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Identification of Yeast V-ATPase Mutants by Western Blot Analysis of Whole Cell Lysates Karlett Parra-Belky Department of Chemistry, Ball State University, Muncie, IN 47306;
[email protected] Background The diverse and fast-growing applications of biochemical tools in health-related fields and biotechnology make it imperative to implement such biochemical techniques in undergraduate biochemistry labs. We have designed a lab to identify yeast-cell strains using electrophoretic and immunochemical techniques. These techniques are used in basic research (1) and clinical confirmatory tests for HIV infection (2). The budding yeast Saccharomyces cerevisiae represents an ideal system to illustrate ongoing biotechnology research because the cells are innocuous, the entire genome has been sequenced, its genes have been cloned, and information on its 6300 proteins is available in the protein database (3). Yeast research has provided essential background for understanding complex biochemical processes because yeast cells are structurally and functionally similar to other eukaryotic cells (4). In addition, the yeast proton pump vacuolar H+-ATPase (V-ATPase) resembles its human counterpart, which is implicated in kidney function (6), deafness (6), and spermatozoa maturation (7). The laboratory presented here uses three yeast strains to illustrate the link between genetic engineering and biochemical research. The strains used included a wild type and two mutants for the V-ATPase (1). Mutants lack one structural gene of the multisubunit V-ATPase complex. Mutants are easily identified and distinguishable from wild-type cells because mutants do not express the protein encoded by the deleted gene, whereas wild-type cells express all the V-ATPase subunits. The multimeric complex consists of 13 different subunits (named A–H, a, c, c′, c′′, and d), including peripheral- and integral-membrane proteins (Figure 1, ref 5). The molecular weights of the subunits range from 13 kDa to 100
kDa, making this complex an attractive system to identify particular subunits according to their molecular weight. Students use electrophoresis to separate a heterogeneous mixture of total cellular proteins isolated from each yeast strain. Proteins are separated by their molecular weight using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and specific V-ATPase subunits identified in the mixture by Western blotting. At the end of three, threehour lab sessions, students are able to clearly interpret the results by identifying each strain on the basis of the subunit’s antigenic properties and molecular mass. Experimental Procedure Students receive a liquid culture of three unknown cell strains grown overnight in YEPD (yeast extract–peptone–dextrose) medium. Vacuolar membrane ATPase (vma) mutants used are vma1∆ and vma2∆, which lack the genes encoding for the V-ATPase subunits A (69 kDa) and B (60 kDa), respectively (1). Students begin the experiment by converting cells to spheroplasts by enzymatic digestion of the cell wall. Each strain is resuspended in buffer containing dithiothreitrol (DTT) for five minutes to reduce sulfhydryl bridges. DTT is removed by washing the cells with two rounds of centrifugation and resuspension of the pellets in buffer, pH 7.5 containing sorbitol. Neutral pH is optimal for zymolase activity and sorbitol offers osmotic support to the cells. Cell wall enzymatic digestion is accomplished by adding zymolase and rocking at 30 ⬚C for 30 min. Zymolase contains a mixture of proteases that could degrade cellular proteins if it was not removed before lysis. Any trace of the zymolase mixture is removed by washing the spheroplasts in sorbitol. Cell lysis is achieved by resuspending spheroplasts in cracking buffer (prewarmed at 75 ⬚C) containing bromophenol
Subunit Gene Name
Peripheral Subunits
Figure 1. The V-ATPase. V-ATPase structural genes and subunits composition.
Vacuolar
Membrane Subunits
Membrane
A B C D E F G H a c c’ c” d
VMA1 VMA2 VMA5 VMA8 VMA4 VMA7 VMA10 VMA13 VPH1 VMA3 VMA11 VMA16 VMA6
Interior of the vacuole (vacuolar lumen)
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Molecular Weight/kDa 69 60 42 32 27 14 13 54 100 17 17 21 36
In the Laboratory
blue. Cracking buffer contains a mixture of urea, SDS, and -mercaptoethanol, which ensures complete dissolution of cellular membranes and protein denaturation. After 15 min at 75 ⬚C, samples are ready for SDS–PAGE; alternatively, samples can be frozen for later use. Cell lysates are loaded onto precast 12% polyacrylamide minigels next to a mixture of prestained molecular weight marker proteins. Samples are applied in duplicate. Half of the gel is stained with Coomassie Blue and used as reference. Proteins in the other half are electroblotted to a nitrocellulose membrane and used for Western blot analysis. Transfer is performed overnight, although it could be accomplished in three hours. Membranes are stored for one week at 4 ⬚C in blotto solution containing 2% nonfat dry milk. Milk prevents nonspecific binding of the antibodies. Membranes are incubated with a mixture of two primary antibodies for 1.5 h. We used one monoclonal antibody against V-ATPase subunit A (69 kDa), and another that recognizes subunit B (60 kDa). Following primary antibodies incubation, membranes are incubated for an additional 45 min with a secondary antibody (IgG goat anti-mouse) conjugated to alkaline phosphatase. Western blots are then developed by adding the enzyme’s substrates BCIP (5-bromo-4-chloro-3indolyl phosphate) and NTB (nitroblue tetrazolium), which forms a purple precipitate product (reduced NTB) within 10 min where subunits A or B are present. Equipment and Chemicals Power supplies, electrophoretic, and electrotransfer blot cells are purchased only once. Disposable items included precast gels and nitrocellulose membranes. Molecular weight protein markers and antibodies are used in small volumes. BCIP and NTB stock solutions remain fresh at 4 ⬚C for months. Only 1.5 unit of zymolase is used per student and the stock enzyme is stable at ᎑20 ⬚C. Other chemicals are commonly used in biochemical research.
proteolysis and the notion of epitope in antigen–antibody interactions. Prestained molecular weight markers were a convenient control to verify protein transfer to the nitrocellulose membrane and eliminated the need to cut the membrane containing the marker lane for staining. The relative molecular weights of proteins calculated directly on the intact nitrocellulose membrane were consistent with SDS–PAGE (1), indicating that both methods were equivalent for these calculations. Western blotting added specificity to the method allowing identification and characterization of particular protein(s) from a mixture containing thousands of different proteins like a whole-cell lysate. Calibration curves indicate apparent molecular weights of 69 and 60 kDa for subunits A and B, respectively, which are consistent with molecular weights predicted by the genes ORF (open reading frame) nucleotide sequence (67,704 daltons for A and 57,727 daltons for B). These results also allow the instructors opportunity to address the concept of a three-letters genetic code. Coomassie Blue-stained gels showed the broad range molecular weight of cellular proteins separated by SDS– PAGE and served to illustrate the high specificity involved in Western blotting antigen-antibody interactions. Precast minigels allowed reproducibility and better resolution, eliminated the risk of working with liquid-polyacrylamide, and decreased preparation time. Conclusions We report a simple and highly specific protocol to identify strains of the yeast Saccharomyces cerevisiae. Described are powerful tools used in applied biotechnology that we have adapted for undergraduate instruction. Our experiments extended from other protein analyses that have used SDS–PAGE and Western blotting (8). The use of genetically engineered yeast strains helped students better understand molecular engineering applications in
Hazards BCIP and NTB may be harmful if swallowed, inhaled, or absorbed through skin. Gloves should be worn when handling these substrates. DTT and -mercaptoethanol must be handled in the hood. Results and Discussion The results were clear and easy to interpret by all the students (Figure 2A). Unknown yeast cell strain 2 lacked the 60-kDa subunit of the V-ATPase; unknown yeast cell strain 3 lacked the 69-kDa subunit of the V-ATPase; and unknown yeast cell strain 1 had both proteins. Students concluded that 1 was the wild-type strain, 2 was vma2∆, and 3 was vma1∆. The use of monoclonal antibodies simplified data interpretation since cross-reaction of the antibody with other antigens often associated with polyclonal antibodies was eliminated. Students also recognize the importance of washing out traces of zymolase solution before proceeding with cell lysis, because twenty percent of the students detect lower molecular weight protein bands in their results (Figure 2B). These unanticipated observations were meaningful because they exemplified
Figure 2. Western blots of whole cell lysates. SDS–PAGE gel transferred to a nitrocellulose membrane and blotted with monoclonal antibodies against subunits A (69 kDa) and B (60 kDa) of the VATPase complex. A: unknown yeast cell strains 1, 2, and 3 corresponded to wild-type, vma2∆ and vma1∆ strains, respectively. Std.: standard molecular weight markers. B: lower molecular weight bands resulting from subunit B degradation during cell lysis are indicated with an asterisk.
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biochemistry research. Since students prepared their own cell lysates rather than using a prepared mixture of few proteins, they had a firsthand opportunity to understand aspects of protein structure, protein denaturation, and biological membrane composition. Studying different subunits of one multimeric protein allowed for discussion of its structure and function, which helped capture the students’ interest. Students prepare a short report including an introduction describing methods used, their advantages and applications, as well as a description of the structure and cellular functions of the protein under study. In the results, a calibration curve of log(MW) versus RM (relative migration on the gel) is plotted and used to calculate the molecular weights of the V-ATPase subunits detected by Western blots. In their discussion, students compare Western blot membranes to Coomassie Blue-stained gels, identify the V-ATPase subunits and unknown yeast strains, distinguish monoclonal from polyclonal antibodies, and describe potential sources for experimental error. Acknowledgments I would like to thank Patricia Lang for encouraging the publication of this work, Patricia Kane for her generous donation of cell strains and antibodies, Nicole Wick for her assistance, Marcy Towns and Patricia Lang for their helpful revisions of the manuscript, and the Chemistry Department of Ball State University for supporting this project.
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Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Doherty, R. D.; Kane, P. M. J. Biol. Chem. 1993, 268, 16845– 16851. 2. Mylonakis, E.; Paliou, M.; Lally, M.; Flanigan, T. P.; Rich, J. D. Am. J. Med. 2000, 109, 568–576. 3. Saccharomyces Genome Database. http://www.genomewww.stanford.edu/Saccharomyces/ (accessed Aug 2002). 4. Resnick, M. A.; Cox, B. S. Mutat. Res. 2000, 451, 1–11. 5. Stevens, T.; Forgac, M. Annu. Rev. Cell Dev. Biol. 1997, 13, 779–808. 6. Karet, F. E.; Finberg, K. E.; Nelson, R. D.; Nayir, A.; Mocan, H.; Sanjad, S. A.; Rodriguez-Soriano, J.; Santos, F.; Cremers, C. W.; Di Pietro, A.; Hoffbrand, B. I.; Winiarski, J.; Bakkaloglu, A.; Ozen, S.; Dusunsel, R.; Goodyer, P.; Hulton, S. A.; Wu, D. K.; Skvorak, A. B.; Morton, C. C.; Cunningham, M. J.; Jha, V.; Lifton, R. P. Nat. Genet. 1999, 21, 84–90. 7. Brown, D.; Breton, S. J. Exp. Biol. 2000, 203, 137–145. 8. Fenk, Christopher J.; Grooms, Stephanie Y. J. Chem. Educ. 2000, 77, 373–374.
Journal of Chemical Education • Vol. 79 No. 11 November 2002 • JChemEd.chem.wisc.edu
