Name: protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80
Uploaded: 2015-05-03T14:00:00
Description: Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

1. Growth of Yeast Cells
<ol>
	<li>Inoculate yeast cells in 30 ml of YPD (Table 1) in a small flask. Incubate at 30 &#176;C overnight with shaking.</li>
	<li>Dilute the cells to an optical density of 0.2 at 600 nm (OD600 = 0.2) in 50 ml of YPD and incubate at 30 &#176;C with shaking.</li>
	<li>Grow the culture to an optical density of 1.0 at 600 nm (OD600 = 1.0).</li>
	<li>Centrifuge cells for 5 min at 2,000 x g and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 4...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospho-regulation+of+kinetochore-microtubule+attachments+by+the+Aurora+kinase+Ipl1p.">Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p.</a> Cell. 111, 163-172 (2002).</li><li>Liu, D., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+kinetochore-microtubule+attachments+by+Aurora+B+kinase.">Regulation of kinetochore-microtubule attachments by Aurora B kinase.</a> Biochem Soc Trans. 37, 976-980 (2009).</li><li>Montpetit, B., Hazbun, T. 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Epitope tags such as HA, Myc, Flag, and GST are widely used for biochemical analysis of proteins. Construction of strains with HF-Smt3 and Myc-tagged kinetochore proteins, such as Ndc10 and Ndc80, facilitates the detection of PTMs such as sumoylation and ubiquitination. HF-Smt3 pull down assay allows the detection of sumoylated kinetochore proteins, Ndc10 and Ndc80 (Figure 1). The affinity purification protocol and western blot analysis using anti-Flag antibody establish the specificity of interaction be...

Ubiquitination and sumoylation allow the conjugation of ubiquitin and Small Ubiquitin-like MOdifier (SUMO; Smt3 in S. cerevisiae 1) to a target protein, respectively. PTMs of kinetochore proteins affect their cellular levels and protein-protein interactions during different cell cycle phases to ensure faithful chromosome segregation. For example, cellular levels of Cse4/CENP-A and outer kinetochore protein Dsn1 are regulated by ubiquitin-mediated proteolysis for ensuring genome stability 2-5. Destabilization of incorrect kinetochore-microtubule attachments requires the Ipl1/Aurora B kinase, which phosphorylates Dam1 and Ndc80 complexes that directly interact with microtubules 6-8. Despite the identification of over seventy kinetochore proteins, there are very few studies that investigate the modifications of these proteins with PTMs, e.g., ubiquitin and SUMO. A major limitation is the ability to preserve the PTMs during purification and the paucity of custom antibodies for detection of PTMs such as sumoylation, phosphorylation, methylation, and others. Characterization of sumoylated kinetochore proteins Ndc10, Cep3, Bir1, and Ndc80 utilized a custom antibody 9. Additionally, Ndc10 has been implicated as a substrate for ubiquitination 10. Human Hec1 (Ndc80 in S. cerevisiae) is also substrate for ubiquitination, regulated by APC/C-hCdh1 E3 ligase 11. Therefore, Ndc10 and Ndc80 are good candidates for optimization of the protocol to detect both sumoylation and ubiquitination in S. cerevisiae.
To facilitate the identification of sumoylation, we constructed strains that express HF-Smt3 and Myc-tagged Ndc10 or Ndc80. The use of epitope tags (HF: His6-Flag) minimizes the background due to cross-reactivity that is frequently observed in polyclonal serum raised against a candidate protein. We devised a protocol to affinity purify HF-Smt3 conjugates and then used commercial anti-Flag and anti-Myc antibodies to detect the presence of sumolyated Ndc10 and Ndc80 in the purified Smt3 preparation. For ubiquitination, we devised a modified immunoprecipitation protocol that preserves ubiquitination of the Myc-tagged kinetochore proteins and performed western blot analysis with commercial anti-Ub antibody to detect ubiquitination of Ndc10 and Ndc80.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass beads</td>
			<td>BioSpec Products</td>
			<td align="right">11079105</td>
			<td>0.5 mm dia.</td>
		</tr>
		<tr>
			<td>Mini beadbeater</td>
			<td>BioSpec Products</td>
			<td>#693</td>
			<td>8 cell disrupter</td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td align="right">30430</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A7470</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Monoclonal anti-Flag M2 antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>F1804</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>c-Myc antibody (A-14)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-789</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Purified mouse antibody monoclonal 9E10</td>
			<td>Covance</td>
			<td>MMS-150P</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Ubiquitin (P4G7) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-258R</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>ECL Rabbit IgG, HRP-linked whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA934V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>ECL Mouse IgG, HRP-Linked Whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA931V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>DC protein assay</td>
			<td>Bio-Rad</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Novex</td>
			<td>LC2001</td>
			<td>0.45 mm pore size</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Novex</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10 well</td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS Running Buffer</td>
			<td>Novex</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Novex</td>
			<td>NP0006-1</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td align="right">34078</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS pH7.4</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue sensitive X-Ray film</td>
			<td>Dbio</td>
			<td>DBOF30003</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic developer</td>
			<td>Kodak</td>
			<td>M35AX-OMAT</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Selleck Chemicals</td>
			<td>S2619</td>
			<td>25 mg</td>
		</tr>
	</tbody>
</table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

To detect sumoylation of kinetochore proteins Ndc80 and Ndc10, strains with HF-Smt3 and Myc-tagged kinetochore proteins (Ndc80 or Ndc10) were constructed (Table 2), as previously described 9,12. HF-Smt3 conjugates were affinity purified using Ni-NTA beads. Western blot analysis of purified HF-Smt3 with an anti-Flag antibody allowed detection of sumoylated forms of SUMO substrates that were absent in the control strain without HF-Smt3 (Figure 1A and 1B, left pa...

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

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