis in indicated diploid <i>pat1-114/pat1-114</i> cells was induced by shifting nitrogen-starved cultures to restrictive temperature (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001597#s4" target="_blank">Materials and Methods</a>). Meiotic cells refer to cells 8 h after the temperature shift.</p

A systematic genomic screen implicates nucleocytoplasmic transport and membrane growth in nuclear size control

<div><p>How cells control the overall size and growth of membrane-bound organelles is an important unanswered question of cell biology. Fission yeast cells maintain a nuclear size proportional to cellular size, resulting in a constant ratio between nuclear and cellular volumes (N/C ratio). We have conducted a genome-wide visual screen of a fission yeast gene deletion collection for viable mutants altered in their N/C ratio, and have found that defects in both nucleocytoplasmic mRNA transport and lipid synthesis alter the N/C ratio. Perturbing nuclear mRNA export results in accumulation of both mRNA and protein within the nucleus, and leads to an increase in the N/C ratio which is dependent on new membrane synthesis. Disruption of lipid synthesis dysregulates nuclear membrane growth and results in an enlarged N/C ratio. We propose that both properly regulated nucleocytoplasmic transport and nuclear membrane growth are central to the control of nuclear growth and size.</p></div

Quantitative control of protein palmitoylation by varying palmitoyltransferase levels affects meiotic entry.

<p>(A) Labels are indicated in box. qPCR analysis of <i>erf2</i> and <i>erf4</i> transcripts normalized to <i>act1</i> mRNA levels in indicated <i>pat1-114/pat1-114</i> strains 8 h into synchronous meiosis (top panel). Data represent mean values of experimental triplicates ± SD Palmitoylation of cognate Erf2 substrates were analyzed from the same lysate of each strain (bottom panels). Ras1 and Rho3-HA₃ were immunopurified and their levels were determined by Ras1 and HA immunoblots, respectively. Isp3 palmitoylation was monitored at the lysate level since it accounts for most of the fluorescence at ∼23 kDa. (B) Labels are indicated in box. Overexpression of <i>erf2</i> and/or <i>erf4</i> from thiamine-repressible <i>nmt</i> promoters in the indicated vegetatively growing <i>pat1-114</i> cells was achieved by switching them into thiamine-free medium for 24 h. qPCR analysis (top panel) as well as palmitoylation of Ras1 and Rho3 (bottom panels) were performed as described in (A). Isp3 is not expressed in vegetative cells. Cells were maintained at permissive temperature throughout this experiment. (C, D) DAPI (left) and DIC (right) images of indicated cells 96 h after <i>erf2</i> and/or <i>erf4</i> overexpression. The DHHC→DHHA catalytic <i>erf2</i> mutant was co-overexpressed with <i>erf4</i> from <i>nmt1</i> promoters. Scale bars, 10 µm.</p

N/C ratio of <i>rae1-167</i> mutant cells.

<p>(a) N/C ratio of wild type, wild type + thiolutin (15 μg/ml) (Thiol.), wild type + cycloheximide (100 μg/ml) (CYH), <i>rae1-167</i>, <i>rae1-167pabpΔ</i>, <i>rae1-167atf1Δ</i>, <i>rae1-167mei4Δ</i>, <i>rae1-167</i> + thiolutin (15 μg/ml) (Thiol.) and <i>rae1-167</i> + cycloheximide (100 μg/ml) (CYH) cells in YE4S (25°C or 36°C, 4h, n>30). (b) Cell (brightfield) and nuclear envelope (Cut11-GFP) of indicated strains (36°C, 4h). (c) N/C ratio of <i>rae1-167</i> mutant (36°C, n>30). This phenotype is not observed in all temperature sensitive mutants; neither <i>cdc25-22</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006767#pgen.1006767.g001" target="_blank">Fig 1D</a>) nor <i>cut6-621</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006767#pgen.1006767.g004" target="_blank">Fig 4A</a>) temperature sensitive mutant cells show this phenotype following shift to 36°C for 4h. (d) Average cell volume (grey) and nuclear volume (black) of <i>rae1-167</i> cells (36°C). Dotted line represents nuclear volume required to maintain wild type N/C ratio of 0.08. ****<i>P</i><0.0001. Scale bar: 5 μm.</p

Overproduction of Erf2 and Erf4 in proliferating cells disrupts vegetative growth and induces aberrant meiosis and sporulation in<b><i>pat1-114</i></b><b> cells.</b>

<p><i>erf2 OE erf4 OE</i> refers to strain 7 from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001597#pbio-1001597-g004" target="_blank">Figure 4B</a> that co-overexpresses <i>erf2</i> and <i>erf4</i> at high levels. These cells were grown in the presence of nutrients at permissive temperature, and co-overexpression of <i>erf2</i> and <i>erf4</i> was induced by switching cells to thiamine-free medium (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001597#s4" target="_blank">Materials and Methods</a>). Indicated times or time intervals refer to time after induction of <i>erf2</i> and <i>erf4</i> co-overexpression. (A) Fold change in OD₅₉₅ of cultures during indicated 12 h intervals. OD₅₉₅ was maintained <0.6. (B) DNA content analysis. (C) Blankophor staining of cells (top panels). Dimensions of septated cells (cell length and width, <i>n</i> = 20) and percentage of septated cells (septation index, <i>n</i>≥200) were determined by measuring and counting blankophor-stained cells (bottom panels, left to right). Error bars, SD. (D) Percentage of cells with 1, 2, or >2 nuclei (<i>n</i>≥200) was determined by DAPI staining. (E) DAPI (top) and DIC (bottom) images of cells under conditions where <i>erf2</i> and <i>erf4</i> overexpression were repressed (+Thiamine) as well as 96 h postinduction of <i>erf2</i> and <i>erf4</i> co-overexpression. Scale bars, 10 µm. (F) Left panel, percentage of cells with spores (<i>n</i>≥200) at indicated times postinduction. Right panel, DIC images of cells 96 h postinduction before (top) and after (bottom) β-glucuronidase digestion, which specifically kills vegetative cells but not spores. Nonoverexpressing <i>pat1-114</i> cells continue vegetative growth under the same conditions (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001597#pbio.1001597.s005" target="_blank">Figure S5</a>).</p

<i>Rae1-167</i> cells accumulate protein in the nucleus.

<p>(a) Protein distribution in wild type and <i>rae1-167</i> cells (25°C or 36°C, 30 mins or 60 mins) stained with FITC. (b) Quantification of FITC fluorescence distribution along the long cell axis, normalised by cell length, in wild type (WT) and <i>rae1-167</i> cells (25°C or 36°C, 60 mins, n = 100). (c) SILAC mass spectrometry analysis. log₂ transformed <i>rae1</i>/WT ratios for individual proteins at 25°C and 36°C in nucleus enriched and whole cell samples. Dotted lines represent 25% higher protein level in <i>rae1-167</i> than WT, and values represent percentage of proteins exceeding this, at 36°C (red) and 25°C (blue). (d) 2D enrichment analysis of nucleus enriched samples in (c).</p

Identification of N/C ratio mutants.

<p>(a) Screen schematic. (b) N/C ratio of wild type and 14 gene deletion strains identified as having an increased N/C ratio (25°C, n>30). (c) Cell (brightfield) and nuclear envelope (Cut11-GFP) of wild type, <i>dss1Δ</i>, <i>caf1Δ</i>, <i>mlo3Δ</i>, <i>trm112Δ</i>, <i>nem1Δ</i> and <i>spo7Δ</i> cells (25°C). (d) N/C ratio of <i>cdc25-22</i> and double mutant (<i>cdc25-22</i> and candidate gene indicated) cells (36°C, 3h, n>30). <i>Cdc25-22yox1Δ</i> cells were not tested due to synthetic lethality. ***<i>P</i><0.001, ****<i>P</i><0.0001. Scale bar: 5 μm.</p

N/C ratio enlargement and nuclear membrane growth.

<p>(a) N/C ratio of wild type, <i>nem1Δ</i>, <i>cut6-621</i>, and <i>nem1Δcut6-621</i> cells (25°C, 4h 36°C, n>30). (b) Cell (brightfield) and nuclear envelope (Cut11-GFP) of the strains in (a). (c) N/C ratio of <i>rae1-167</i>, <i>rae1-167cut6-621</i> and <i>rae1-167nem1Δ</i> cells (25°C, 4h 36°C, n>30). (d) Cell (brightfield) and nuclear envelope (Cut11-GFP) of the strains in (c). **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001. Scale bar: 5 μm.</p