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

Summary

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

Abstract

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.

Introduction

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.

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Protocol

1. Growth of Yeast Cells

1. Inoculate yeast cells in 30 ml of YPD (Table 1) in a small flask. Incubate at 30 °C overnight with shaking.
2. 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 °C with shaking.
3. Grow the culture to an optical density of 1.0 at 600 nm (OD₆₀₀ = 1.0).
4. Centrifuge cells for 5 min at 2,000 x g and discard the supernatant.
5. Resuspend the cell pellet in 40 ml sterile water and centrifuge for 5 min at 2,000 x g to wash the cells. Discard the supernatant and store the cell pellet at -20 °C.

2. Extraction of Proteins

1. Resuspend cells in 0.5 ml of ice-cold guanidine buffer (Table 1) for pull-down assay using Ni-NTA superflow beads or buffer A (Table 1) for immunoprecipitation. Keep tubes on ice at all times.
2. Transfer to 2 ml screw cap tube.
3. Add the same volume of glass beads (Table of Materials).
4. Bead-beat the cells in a mini bead beater (Table of Materials) for 2 min at room temperature, then place on ice for 2-3 min. Repeat this three times.
5. Vortex on high speed at 4 °C for 30-60 min. Check the cells by visualization under the microscope to ensure that the cells are lysed.
   Note: Lysed cells will appear as dark ghosts and lack a boundary or defined shape. Optimally at least 80% of the cells should be lysed.
6. Puncture a hole in the bottom of the tube using a push pin and place in a collection 15 ml conical tube (screw cap should be loose).
7. Centrifuge at 1,000 x g for 1 min to collect the lysate.
8. Transfer the lysate to a micro centrifuge tube.
9. Centrifuge at 15,000 x g for 30 min at 4 °C to collect the extracted proteins.
10. Measure the concentration of the extracted proteins using the protein assay kit (Table of Materials) and normalize all extracts to contain the same amount of protein. Bring the total volume to 1 ml with appropriate buffer.
    Note: Around 5 mg of total protein is obtained from 50 OD₆₀₀ cells.
11. Save 50 µl (250 µg protein if the total protein extracted from step 2.10 is 5 mg) as whole cell extract (WCE). Add 50 µl of 2x Laemmli sample buffer (Table 1) and incubate at 100 °C in a heat block for 3-5 min. Load 10 µl of each sample in a SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).

3. Purification of HF-Smt3 Conjugates

1. Obtain the Ni-NTA superflow beads (Table of Materials) necessary for the experiments (100 µl of beads per sample) by low speed centrifugation (800-1,500 x g) for 1 min. Remove the supernatant.
2. Wash beads 5x with 1 ml of PBS (Table of Materials): Invert top-over-bottom until the beads are resuspended, collect beads by low speed centrifugation, and remove the supernatant.
3. Suspend beads in 1 ml of guanidine buffer (Table 1) and aliquot them into the number of tubes corresponding to samples to be processed. Collect beads by low speed centrifugation, and remove the supernatant. Mix the beads well by inverting top-over bottom.
   Note: Mix beads well by inverting top-over bottom.
4. For each sample, add 950 µl of the remaining WCE (from Step 2.11: Extraction of proteins) to 100 µl of Ni-NTA superflow beads.
5. Incubate on a rocking platform at 4 °C for at least 4 hr or overnight.
6. Centrifuge at 800-1,500 x g for 1 min at 4 °C.
7. Save 50 µl as supernatant (SUP). Add 50 µl of 2x Laemmli sample buffer and incubate at 100 °C in a heat block for 3-5 min. Load 10 µl of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).
8. Wash beads once with 1 ml of guanidine buffer for 5-10 min on a rocking platform.
9. Wash beads 5x with 1 ml of breaking buffer (Table 1) for 5-10 min on a rocking platform.
10. Resuspend beads in 90 µl of 2x Laemmli sample buffer.
11. Add 10 µl of 1 M imidazole.
    Note: Imidazole helps to dissociate the His-tagged protein from the Ni-NTA beads due to competing interaction between imidazole and the beads.
12. Incubate at 100 °C in a heat block for 3-5 min.
13. Vortex, then centrifuge at 13,000 x g for 30 sec. Transfer the supernatant to a fresh tube.
14. Load 10-20 µl of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).
    Note: 10-20 µl corresponds to 0.5-1.0 mg of input, if 5 mg of WCE is used.

4. Immunoprecipitation of Myc-tagged Kinetochore Proteins

1. Obtain anti-c-Myc agarose affinity gel antibody (Table of Materials) necessary for the experiments (25 µl of resin per sample) by low speed centrifugation (800-1,500 x g). Remove the supernatant.
2. Wash resin 5x with 1 ml of buffer A: Invert top-over-bottom until the resin is resuspended, collect resin by low speed centrifugation, and remove the supernatant.
3. Suspend resin in 1 ml of buffer A and aliquot them into the number of tubes corresponding to samples to be processed. Collect resin by low speed centrifugation, and remove the supernatant.
   Note: Mix the resin well by inverting top-over-bottom.
4. Add 950 µl of the remaining WCE (from Step 2.11: Extraction of proteins) to 25 µl of resin.
5. Incubate on a rocking platform at 4 °C overnight.
6. Centrifuge at 800-1,500 x g for 1 min at 4 °C.
7. Save 50 µl as supernatant (SUP). Add 50 µl of 2x Laemmli sample buffer and incubate at 100 °C in a heat block for 3-5 min. Load 10 µl of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).
8. Wash resin 5x with 1 ml of buffer A: Invert top-over-bottom until the resin is resuspended, collect resin by low speed centrifugation, and remove the supernatant.
9. Resuspend the resin in 100 µl of SUMEB sample buffer (Table 1).
   Note: SUMEB sample buffer contains 8 M Urea and 1 % SDS (strong denaturing condition).
10. Incubate at 100 °C in a heat block for 3-5 min.
11. Vortex, then centrifuge at 13,000 x g for 30 sec. Transfer the supernatant to a fresh tube.
12. Load 10-20 µl of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).
    Note: 10-20 µl corresponds to 0.5-1.0 mg of input, if 5 mg of WCE is used.

5. Western Blot Analysis

1. Load the protein extracts on 4-12% Bis-Tris gels (Table of Materials) and perform electrophoresis at 120 V for 90 min (Running buffer: Table of Materials).
2. Transfer the proteins from gel to a nitrocellulose membrane (Table of Materials) using a transfer apparatus at 30 V for 90 min (Transfer buffer: Table of Materials).
3. Block the membrane in 5% milk/1x TBST (Table1) for 1 hr at room temperature.
4. Incubate membrane with primary antibody (Table of Materials) in 5% milk/1x TBST overnight at 4 °C. For primary antibodies, use a dilution of 1:1,000 (anti-Flag and anti-Ub antibodies) or 1:5,000 (anti-Myc antibody).
5. Wash membrane 3x for 10 min each with 1x TBST.
6. Incubate membrane with secondary antibody (Table of Materials) in 5% milk/1x TBST for 1 hr at room temperature. Use a 1:5,000 dilution of secondary antibodies.
7. Wash membrane 3x for 10 min each with 1x TBST.
8. Incubate membrane with ECL working solution (Table of Materials) for 5 min.
9. Expose the membrane to blue sensitive X-Ray film (Table of Materials) and develop using an automatic developer (Table of Materials).

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Results

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...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
Glass beads	BioSpec Products	11079105	0.5 mm diameter
Mini beadbeater	BioSpec Products	#693	8 cell disrupter
Ni-NTA superflow	Qiagen	30430	100 ml
Protease inhibitor cocktail	Sigma-Aldrich	P8215	1 ml
Anti-c-Myc agarose affinity gel antbody produced in rabbit	Sigma-Aldrich	A7470	1 ml
Monoclonal anti-Flag M2 antibody produced in mouse	Sigma-Aldrich	F1804	Primary antibody, dilution 1:1,000
c-Myc antibody (A-14)	Santa Cruz Biotechnology	sc-789	Primary antibody, dilution 1:5,000
Purified mouse antibody monoclonal 9E10	Covance	MMS-150P	Primary antibody, dilution 1:5,000
Ubiquitin (P4G7) monoclonal antibody	Covance	MMS-258R	Primary antibody, dilution 1:1,000
ECL Rabbit IgG, HRP-linked whole Ab	GE Healthcare Life Sciences	NA934V	Secondary antibody, dilution 1:5,000
ECL Mouse IgG, HRP-Linked Whole Ab	GE Healthcare Life Sciences	NA931V	Secondary antibody, dilution 1:5,000
DC protein assay	Bio-Rad	500-0116
Nitrocellulose membrane	Novex	LC2001	0.45 mm pore size
NuPAGE 4-12% Bis-Tris Protein Gels	Novex	NP0321BOX	1.0 mm, 10 well
NuPAGE MES SDS Running Buffer	Novex	NP0002	20x
NuPAGE Transfer Buffer	Novex	NP0006-1	20x
SuperSignal West Pico Chemiluminescent Substrate	Thermo Scientific	34078
10x PBS pH7.4	GIBCO	70011-044
Blue sensitive X-Ray film	Dbio	DBOF30003
Automatic developer	Kodak	M35AX-OMAT
Nocodazole	Sigma-Aldrich	M1404	50 mg
MG-132	Selleck Chemicals	S2619	25 mg