Difference between revisions of "Team:Cologne-Duesseldorf/Test2"

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<h3>The peroxisomal proteome of <i>Saccharomyces cerevisiae</i></h3>
 
<h3>The peroxisomal proteome of <i>Saccharomyces cerevisiae</i></h3>
 
<p>The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (<abbr title="Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J. (2013): Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat--store 'em up or burn 'em down. In: Genetics 193 (1), S. 1–50. DOI: 10.1534/genetics.112.143362.">Kohlwein et al. 2013</abbr>). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.</p>
 
<p>The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (<abbr title="Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J. (2013): Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat--store 'em up or burn 'em down. In: Genetics 193 (1), S. 1–50. DOI: 10.1534/genetics.112.143362.">Kohlwein et al. 2013</abbr>). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.</p>
 +
 +
 +
 +
 +
 +
 +
<table>
 +
  <thead>
 +
    <tr>
 +
      <th>Gene</th>
 +
      <th>Required for growth on oleate</th>
 +
      <th>Expression induced by oleate</th>
 +
      <th>Enzyme/activity</th>
 +
      <th>Molecular mass (kDa)</th>
 +
      <th>Isoelectric point</th>
 +
      <th>Molecules per cell </th>
 +
      <th>Localization</th>
 +
      <th>Function</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td colspan=”9”>ß-Oxidation enzymes</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PCS60 (FAT2)</td>
 +
      <td>No</td>
 +
      <td>Yes</td>
 +
      <td>Medium chain fatty acyl-CoA synthetase</td>
 +
      <td>60.5</td>
 +
      <td>9.98</td>
 +
      <td>8.770</td>
 +
      <td>Peripheral peroxisomal membrane and matrix</td>
 +
      <td>Activates fatty acids with a preference for medium chain lengths, C9-C13</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FAT1</td>
 +
      <td>No</td>
 +
      <td>-</td>
 +
      <td> Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter</td>
 +
      <td>77.1</td>
 +
      <td>8.47</td>
 +
      <td>16,900</td>
 +
      <td>Lipid droplet, ER, peroxisome Three predicted TM</td>
 +
      <td>Activates fatty acids with a preference for very long chain lengths, C20–C26</td>
 +
    </tr>
 +
    <tr>
 +
      <td>POX1</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Acyl-CoA- oxidase</td>
 +
      <td>84.0</td>
 +
      <td>8.73</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Oxidation of acyl-CoA</td>
 +
    </tr>
 +
    <tr>
 +
      <td>CTA1</td>
 +
      <td>No</td>
 +
      <td>Yes</td>
 +
      <td>Catalase</td>
 +
      <td>58.6</td>
 +
      <td>7.46</td>
 +
      <td>623</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Degrades hydrogen peroxide produced by Pox1</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FOX2 (POX2)</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase</td>
 +
      <td>98.7</td>
 +
      <td>9.75</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>-</td>
 +
    </tr>
 +
    <tr>
 +
      <td>POT1 (FOX3, POX3)</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>3-Ketoacyl-CoA thiolase</td>
 +
      <td>44.7</td>
 +
      <td>7.56</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA</td>
 +
    </tr>
 +
    <tr>
 +
      <td>DCI1 (ECI2)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative)</td>
 +
      <td>30.1</td>
 +
      <td>8.83</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated</td>
 +
    </tr>
 +
    <tr>
 +
      <td>SPS19 (SPX1)</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>2,4-Dienoyl-CoA reductase</td>
 +
      <td>31.1</td>
 +
      <td>9.67</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
 +
    </tr>
 +
    <tr>
 +
      <td>ECI1</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Δ3, Δ2-enoyl-CoA isomerase</td>
 +
      <td>31.7</td>
 +
      <td>8.21</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
 +
    </tr>
 +
    <tr>
 +
      <td>TES1 (PTE1)</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Acyl-CoA thioesterase</td>
 +
      <td>40.3</td>
 +
      <td>9.58</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
 +
    </tr>
 +
    <tr>
 +
      <td>MDH3</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Malate dehydrogenase</td>
 +
      <td>37.3</td>
 +
      <td>10.00</td>
 +
      <td>3,300</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
 +
      </td>
 +
    </tr>
 +
    <tr>
 +
      <td>IDP3</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>NADP+ dependent isocitrate dehydrogenase</td>
 +
      <td>47.91</td>
 +
      <td>10.02</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH</td>
 +
    </tr>
 +
    <tr>
 +
      <td>CAT2</td>
 +
      <td>No</td>
 +
      <td>No</td>
 +
      <td>Carnitine acetyl-CoA transferase</td>
 +
      <td>77.2</td>
 +
      <td>8.34</td>
 +
      <td>470</td>
 +
      <td>Peroxisome mitochondria</td>
 +
      <td>Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes</td>
 +
    </tr>
 +
    <tr>
 +
      <td colspan=”9”>Glyoxylate cycle</td>
 +
    </tr>
 +
    <tr>
 +
      <td>CIT2</td>
 +
      <td>No</td>
 +
      <td>-</td>
 +
      <td>Citrate synthase</td>
 +
      <td>51.4</td>
 +
      <td>6.34</td>
 +
      <td>2,310</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Condensation of acetyl CoA and oxaloacetate to form citrate</td>
 +
    </tr>
 +
    <tr>
 +
      <td>MDH3</td>
 +
      <td>Yes</td>
 +
      <td>Yes</td>
 +
      <td>Malate dehydrogenase</td>
 +
      <td>37.3</td>
 +
      <td>10.00</td>
 +
      <td>3,300</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+</td>
 +
    </tr>
 +
    <tr>
 +
      <td>MLS1</td>
 +
      <td>Yes</td>
 +
      <td>-</td>
 +
      <td>Malate synthase</td>
 +
      <td>62.8</td>
 +
      <td>7.18</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal protein</td>
 +
      <td>Required for utilization of nonfermentable carbon sources</td>
 +
    </tr>
 +
    <tr>
 +
      <td colspan=”9”> Other peroxisome-associated enzyme activities</td>
 +
    </tr>
 +
    <tr>
 +
      <td> GPD1 (DAR1, HOR1, OSG1, OSR5</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> NAD-dependent glycerol-3-phosphate dehydrogenase</td>
 +
      <td>42.9</td>
 +
      <td>5.26</td>
 +
      <td>807</td>
 +
      <td>Peroxisome, cytosol, nucleus</td>
 +
      <td> Key enzyme of glycerol synthesis, essential for growth under osmotic stress</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PNC1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Nicotinamidase</td>
 +
      <td>25.0</td>
 +
      <td>6.23</td>
 +
      <td>7,720</td>
 +
      <td>Peroxisome, cytosol</td>
 +
      <td>Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway</td>
 +
    </tr>
 +
    <tr>
 +
      <td>NPY1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>NADH diphosphatase</td>
 +
      <td>43.5</td>
 +
      <td>6.26</td>
 +
      <td>846</td>
 +
      <td>Peroxisome cytosol</td>
 +
      <td>Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides</td>
 +
    </tr>
 +
    <tr>
 +
      <td>STR3</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> Cystathionine β-lyase</td>
 +
      <td>51.8</td>
 +
      <td>7.96</td>
 +
      <td>ND</td>
 +
      <td>Peroxisome</td>
 +
      <td>Converts cystathionine into homocysteine</td>
 +
    </tr>
 +
    <tr>
 +
      <td>STR3</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Cystathionine ß-lyase</td>
 +
      <td>51.8</td>
 +
      <td>7.96</td>
 +
      <td>ND</td>
 +
      <td>Peroxisome</td>
 +
      <td>Converts cystathionine into homocysteine</td>
 +
    </tr>
 +
    <tr>
 +
      <td>GTO1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> ω-Class glutathione transferase </td>
 +
      <td>41.3</td>
 +
      <td>9.53</td>
 +
      <td>-</td>
 +
      <td>Peroxisome</td>
 +
      <td>Induced under oxidative stress</td>
 +
    </tr>
 +
    <tr>
 +
      <td>AAT2(ASP5)</td>
 +
      <td>-</td>
 +
      <td>Yes</td>
 +
      <td>Aspartate aminotransferase</td>
 +
      <td>46.1</td>
 +
      <td>8.50</td>
 +
      <td>7,700</td>
 +
      <td>Cytosol, peroxisome</td>
 +
      <td>Involved in nitrogen metabolism</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PCD1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives</td>
 +
      <td>39.8</td>
 +
      <td>6.59</td>
 +
      <td>238</td>
 +
      <td>Peroxisome</td>
 +
      <td>May function to remove potentially toxic oxidized CoA disulfide from peroxisomes</td>
 +
    </tr>
 +
    <tr>
 +
      <td>LPX1</td>
 +
      <td>-</td>
 +
      <td>Yes</td>
 +
      <td>Triacylglycerol lipase</td>
 +
      <td>43.7</td>
 +
      <td>8.16</td>
 +
      <td>2,350</td>
 +
      <td>Peroxisomal matrix</td>
 +
      <td>-</td>
 +
    </tr>
 +
    <tr>
 +
      <td colspan=”9”>Peroxisomal transporters</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PXA1 (LPI1, PAL1, PAT2, SSH2</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
 +
      <td>100.0</td>
 +
      <td>10.34</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Import of long-chain fatty acids into peroxisomes</td>
 +
    </tr>
 +
    <tr>
 +
      <td>PXA2 (PAT1)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
 +
      <td>97.1</td>
 +
      <td>9.47</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Import of long-chain fatty acids into peroxisomes</td>
 +
    </tr>
 +
    <tr>
 +
      <td>ANT1(YPR118C)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Adenine nucleotide transporter</td>
 +
      <td>36.4</td>
 +
      <td>10.6</td>
 +
      <td>2,250</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Involved in β-oxidation of medium-chain fatty acids</td>
 +
    </tr>
 +
    <tr>
 +
      <td colspan=”9”>Peroxins</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex1 (PAS1)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>AAA ATPase</td>
 +
      <td>117.3</td>
 +
      <td>6.93</td>
 +
      <td>2,100</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Involved in recycling of Pex5, forms heterodimer with Pex6</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex2 (RT1, PAS5)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>E3 ubiquitin ligase</td>
 +
      <td>30.8</td>
 +
      <td>9.02</td>
 +
      <td>339</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex3 (PAS3)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>50.7</td>
 +
      <td>6.29</td>
 +
      <td>1,400</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Required for proper localization of PMPs</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex4 (PAS2, UBC10)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Ubiquitin conjugating enzyme</td>
 +
      <td>21.1</td>
 +
      <td>5.36</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Involved in matrix protein import</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex5 (PAS10)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Soluble PTS1 receptor</td>
 +
      <td>69.3</td>
 +
      <td>4.79</td>
 +
      <td>2,070</td>
 +
      <td>Cytosol and peroxisomal matrix</td>
 +
      <td>Required for import of PTS1-containing peroxisomal proteins, contains TPR domains</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex6 (PAS8)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>AAA ATPase</td>
 +
      <td>115.6</td>
 +
      <td>5.44</td>
 +
      <td>1,630</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Involved in recycling of Pex5, forms heterodimer with Pex1</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex7 (PAS7, PEB1)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Soluble PTS2 receptor</td>
 +
      <td>42.3</td>
 +
      <td>8.34</td>
 +
      <td>589</td>
 +
      <td>Cytosol and peroxisomal matrix</td>
 +
      <td>Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat </td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex8 (PAS6)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td> Intra peroxisomal organizer of the peroxisomal import machinery </td>
 +
      <td>68.2</td>
 +
      <td>7.62</td>
 +
      <td>538</td>
 +
      <td>Peroxisomal matrix and luminal membrane face</td>
 +
      <td>Pex5-cargo dissociation</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex9</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>PTS-receptor</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex10</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>E3 ubiquitin ligase</td>
 +
      <td>39.1</td>
 +
      <td>9.88</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12 </td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex11 (PMP24, PMP 27)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>26.9</td>
 +
      <td>10.65</td>
 +
      <td>1,630</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Involved in peroxisome fission, required for medium-chain fatty acid oxidation </td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex12 (PAS11)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>E3 ubiquitin ligase</td>
 +
      <td>46.0</td>
 +
      <td>9.86</td>
 +
      <td>907</td>
 +
      <td>-</td>
 +
      <td>RING finger protein, forms complex with Pex2 and Pex10</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex13 (PAS20)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Component of docking complex for Pex5 and Pex7</td>
 +
      <td>42.7</td>
 +
      <td>9.83</td>
 +
      <td>7,900</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Forms complex with Pex14 and Pex17</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex14</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Central component of the receptor docking complex</td>
 +
      <td>38.4</td>
 +
      <td>4.61</td>
 +
      <td>2,570</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Interacts with Pex13</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex15 (PAS21)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>43.7</td>
 +
      <td>8.42</td>
 +
      <td>1,070</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex17 (PAS9)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Component of docking complex for Pex5 and Pex7</td>
 +
      <td>23.2</td>
 +
      <td>10.24</td>
 +
      <td>656</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Forms complex with Pex13 and Pex14</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex18</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Required for PTS2 import</td>
 +
      <td>32.0</td>
 +
      <td>4.78</td>
 +
      <td>ND</td>
 +
      <td>Interacts with Pex7 partially redundant with Pex21</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex19 (PAS12)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Chaperone and import receptor for newly synthesized PMP</td>
 +
      <td>38.7</td>
 +
      <td>4.08</td>
 +
      <td>5,350</td>
 +
      <td>Cytosol, peroxisome, farnesylated</td>
 +
      <td>Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex21</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Required for PTS2 protein import</td>
 +
      <td>33.0</td>
 +
      <td>6.67</td>
 +
      <td>ND</td>
 +
      <td>Cytosol</td>
 +
      <td>Interacts with Pex7, partially redundant with Pex18</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex22(YAF5)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Required for import of peroxisomal proteins</td>
 +
      <td>19.9</td>
 +
      <td>8.33</td>
 +
      <td>259</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Recruits Pex4 to the peroxisomal membrane</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex25</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells</td>
 +
      <td>44.9</td>
 +
      <td>9.77</td>
 +
      <td>2,420</td>
 +
      <td>Peripheral peroxisomal membrane</td>
 +
      <td>Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex27</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome size and number</td>
 +
      <td>44.1</td>
 +
      <td>10.49</td>
 +
      <td>382</td>
 +
      <td>Peripheral peroxisomal membrane</td>
 +
      <td>Interacts with homologous protein Pex25</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex28</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome size, number and distribution</td>
 +
      <td>66.1</td>
 +
      <td>7.09</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>May act upstream of Pex30, Pex31 and Pex 32</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex29</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>63.5</td>
 +
      <td>6.8</td>
 +
      <td>5,040</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>May act upstream of Pex30, Pex31 and Pex32</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex30</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome number</td>
 +
      <td>59.5</td>
 +
      <td>5.59</td>
 +
      <td>4,570</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Negative regulator, partially functionally redundant with Pex31 and Pex32</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex31</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome number</td>
 +
      <td>52.9</td>
 +
      <td>10.15</td>
 +
      <td>238</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Negative regulator, partially functionally redundant with Pex30 and Pex32 </td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex32</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome number</td>
 +
      <td>48.6</td>
 +
      <td>9.14</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Negative regulator partially functionally redundant with Pex30 and Pex31</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Pex34</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in the regulation of peroxisome number</td>
 +
      <td>16.6</td>
 +
      <td>10.30</td>
 +
      <td>ND</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>-</td>
 +
    </tr>
 +
    <tr>
 +
      <td colspan=”9”>Peroxisome fission and inheritance</td>
 +
    </tr>
 +
    <tr>
 +
      <td>DYN2 (SLC1)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Light chain dynein</td>
 +
      <td>10.4</td>
 +
      <td>9.03</td>
 +
      <td>1,310</td>
 +
      <td>Cytosol</td>
 +
      <td>Microtubule motor protein</td>
 +
    </tr>
 +
    <tr>
 +
      <td>SEC20</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>v-SNARE</td>
 +
      <td>43.9</td>
 +
      <td>5.92</td>
 +
      <td>4,910</td>
 +
      <td>Golgi, ER</td>
 +
      <td>Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20</td>
 +
    </tr>
 +
    <tr>
 +
      <td>SEC39(DSL3)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Component of the Ds11p-tethering complex</td>
 +
      <td>82.4</td>
 +
      <td>4.65</td>
 +
      <td>1,840</td>
 +
      <td>ER, nuclear envelope</td>
 +
      <td>Proposed to be involved in protein secretion</td>
 +
    </tr>
 +
    <tr>
 +
      <td>DSL1 (RNS1)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Component of the ER target site that interacts with coatomer</td>
 +
      <td>88.1</td>
 +
      <td>4.69</td>
 +
      <td>8,970</td>
 +
      <td>Peripheral ER, Golgi membrane</td>
 +
      <td>Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1</td>
 +
    </tr>
 +
    <tr>
 +
      <td>FIS1 (MDV2)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Required for peroxisome fission</td>
 +
      <td>17.7</td>
 +
      <td>9.87</td>
 +
      <td>2,410</td>
 +
      <td>Peroxisomal membrane mitochondria</td>
 +
      <td>Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission</td>
 +
    </tr>
 +
    <tr>
 +
      <td>DNM1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
 +
      <td>85.0</td>
 +
      <td>5.25</td>
 +
      <td>9,620</td>
 +
      <td>-</td>
 +
      <td>Also involved in mitochondrial fission</td>
 +
    </tr>
 +
    <tr>
 +
      <td> VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
 +
      <td>78.7</td>
 +
      <td>8.15</td>
 +
      <td>5,960</td>
 +
      <td>-</td>
 +
      <td>Also involved in vacuolar protein sorting</td>
 +
    </tr>
 +
    <tr>
 +
      <td> VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7)</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Phosphatidylinositol 3-kinase</td>
 +
      <td>100.9</td>
 +
      <td>7.79</td>
 +
      <td>1,080</td>
 +
      <td>-</td>
 +
      <td>Forms complex with Vps15</td>
 +
    </tr>
 +
    <tr>
 +
      <td>INP1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Involved in retention of peroxisomes in mother cells</td>
 +
      <td>47.3</td>
 +
      <td>8.34</td>
 +
      <td>639</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>Recruited to the peroxisome by binding to Pex3</td>
 +
    </tr>
 +
    <tr>
 +
      <td>INP2</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>Myo2 receptor, involved in peroxisome inheritance</td>
 +
      <td>81.5</td>
 +
      <td>9.41</td>
 +
      <td>736</td>
 +
      <td>Peroxisomal membrane</td>
 +
      <td>-</td>
 +
    </tr>
 +
    <tr>
 +
      <td>RHO1</td>
 +
      <td>-</td>
 +
      <td>-</td>
 +
      <td>GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome</td>
 +
      <td>23.2</td>
 +
      <td>6.07</td>
 +
      <td>ND</td>
 +
      <td>-</td>
 +
      <td>Involved in <em>de novo</em> peroxisome formation recruited to peroxisomes by Pex25</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
  
  

Revision as of 03:31, 2 November 2017

For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.

Introduction

In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.

Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.

All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding the proteome, metabolome and the entire peroxisomal environment.

Design of yeast multi -knockout strains

The Crispr Cas9 System

The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:

  • Markerless integration of multiple genetic cassettes into selected genomic loci
  • Multiplexed and iterative gene knockouts without the need to recycle a marker
  • Precise genome editing – nucleotide substitutions, etc.

We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from (Lee et al. 2015) and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.

Figure 1: Plasmid construction for the gRNA expression plasmid
Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (Lee et al. 2015)

Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).

Figure 2: Plasmid construction for the expression plasmid containing Cas9 and gRNA´s
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)

The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.

Figure 3: Design of repair DNA sequences for homologous recombination after inducing double strand break by Cas9
Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes. Adapted from (Lee et al. 2015)

The peroxisomal proteome of Saccharomyces cerevisiae

The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (Kohlwein et al. 2013). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.

Gene Required for growth on oleate Expression induced by oleate Enzyme/activity Molecular mass (kDa) Isoelectric point Molecules per cell Localization Function
ß-Oxidation enzymes
PCS60 (FAT2) No Yes Medium chain fatty acyl-CoA synthetase 60.5 9.98 8.770 Peripheral peroxisomal membrane and matrix Activates fatty acids with a preference for medium chain lengths, C9-C13
FAT1 No - Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter 77.1 8.47 16,900 Lipid droplet, ER, peroxisome Three predicted TM Activates fatty acids with a preference for very long chain lengths, C20–C26
POX1 Yes Yes Acyl-CoA- oxidase 84.0 8.73 ND Peroxisomal matrix Oxidation of acyl-CoA
CTA1 No Yes Catalase 58.6 7.46 623 Peroxisomal matrix Degrades hydrogen peroxide produced by Pox1
FOX2 (POX2) Yes Yes Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase 98.7 9.75 ND Peroxisomal matrix -
POT1 (FOX3, POX3) Yes Yes 3-Ketoacyl-CoA thiolase 44.7 7.56 ND Peroxisomal matrix Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA
DCI1 (ECI2) - - Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative) 30.1 8.83 ND Peroxisomal matrix Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated
SPS19 (SPX1) Yes Yes 2,4-Dienoyl-CoA reductase 31.1 9.67 ND Peroxisomal matrix Auxiliary enzyme of fatty acid β-oxidation
ECI1 Yes Yes Δ3, Δ2-enoyl-CoA isomerase 31.7 8.21 ND Peroxisomal matrix Auxiliary enzyme of fatty acid β-oxidation
TES1 (PTE1) Yes Yes Acyl-CoA thioesterase 40.3 9.58 ND Peroxisomal matrix Auxiliary enzyme of fatty acid β-oxidation
MDH3 Yes Yes Malate dehydrogenase 37.3 10.00 3,300 Peroxisomal matrix Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
IDP3 Yes Yes NADP+ dependent isocitrate dehydrogenase 47.91 10.02 ND Peroxisomal matrix Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH
CAT2 No No Carnitine acetyl-CoA transferase 77.2 8.34 470 Peroxisome mitochondria Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes
Glyoxylate cycle
CIT2 No - Citrate synthase 51.4 6.34 2,310 Peroxisomal matrix Condensation of acetyl CoA and oxaloacetate to form citrate
MDH3 Yes Yes Malate dehydrogenase 37.3 10.00 3,300 Peroxisomal matrix Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+
MLS1 Yes - Malate synthase 62.8 7.18 ND Peroxisomal protein Required for utilization of nonfermentable carbon sources
Other peroxisome-associated enzyme activities
GPD1 (DAR1, HOR1, OSG1, OSR5 - - NAD-dependent glycerol-3-phosphate dehydrogenase 42.9 5.26 807 Peroxisome, cytosol, nucleus Key enzyme of glycerol synthesis, essential for growth under osmotic stress
PNC1 - - Nicotinamidase 25.0 6.23 7,720 Peroxisome, cytosol Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway
NPY1 - - NADH diphosphatase 43.5 6.26 846 Peroxisome cytosol Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides
STR3 - - Cystathionine β-lyase 51.8 7.96 ND Peroxisome Converts cystathionine into homocysteine
STR3 - - Cystathionine ß-lyase 51.8 7.96 ND Peroxisome Converts cystathionine into homocysteine
GTO1 - - ω-Class glutathione transferase 41.3 9.53 - Peroxisome Induced under oxidative stress
AAT2(ASP5) - Yes Aspartate aminotransferase 46.1 8.50 7,700 Cytosol, peroxisome Involved in nitrogen metabolism
PCD1 - - Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives 39.8 6.59 238 Peroxisome May function to remove potentially toxic oxidized CoA disulfide from peroxisomes
LPX1 - Yes Triacylglycerol lipase 43.7 8.16 2,350 Peroxisomal matrix -
Peroxisomal transporters
PXA1 (LPI1, PAL1, PAT2, SSH2 - - Subunit of a heterodimeric ATP-binding cassette transporter complex 100.0 10.34 ND Peroxisomal membrane Import of long-chain fatty acids into peroxisomes
PXA2 (PAT1) - - Subunit of a heterodimeric ATP-binding cassette transporter complex 97.1 9.47 ND Peroxisomal membrane Import of long-chain fatty acids into peroxisomes
ANT1(YPR118C) - - Adenine nucleotide transporter 36.4 10.6 2,250 Peroxisomal membrane Involved in β-oxidation of medium-chain fatty acids
Peroxins
Pex1 (PAS1) - - AAA ATPase 117.3 6.93 2,100 Peroxisomal membrane Involved in recycling of Pex5, forms heterodimer with Pex6
Pex2 (RT1, PAS5) - - E3 ubiquitin ligase 30.8 9.02 339 Peroxisomal membrane RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import
Pex3 (PAS3) - - - 50.7 6.29 1,400 Peroxisomal membrane Required for proper localization of PMPs
Pex4 (PAS2, UBC10) - - Ubiquitin conjugating enzyme 21.1 5.36 ND Peroxisomal membrane Involved in matrix protein import
Pex5 (PAS10) - - Soluble PTS1 receptor 69.3 4.79 2,070 Cytosol and peroxisomal matrix Required for import of PTS1-containing peroxisomal proteins, contains TPR domains
Pex6 (PAS8) - - AAA ATPase 115.6 5.44 1,630 Peroxisomal membrane Involved in recycling of Pex5, forms heterodimer with Pex1
Pex7 (PAS7, PEB1) - - Soluble PTS2 receptor 42.3 8.34 589 Cytosol and peroxisomal matrix Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat
Pex8 (PAS6) - - Intra peroxisomal organizer of the peroxisomal import machinery 68.2 7.62 538 Peroxisomal matrix and luminal membrane face Pex5-cargo dissociation
Pex9 - - PTS-receptor - - - - -
Pex10 - - E3 ubiquitin ligase 39.1 9.88 ND Peroxisomal membrane RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12
Pex11 (PMP24, PMP 27) - - - 26.9 10.65 1,630 Peroxisomal membrane Involved in peroxisome fission, required for medium-chain fatty acid oxidation
Pex12 (PAS11) - - E3 ubiquitin ligase 46.0 9.86 907 - RING finger protein, forms complex with Pex2 and Pex10
Pex13 (PAS20) - - Component of docking complex for Pex5 and Pex7 42.7 9.83 7,900 Peroxisomal membrane Forms complex with Pex14 and Pex17
Pex14 - - Central component of the receptor docking complex 38.4 4.61 2,570 Peroxisomal membrane Interacts with Pex13
Pex15 (PAS21) - - - 43.7 8.42 1,070 Peroxisomal membrane Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP
Pex17 (PAS9) - - Component of docking complex for Pex5 and Pex7 23.2 10.24 656 Peroxisomal membrane Forms complex with Pex13 and Pex14
Pex18 - - - Required for PTS2 import 32.0 4.78 ND Interacts with Pex7 partially redundant with Pex21
Pex19 (PAS12) - - Chaperone and import receptor for newly synthesized PMP 38.7 4.08 5,350 Cytosol, peroxisome, farnesylated Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning
Pex21 - - Required for PTS2 protein import 33.0 6.67 ND Cytosol Interacts with Pex7, partially redundant with Pex18
Pex22(YAF5) - - Required for import of peroxisomal proteins 19.9 8.33 259 Peroxisomal membrane Recruits Pex4 to the peroxisomal membrane
Pex25 - - Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells 44.9 9.77 2,420 Peripheral peroxisomal membrane Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27
Pex27 - - Involved in the regulation of peroxisome size and number 44.1 10.49 382 Peripheral peroxisomal membrane Interacts with homologous protein Pex25
Pex28 - - Involved in the regulation of peroxisome size, number and distribution 66.1 7.09 ND Peroxisomal membrane May act upstream of Pex30, Pex31 and Pex 32
Pex29 - - 63.5 6.8 5,040 Peroxisomal membrane May act upstream of Pex30, Pex31 and Pex32
Pex30 - - Involved in the regulation of peroxisome number 59.5 5.59 4,570 Peroxisomal membrane Negative regulator, partially functionally redundant with Pex31 and Pex32
Pex31 - - Involved in the regulation of peroxisome number 52.9 10.15 238 Peroxisomal membrane Negative regulator, partially functionally redundant with Pex30 and Pex32
Pex32 - - Involved in the regulation of peroxisome number 48.6 9.14 ND Peroxisomal membrane Negative regulator partially functionally redundant with Pex30 and Pex31
Pex34 - - Involved in the regulation of peroxisome number 16.6 10.30 ND Peroxisomal membrane -
Peroxisome fission and inheritance
DYN2 (SLC1) - - Light chain dynein 10.4 9.03 1,310 Cytosol Microtubule motor protein
SEC20 - - v-SNARE 43.9 5.92 4,910 Golgi, ER Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20
SEC39(DSL3) - - Component of the Ds11p-tethering complex 82.4 4.65 1,840 ER, nuclear envelope Proposed to be involved in protein secretion
DSL1 (RNS1) - - Component of the ER target site that interacts with coatomer 88.1 4.69 8,970 Peripheral ER, Golgi membrane Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1
FIS1 (MDV2) - - Required for peroxisome fission 17.7 9.87 2,410 Peroxisomal membrane mitochondria Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission
DNM1 - - GTPase, dynamin like protein involved in peroxisome fission 85.0 5.25 9,620 - Also involved in mitochondrial fission
VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26) - - GTPase, dynamin like protein involved in peroxisome fission 78.7 8.15 5,960 - Also involved in vacuolar protein sorting
VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7) - - Phosphatidylinositol 3-kinase 100.9 7.79 1,080 - Forms complex with Vps15
INP1 - - Involved in retention of peroxisomes in mother cells 47.3 8.34 639 Peroxisomal membrane Recruited to the peroxisome by binding to Pex3
INP2 - - Myo2 receptor, involved in peroxisome inheritance 81.5 9.41 736 Peroxisomal membrane -
RHO1 - - GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome 23.2 6.07 ND - Involved in de novo peroxisome formation recruited to peroxisomes by Pex25