We thank all members of the Kerscher lab for their support. We thank Mark Hochstrasser for yeast strain MHY3765. This work was supported by NSF grant 1051970 (to OK) and a Howard Hughes Medical Institute Undergraduate Travel grant and Monroe Scholars Program Grant (to ES).

Purification of 6xHIS-tagged proteins expressed in budding yeast cells under native conditions
1. Growth of Yeast Cells and Induction of Protein Expression
(Modified from 2)
OPTIONAL: Use logarithmically growing yeast cultures expressing protein of interest instead of the galactose-induced cultures described below.
<ol>
	<li>Transform cells of a Gal+ yeast strain with a plasmid encoding a galactose-inducible 6xHIS-tagged protein of choice. For example, see reagents list.</li>
	<li>Inoculate transformants in 5 ml of appropriate selective media (e.g. SD-uracil) containing 2% sucrose. Incubate at 30 &#176;C O/N, rotating.</li>
	<li>Dilute O/N culture so that the optical density at 600 nm measures 0.3 (OD600 = 0.3) in 33 ml of selective media with 2% sucrose. Grow at 30 &#176;C, shaking (&#916;150 rpm).</li>
	<li>When the culture has reached OD600 = 0.8-1.5, induce expression of the desired protein by adding 17 ml of 3x YEP with 6% galactose (Recipe in Table 1), for a final concentration of 1x YEP with 2% galactose. Total culture volume is now 50 ml. Incubate, shaking, at 30 &#176;C for an additional 5-6 hr. 
	Note: the culture volume can be varied. In step 1.3, dilute into two-thirds of the desired final volume. In step 1.4, add one-third the final volume of 3x YEP/6% galactose.</li>
	<li>Measure the OD600 of the induced culture and centrifuge &#916;150-200 OD600 units of cells for 5 min at 5,000 rpm at 4 &#176;C.</li>
	<li>Resuspend cell pellet with 1 ml ice-cold 1x PBS with 1x protease inhibitor cocktail and transfer to a 2 ml screw cap tube.</li>
	<li>Centrifuge cells for 1 min at 15,000 rpm at 4 &#176;C. Decant supernatant.</li>
	<li>Snap freeze cell pellet in liquid nitrogen, followed by immediate cell lysis or storage at -80 &#176;C until further use.</li>
</ol>
2. Homogenization of Yeast Cells and Extraction of Proteins
<ol>
	<li>To the frozen cell pellet from the previous step, add 200 &#181;l of acid-washed glass beads and 500 &#181;l of ice-cold lysis buffer (Recipe in Table 1 or use cell lysis buffer of choice).</li>
	<li>Briefly pipette up and down. It is not required to fully resuspend the cell pellet. Keep tubes on ice at all times. 
	Note: The end of the pipette tip may be cut off before pipetting up and down.</li>
	<li>At 4 &#176;C, place the tube(s) with cells into the bead mill, balance, lock, and run the machine as per manufacturer&#39;s instructions.</li>
	<li>Run the bead beater containing the tube(s) for 20 sec at 5.5 m/sec, then place on slushy ice for 1 min. Repeat 6x&#160;in total.</li>
	<li>Clear the extracted proteins by centrifugation for 15 min at 15,000 rpm at 4 &#176;C. OPTIONAL: remove small particulates by centrifugation through a SpinX filter.</li>
	<li>Prepare a sample of the whole cell extract (WCE) to confirm presence of the protein of interest by Western Blot:
	<ol>
		<li>Add WCE corresponding with 2 OD600 units of cells (e.g.&#160;5 &#181;l of cleared extract if 200 OD600 units of cells were harvested) to 800 &#181;l 20% trichloroacetic acid (TCA). Vortex to mix.</li>
		<li>Centrifuge for 2.5 min at 15,000 rpm at 4 &#176;C. Decant the supernatant, but be careful to retain the pellet.</li>
		<li>Add 800 &#181;l of 2% TCA to the pellet and vortex the tube, followed by centrifugation for 2.5 min at 15,000 rpm at 4 &#176;C. Decant the supernatant, but be careful to retain the pellet.</li>
		<li>Add 100 &#181;l of TCA Sample Buffer (Recipe in Table 1), vortex to dissolve pellet. Note: If the sample buffer becomes acidic (turns yellow) after addition to the pellet, add aliquots of &#916;10 &#181;l Tris base [1M] until the sample is blue again.</li>
		<li>Incubate in a 100 &#176;C heat block for 2-5 min.</li>
		<li>Vortex again to fully dissolve if remnants of pellet are still present. Pellets prepared by this method are notoriously difficult to fully dissolve. It may take &#916;10 min of vortexing to completely dissolve pellets.</li>
		<li>Store sample at -80 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Snap freeze aliquots of remaining cleared WCE in liquid nitrogen and store at -80 &#176;C until further use.</li>
</ol>
3. Batch Purification of Proteins from Yeast Cell Homogenates
Note: This purification method was optimized for purification of 6xHIS-tagged proteins on Co2+ metal affinity resin.
<ol>
	<li>Resin Equilibration
	<ol>
		<li>For a sample of cleared WCE prepared from &#916;20-40 OD600 units of cells, add 50-100 &#181;l of affinity resin to a microcentrifuge tube. Proteins extracted from cells that did not express the desired protein or a nonspecific resin, such as amylose, may be used as controls for nonspecific binding.</li>
		<li>Wash resin 5x with 1 ml of wash buffer: invert top-over-bottom until resin is resuspended, and then spin for 1 min at 5,000 rpm at 4 &#176;C. Aspirate the supernatant. 
		Note: if performing extraction and purification on the same day, resin equilibration can be performed prior to extraction.</li>
	</ol>
	</li>
	<li>Protein Binding for Affinity Purification
	<ol>
		<li>Add 100-200 &#181;l of cleared lysate (&#916;20-40 OD600 units) to 50-100 &#181;l washed beads, and bring the total volume to 1 ml with lysis buffer.</li>
		<li>Incubate with nutation or rocking at 4 &#176;C for 2-5 hr.</li>
		<li>Spin for 1 min at 5,000 rpm at 4 &#176;C.</li>
		<li>If desired, save a sample of the remaining supernatant for subsequent analysis of unbound proteins. TCA precipitate as detailed above for the WCE (step 2.6).</li>
		<li>Wash resin with bound proteins 5x with 1 ml of wash buffer, followed by a spin for 1 min at 5,000 rpm at 4 &#176;C. Keep samples cold during washes.</li>
	</ol>
	</li>
	<li>Elution of Bound Proteins
	<ol>
		<li>Add 150 &#181;l elution buffer to resin, nutate at 4 &#176;C for 5 min, spin for 1 min at 5,000 rpm at 4 &#176;C and save the supernatant in a new tube. OPTIONAL: Repeat 2x and pool elutions.</li>
		<li>Prepare eluted sample for Western blot: To 25 &#181;l of eluted proteins, add 25 &#181;l 2x lithium dodecyl sulfate (LDS) sample buffer with 2 &#181;l &#946;-mercaptoethanol (BME) and incubate in a 100 &#176;C heat block for 2 min. 
		Note: Standard 2X Laemmli sample buffer may also be used.</li>
		<li>Snap freeze excess eluted protein in liquid nitrogen. 
		OPTIONAL: strip remaining proteins from resin with an equal volume of 2x LDS sample buffer at 65 &#176;C for 5 min, remove the LDS-eluted proteins to a new tube, then add 2 &#181;l BME to the eluted proteins, not to the resin. This order is to prevent removal of any ions or antibodies that are conjugated to the beads.</li>
		<li>Store samples at -80 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Western Blot and probe with appropriate antibodies to visualize proteins.
	<ol>
		<li>Load 10-20 &#181;l (&#916;1-3 OD600 units) of each sample and 3-10 &#181;l of a protein ladder in an SDS-PAGE gel of choice. Gels routinely used for this protocol include 4-12% Bis-Tris and 8% Tris-Glycine.</li>
		<li>Run gel at 200 V for 50 min.</li>
		<li>Transfer proteins from gel to a PVDF membrane by semi-dry transfer at 19 V for 20-30 min (recipe in Table 1).</li>
		<li>Block membrane in 4% milk/1x Tris buffered saline-TWEEN (TBST) for 1 hr at RT or O/N at 4 &#176;C (recipe in Table 1).</li>
		<li>Incubate membrane with primary antibody to the epitope-tagged protein of interest in 4% milk/1x TBST for 1-3 hr at RT or O/N at 4 &#176;C.</li>
		<li>Wash membrane 3x for 5 min each with 1x TBST.</li>
		<li>Incubate membrane with appropriate secondary horseradish peroxidase (HRP) -conjugated antibody for 1-3 hr at RT.</li>
		<li>Wash membrane 3x for 15 min each in a large volume of 1x TBST.</li>
		<li>Cover membrane with ECL substrate and wrap in saran wrap.</li>
		<li>Expose membrane to film and develop to visualize proteins.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Klis, F. M., Boorsma, A., De Groot, P. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+construction+inSaccharomyces+cerevisiae.">Cell wall construction inSaccharomyces cerevisiae.</a> Yeast. 23 (3), 185-202 (2006).</li><li>Gelperin, D. M., White, M. A., 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=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes Dev. 19 (23), 2816-2826 (2005).</li><li>Caserta, E., Haemig, H. A. H., 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=In+Vivo+and+In+Vitro+Analyses+of+Regulation+of+the+Pheromone-Responsive+prgQ+Promoter+by+the+PrgX+Pheromone+Receptor+Protein.">In Vivo and In Vitro Analyses of Regulation of the Pheromone-Responsive prgQ Promoter by the PrgX Pheromone Receptor Protein.</a> J. Bacteriol. 194 (13), 3386-3394 (2012).</li><li>Ghaemmaghami, S., Huh, W. -. K., 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=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Hannich, J. T., Lewis, A., 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=Defining+the+SUMO-modified+proteome+by+multiple+approaches+in+Saccharomyces+cerevisiae.">Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.</a> J. Biol. Chem. 280 (6), 4102-4110 (2005).</li><li>Xie, Y., Rubenstein, E. M., Matt, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-independent+in+vivo+activity+of+a+SUMO-targeted+ubiquitin+ligase+toward+a+short-lived+transcription+factor.">SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor.</a> Genes Dev. 24 (9), 893-903 (2010).</li><li>Daum, G. G., B&#246;hni, P. C. P., Schatz, G. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to protein purification. , (1990).</li><li>Puig, O., Caspary, F., 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=The+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Conzelmann, A., Riezman, H., Desponds, C., Bron, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+125-kd+membrane+glycoprotein+of+Saccharomyces+cerevisiae+is+attached+to+the+lipid+bilayer+through+an+inositol-containing+phospholipid.">A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid.</a> EMBO J. 7 (7), 2233 (1988).</li><li>Dertzbaugh, M. T. M., Cox, L. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+affinity+of+cholera+toxin+for+Ni2++ion.">The affinity of cholera toxin for Ni2+ ion.</a> Protein Eng. 11 (7), 577-581 (1998).</li><li>Hautbergue, G. G., Goguel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+C-type+cyclin+Ctk2p+is+phosphorylated+and+rapidly+degraded+by+the+ubiquitin-proteasome+pathway.">The yeast C-type cyclin Ctk2p is phosphorylated and rapidly degraded by the ubiquitin-proteasome pathway.</a> Mol. Cell. Biol. 19 (4), 2527-2534 (1999).</li><li>Elmore, Z. C., Donaher, M., Matson, B. C., Murphy, H., Westerbeck, J. 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The authors have nothing to disclose.

This protocol focuses on the preparation of intact, native, and post-translationally modified proteins from budding yeast cells for down-stream applications. Before attempting this protocol, it is critical to determine if the protein of interest can be readily detected in protein extracts prepared under denaturing conditions12. If polyclonal antibodies are not available it may be advantageous to epitope tag the protein of interest so that the fusion protein can be detected on Western blots. In the present prot...

The awesome power of yeast genetics is legendary, but the preparation and analysis of native proteins from budding yeast, Saccharomyces cerevisiae, is often fraught with problems. The latter is due to the considerable mechanical strength and elasticity of the yeast cell wall1. Different means have been described for the enzymatic, chemical, mechanical, and pressure-based disruption of yeast cells to obtain whole-cell protein extract 2-6. These techniques vary widely in their efficacy to yield cell-representative, native protein extracts that can be used for subsequent analyses or purification steps. For example, the yeast cell wall can be removed with lytic enzymes (e.g. zymolyase) and resulting spheroblasts can be disrupted by shearing, detergents, or osmotic lysis to release proteins. This approach has been successfully employed as the starting point for many subcellular fractionations but it requires lengthy incubations that are not compatible with the stability of some proteins7.
Proprietary yeast lysis reagents (such as detergents) for the chemical extraction of proteins of yeast cells are commercially available but the efficacy of these reagents in protein extraction and their effect on subsequent biochemical characterization of proteins is not always clear8. High pressure homogenizers, often referred to as French presses, effectively break yeast cells by first subjecting them to high pressure and then extruding them through a small opening in a pressure cell. This technique produces high quality extracts but the equipment is very expensive and may not be suitable for small quantities of cells or multiple samples9. Therefore, mechanical disruption of yeast cells in a bead mill is often the method of choice for native yeast protein preparations10. This technique involves mechanical disruption of the yeast cell wall with acid-washed glass beads, which can be conducted with a variety of shakers, vortexers or bead mills. Notably, this method can be used to simultaneously process multiple smaller samples (1 ml of cells or less). Many different beads or bead mill disruption matrices are now commercially available to disrupt almost any kind of cell type in 2 ml tubes. Considering the other techniques and equipment, a bead mill has the added advantage that the disruption of yeast cells occurs very fast, which helps to preserve post-translational modifications such as sumoylation, especially when the appropriate buffers with protease and/or proteasome inhibitors are utilized and the temperature of extracts is controlled.
This protocol focuses on the fast, effective, and reliable extraction of endogenous and over-expressed proteins under gentle conditions with the ultimate goal to preserve protein function, interactions, and post-translational modifications. Growth media, cell lysis buffer compositions, and bead mill settings are optimized to maintain protein interactions and post-translational modifications such as sumoylation and ubiquitylation.

<table><tbody><tr><td>Omni Bead Ruptor 24</td><td>Omni International</td><td>19-010</td><td></td></tr><tr><td>Yeast ORF strain in BG1805</td><td>ThermoScientific</td><td>YSC3869</td><td>GAL+ yeast strain that can be used for induction and extraction</td></tr><tr><td>pYES2.1 TOPO vector</td><td>Life Technologies</td><td>K4150-01</td><td>GAL+ yeast strain that can be used for induction and extraction; contains a V5/6xHIS tag</td></tr><tr><td>Halt Protease Inhibitor Single-Use Cocktail (100x)</td><td>Thermo Scientific</td><td>1860932</td><td></td></tr><tr><td>2.0 ml Skirted Tube with Tethered Screw Cap</td><td>BioExpress</td><td>C-3369-3</td><td>Make sure that the tube properly fits in the bead ruptor</td></tr><tr><td>Glass beads, acid-washed, 425-600 &amp;micro;m (30-40 US sieve)</td><td>Sigma-Aldrich</td><td>G8772</td><td></td></tr><tr><td>Corning Costar Spin-X centrifuge tube filters</td><td>Sigma-Aldrich</td><td>CLS8161</td><td>Use for additional filtering of clarified lysate</td></tr><tr><td>TALON Metal Affinity Resin</td><td>Clontech</td><td>635502</td><td>For the purification of 6xHIS tagged proteins</td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Life Technologies</td><td>NP0007</td><td>To prepare and/or elute samples prior to SDS-PAGE and Western Blotting</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Gel</td><td>Life Technologies</td><td>NP0321BOX</td><td>Precast gels often used for SDS-PAGE prior to Western Blotting</td></tr><tr><td>Simply Blue</td><td>Life Technologies</td><td>#LC6060</td><td>Protein gel stain</td></tr><tr><td>Mammalian Lysis Buffer</td><td>Promega</td><td>G9381</td><td>Alternative commercial lysis buffer</td></tr><tr><td>Anti-V5 agarose</td><td>Sigma</td><td>A7345</td><td>Method of immunoprecipitation</td></tr><tr><td>ECL</td><td>Millipore</td><td>WBKL S0 050</td><td></td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>BIS-TRIS gels</td><td>Life Technologies</td><td>NP0321BOX</td><td></td></tr><tr><td>anti-myc antibody</td><td>Covance</td><td>mms-150R</td><td></td></tr><tr><td>secondary antibody</td><td>Abcam</td><td>ab97040</td><td>Goat pAb to mouse IgG (HRP)</td></tr></tbody></table>

budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

Our representative results reveal that the described bead-beating and protein extraction protocol is useful for the reproducible preparation of proteins for a variety of downstream applications (summarized in Table 2). SDS-PAGE followed by Coomassie staining of WCEs show that a wide range of proteins (~12 to &#62;250 kDa) can be reproducibly extracted from yeast cells (Figure 1A). Discrete bands over a range of molecular weights are indicative of high quality protein extracts. The qualit...

Watch this Scientific Journal Video about Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications at JoVE.com

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

budding-yeast-protein-extraction-purification-for-study-function

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

24.8K Views.  The College of William & Mary. The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Video: Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biochemistry

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...

Genetics

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

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.

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 ...

An Introduction to Saccharomyces cerevisiae

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans).
This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer&rsquo;s and Parkinson&rsquo;s diseases.

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

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Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

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

An Introduction to Saccharomyces cerevisiae