Relationships between Cell Cycle Regulator Gene Copy Numbers and Protein Expression Levels in <i>Schizosaccharomyces pombe</i>

<div><p>We previously determined the copy number limits of overexpression for cell division cycle (<i>cdc</i>) regulatory genes in the fission yeast <i>Schizosaccharomyces pombe</i> using the “genetic tug-of-war” (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in <i>S. pombe</i>. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.</p></div

Relationships between native and TAP-tagged gene copy number limit.

<p>Genes whose copy numbers varied between native and TAP-tagged are shown. Circles indicate those genes whose copy numbers were determined under −leucine conditions, and squares indicate genes whose copy numbers were determined under +leucine conditions. Copy numbers of native genes were obtained from previously published results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319-Moriya2" target="_blank">[2]</a>. The averages of more than three independent experiments are shown. The original data with standard deviations are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s004" target="_blank">Table S2</a>.</p

Relationships between copy numbers and fold increase in protein level.

<p>ND: Not done,</p>*<p>The number shown is the plasmid copy number determined plus 1 (genomic copy).</p

Control Experiments with Frame Shift Mutants in the gTOW Experiment

<div><p>(A) A scatter plot showing the correlation of maximum growth rates and plasmid copy numbers with wild-type <i>CDC</i> genes and their frame shift mutants determined in the gTOW experiment.</p><p>(B) Plasmid copy numbers with wild-type and frame shift mutants in the gTOW experiment in the uracil− condition. The data used in the graphs are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020111#pgen-0020111-st002" target="_blank">Table S2</a>.</p></div

Quantifying Cdc–TAP protein levels expressed by a single chromosomal copy with an increase in gene copy number.

<p><b>A</b>. <i>S. pombe</i> strains for determining the increases in protein levels expressed by a single chromosomal copy with an increase in gene copy number. Each <i>cdc–</i>TAP strain was transformed with either an empty vector or the corresponding target plasmid and then cultured in medium with or without leucine (as indicated). <b>B</b>–<b>E</b>. Quantitative results for Cdc16–TAP, Sid2–TAP, Cdc10–TAP, and Cig1–TAP. TAP-tagged protein levels were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#s4" target="_blank">Methods</a>. Copy number* is the copy number of a Target plasmid plus 1 (chromosomal copy).</p

Quantifying Cdc–TAP protein levels with increased gene copy numbers.

<p><b>A</b>. <i>S. pombe</i> strains for determining increased protein levels expressed by a TAP plasmid and a chromosomal copy with an increase in gene copy number. Each <i>cdc–</i>TAP strain was transformed with either an empty vector or the corresponding <i>cdc–</i>TAP plasmid and then cultured in medium with or without leucine. <b>B</b>–<b>E</b> are examples of these quantitative results. The levels of TAP-tagged proteins were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#s4" target="_blank">Methods</a>. Copy number* indicates the copy number of each TAP plasmid plus 1 (chromosomal copy). Circled numbers indicate the fold-dilutions used to measure the intensity of a Cdc–TAP protein. Total proteins were visualized using Coomassie® G-250 staining. <b>B</b>. Pyp3^1–96–TAP used as a control. <b>C</b>. Csk1–TAP; an example for which the protein level increase and the copy number were well correlated. <b>D</b> and <b>E</b>. Cdc16–TAP and Sid2–TAP; examples for which the protein levels did not increase with an increase in copy number. <b>F</b> and <b>G</b>. Cdc10–TAP and Cig1–TAP; examples for which protein level increases exceeded copy number increases. All Cdc–TAP analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s002" target="_blank">Figure S2</a> and the quantitative results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone-0073319-t001" target="_blank">Table 1</a>.</p

Relationships between fold-increases in protein levels and copy numbers.

<p><b>A</b>. A scatter plot between the fold increase in protein level and the copy number. Squares indicate the results of control experiments using Pyp3^1–96–TAP. Genes that showed high variations between protein level increases and copy numbers are indicated. <b>B</b> and <b>C</b>. Fold increase in the gene copy number, the mRNA and protein levels of indicated gene in the +leucine (<b>B</b>) and –leucine (<b>C</b>) conditions. The original data for the gene copy number and protein increase are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone-0073319-t001" target="_blank">Table 1</a>, and the data of the mRNA increase are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073319#pone.0073319.s005" target="_blank">Table S3</a>.</p

Supplemental Material for Scharff-Poulsen, Moriya, and Johnston, 2018

Supplementary FIG Y1: Sequence alignment<br>Sequences of rat GLUT5, human GLUT1, human GLUT3, <i>Escherichia coli</i> XylE, and Staphylococcus epidermis GlcP, for which the 3D structures are known, are aligned with <i>Saccharomyces cerevisiae </i>Hxt1 and Rgt2. Numbering of amino acids is shown to the left of the sequences. Strictly conserved residues are highlighted in red-filled boxes, and highly conserved residues are shown in yellow-filled boxes. Boxes above the alignment indicate transmembrane helices (TM1 through TM12) and intracellular helices (ICH1 through ICH5) in rGLUT5. The substitutions that confer constitutive signaling of Rgt2 are indicated by arrows below the alignment.<br> <br>Supplementary FIG Y2: Structural alignment<br>Structural alignment of Bovine GLUT5 (4YB9) and <i>E. coli</i> XylE (4QIQ) in inward-open conformations. The GLUT5 (violet) and XylE (yellow) structures were aligned using PyMOL and are shown in a side-view. Visible transmembrane helices (TM) and intracellular helices (ICH) are indicated. The alignment shows that the two structures are very similar to each other.<br> <br>Supplementary FIG Y3: Imaging of live <i>S. cerevisiae </i>cells expressing GFP-fusions of Hxt1 wild type (upper panel) and of Hxt1-D82V (lower panel) from the GAL1 promoter. Cells were grown and induced with galactose as described in (Scharff-Poulsen and Pedersen 2013). Comparison of GFP fluorescence and phase contrast images show that the GFP fusions are highly expressed and accumulate in the plasma membrane of the cells.<br><br

gTOW Is a Genetic Selection Method That Determines the Upper Limit Copy Number of Target Genes

<div><p>(A) Principle of the gTOW method. The cells with plasmid copy number close to the upper limit of each target gene are concentrated because of the genetic tug-of-war that emerges due to the high copy selection bias due to <i>leu2d</i> and the low copy selection bias due to the target gene. See text for details.</p><p>(B) A scatter plot shows the correlation of maximum growth rates and plasmid copy numbers of each <i>CDC</i> gene determined in the gTOW experiment. The data used in the graphs are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020111#pgen-0020111-st001" target="_blank">Tables S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020111#pgen-0020111-st002" target="_blank">S2</a>.</p></div

Determination of Upper Limit Gene Copy Number with Leucine Supplementation

<p>Growth and copy number of plasmids in the gTOW experiment with various leucine concentrations are shown. The data used in the graphs are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020111#pgen-0020111-st002" target="_blank">Table S2</a>.</p