Shp1 functions in growth and mitotic progression require Cdc48 binding.

<p>(a) Schematic of <i>shp1</i> mutations in Cdc48 binding motifs engineered for this study. Wild-type Shp1 is shown at the top, with defined domains and motifs labeled. UBA, ubiquitin-associated domain; SEP, Shp1, eyc, and p47 domain; UBX, ubiquitin regulatory X domain; BS1, binding site 1. Key BS1 and UBX residues mutated in Cdc48 binding-deficient <i>shp1</i> alleles are indicated in bold in the sequence and by asterisks in the outlines of the Shp1 variant proteins shown below. (b) Simultaneous mutation of the R-FPR motif in the UBX domain and of binding site(s) 1 abolishes Cdc48 binding <i>in vivo</i>. Lysates of cells expressing the indicated <i>shp1</i> alleles were subjected to immunoprecipitation with a Shp1 antibody and analyzed for Cdc48 co-immunoprecipitation by Western blot. (c) <i>shp1</i> mutants defective in Cdc48 binding are temperature sensitive. Wild-type (WT) and <i>shp1-7</i> mutant cells carrying the indicated centromeric plasmids were analyzed for growth at the indicated temperatures as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g001" target="_blank">Fig. 1a</a>. (d, e) <i>shp1</i> mutants defective in Cdc48 binding are delayed in mitotic progression. Asynchronous WT and <i>shp1-a1</i> cultures were analyzed by FACS (d) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g001" target="_blank">Fig. 1b</a>, and WT and <i>shp1-a1</i> cultures synchronized by α-factor arrest/release were analyzed by Western blot against the mitotic cyclin Clb2 (e) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g001" target="_blank">Fig. 1d</a>.</p

The mitotic phenotype of <i>shp1</i> mutants is caused by reduced Glc7 activity.

<p>(a) <i>shp1</i> mutants tolerate over-expression of <i>GLC7</i>. Wild-type (WT) and <i>shp1-7</i> and <i>shp1-a1</i> mutant cells expressing <i>GLC7</i> from an integrative plasmid under the control of the inducible <i>P_MET25</i> promoter (<i>P_MET-GLC7</i>) were analyzed for growth at 25°C in the presence (+Met (off)) and absence (−Met (on)) of methionine in the growth medium. The respective strains carrying an empty integrative plasmid (<i>P_MET</i>) served as control. (b) Over-expression of <i>GLC7</i> suppresses the mitotic delay of <i>shp1</i> mutants. The strains described in panel (a) were analyzed for cell cycle distribution by FACS in the absence and presence of methionine as indicated. (c) Over-expression of <i>GLC7</i> suppresses the chromosome segregation defect of <i>shp1</i> mutants. Sister chromatid separation of wild-type, <i>shp1-7</i> and <i>shp1-a1</i> mutant cells expressing <i>GLC7</i> under the control of the inducible <i>P_MET25</i> promoter was analyzed at 25°C in the presence (<i>P_MET-GLC7</i> off) and absence (<i>P_MET-GLC7</i> on) of methionine in the growth medium. Large-budded cells (n>300 for each condition) were sorted into four classes based on the relative orientation of the ^GFPLacI-marked chromosomes III and the spindle pole body (SPB) marker Spc42^Mars: I, normal metaphase spindle; II, normal anaphase spindle; III, meta-/anaphase spindle with segregation defect; IV, aberrant number of SPBs. Error bars indicate binomial standard errors. The distribution of the five cell types over the four classes is non-random with high statistic significance according to a Pearson's chi-squared test of independence (Χ²₍₁₂₎ = 123.931; p>0.001). All pairwise differences within classes I–III between (i) wild-type and <i>shp1</i> mutants without over-expression of <i>GLC7</i>, and (ii) <i>shp1</i> mutants with and without <i>GLC7</i> over-expression are statistically significant with p<0.01 according to Fisher's exact test. (d) Representative examples of large-budded cells falling into the four classes analyzed in panel (c). Upper row, fluorescence microscopy of ^GFPLacI-marked chromosomes III (chr. III) and Spc42^Mars-marked SPBs; lower row, differential interference contrast (DIC) microscopy. The asterisks mark an additional unbudded cell in class I that was not included in the analysis.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

<i>shp1</i> null mutants exhibit growth defects and mitotic delay.

<p>(a) <i>shp1</i> null mutants are cold- and temperature-sensitive. 5-fold serial dilutions of wild-type (WT), <i>shp1-7</i> and <i>Δshp1</i> cultures were spotted on YPD plates and incubated at the indicated temperatures for 3 days. (b) <i>shp1</i> null cells accumulate and terminally arrest in G2/M at 25°C and 14°C, respectively. Asynchronously growing WT and <i>Δshp1</i> cultures at 25°C were split and incubated for 14 h at 25°C or 14°C as indicated. Cells were fixed and analyzed for DNA content by staining with propidium iodide and flow cytometry. The peaks for single (1n) and double (2n) DNA content are labeled. (c, d) <i>shp1</i> null cells are delayed in mitotic progression. Exponentially growing WT and <i>Δshp1</i> strains expressing <i>CLN2^3HA</i> were arrested in G1 with α-factor and released. Samples were taken every 20 min. (c) FACS analysis was performed as in (b). (d) Clb2 and Cln2^3HA levels were analyzed by Western blot.</p

<i>shp1</i> mutants exhibit reduced Glc7 activity.

<p>(a) Synthetic lethality of <i>glc7-129</i> with <i>shp1</i> mutants defective in Cdc48 binding. <i>glc7-129 shp1-7</i> double mutant cells carrying the <i>URA3</i>-based <i>SHP1</i> expression plasmid <i>YC33-SHP1</i> and a <i>LEU2</i>-based centromeric plasmid for the expression of the indicated wild-type and mutant <i>SHP1</i> alleles were spotted in serial dilutions onto control plates (SC-Leu) or plates containing 5-fluoro orotic acid (5′FOA) to counterselect against <i>YC33-SHP1</i>. The ability of the <i>shp1</i> mutant gene products to bind Cdc48 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g002" target="_blank">Fig. 2b</a> is indicated at the right. (b) Synthetic lethality of <i>shp1-7</i> and <i>sds22-6</i>. Growth of haploid progeny of one tetrad from the cross of <i>shp1-7</i> with <i>sds22-6</i> carrying <i>YC-SHP1</i> was analyzed on control (YPD) and 5′FOA plates as described above. (c) Positive genetic interaction between <i>ipl1-321</i> and <i>shp1</i> mutants defective in Cdc48 binding. Growth of haploid progeny of one tetrad from the cross of <i>shp1-7</i> with <i>ipl1-321</i> carrying a centromeric plasmid for the expression of the indicated wild-type and mutant <i>SHP1</i> alleles was analyzed at the indicated temperatures. The ability of the <i>shp1</i> mutant gene products to bind Cdc48 is indicated at the right. (d) Hyper-phosphorylation of histone H3 in <i>shp1-7</i>. The phosphorylation state of histone H3 in the indicated WT and mutant strains at 35°C was analyzed by Western blot using an antibody recognizing phosphorylated residue Ser10 (pH3) and total H3, respectively. The ratio of the signal intensities (pH3/total H3) is given at the bottom.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Glc7 nuclear localization is not severely affected in <i>shp1</i> mutants.

<p>(a, b) Functionality of epitope-tagged Glc7 variants. Growth of wild-type (WT) and strains expressing the indicated carboxyl-terminally epitope-tagged Glc7 fusion proteins from the chromosomal <i>GLC7</i> locus as the sole source of Glc7 was analyzed by (a) plate assay and (b) FACS. (c, d) Physical interaction between Glc7 and Shp1. (c) Lysates of strains expressing Shp1^3HA and Glc7^3myc as indicated were subjected to immunoprecipitation (IP) with anti-myc antibody and analyzed for co-precipitation of Shp1^3HA. The asterisk marks a cross-reactive band of the Glc7 antibody. (d) Lysates of strains expressing Glc7^3myc and Shp1^3HA as indicated were subjected to immunoprecipitation with anti-HA antibody and analyzed for co-precipitation of Glc7^3myc. The asterisk marks the immunoglobulin heavy chain of the HA antibody. Quantification of the Glc7 signal in the IP lanes relative to the heavy chain signal revealed a more than eight-fold difference between the Shp1^3HA lane and the negative control. (e, f) Nuclear localization of Glc7^GFP in <i>shp1</i> mutants. WT, <i>shp1-7</i> and <i>shp1-b1</i> cells expressing Glc7^GFP as sole source of Glc7 were analyzed by confocal spinning disk microscopy. (e) Representative z-stack projections generated with ImageJ. Scale bars: 5 µm. (f) Quantification of the GFP signal in equal areas of nucleus <i>versus</i> cytosol in single z-slices of confocal images. (g) Normal binding of Glc7 to Sds22 in <i>shp1-7</i>. Lysates of WT and <i>shp1-7</i> cells expressing Sds22^3myc as indicated were subjected to immunoprecipitation with anti-myc antibody and analyzed for co-precipitation of untagged Glc7. The asterisk marks the immunoglobulin light chain of the myc antibody.</p

Impaired interaction between Glc7 and Glc8 in <i>shp1</i>.

<p>(a) Synthetic lethality of <i>shp1-7 Δglc8</i>. Growth of haploid progeny of one tetrad from the cross of <i>shp1-7</i> with <i>Δglc8</i> carrying <i>YC33-SHP1</i> was analyzed on control (YPD) and 5′FOA plates as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g004" target="_blank">Fig. 4b</a>. (b) Synthetic lethality of <i>Δglc8</i> with <i>shp1</i> mutants defective in Cdc48 binding. <i>Δglc8 shp1-7</i> double mutant cells carrying <i>YC33-SHP1</i> and a <i>LEU2</i>-based centromeric plasmid for the expression of the indicated wild-type and mutant <i>SHP1</i> and <i>GLC8</i> alleles were analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056486#pone-0056486-g004" target="_blank">Fig. 4a</a>. (c, d, e) Reduced physical interaction between Glc7 and Glc8 in <i>shp1-7</i>. Lysates of asynchronous (c) or α-factor-arrested (d) wild-type and <i>shp1-7</i> cells expressing Glc8^3HA were subjected to immunoprecipitation with anti-HA antibody and analyzed for co-precipitation of endogenous, untagged Glc7. The asterisks mark a cross-reactive band of the Glc7 antibody (Glc7 blots) and the immunoglobulin light chain of the HA antibody (Glc8 blots), respectively. In panel (c), irrelevant lanes were removed from the figure. However, all lanes shown were on the same Western blot and exposed and processed identically. (e) Quantification of three independent experiments as in panel (c), showing the ratio of the Glc7 and Glc8 signal intensities. (f) Overexpression of <i>GLC8</i> partially suppresses the temperature sensitivity of <i>shp1-7</i>. Wild-type (WT) and <i>shp1-7</i> cells expressing the indicated <i>GLC8^3HA</i> alleles from an integrative plasmid under the control of the inducible <i>P_MET25</i> promoter were analyzed for growth at the indicated temperatures in the presence (+Met (off)) and absence (−Met (on)) of methionine in the growth medium.</p

Densitometry for yeast cycloheximide chase experiments.

(XLSX)</p

Overexpression of <i>RECQL4</i> results in increased RAD51 foci and decreased tail moment.

(A) Overexpression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. U2OS cells were transfected with an empty plasmid or a plasmid expressing RECQL4 or RECQL4-K508A under a CMV promoter. The cells were either mock or cisplatin treated for one hour and after a two-hour recovery, imaged for RAD51 foci or DAPI by immunofluorescence. RAD51 foci was quantified from 200 cells per condition for each experiment. The experiment was performed three to five times and the median was graphed (Unprocessed foci count in S9 Data). Representative images are shown. (B) Overexpression of RECQL4 results decreased tail moment following cisplatin exposure, which is dependent on its helicase activity. U2OS cells were treated similarly to the immunofluorescence experiment, before being harvested for neutral comet assay. At least 40 comets were counted per condition for each experiment. The experiment was performed four times and the mean and standard deviation was graphed (Unprocessed tail moments in S10 Data).</p