Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

Abstract

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

A research‐based laboratory course designed to strengthen the research‐teaching nexus

“…That their secondary and tertiary structures were remarkably similar, in spite of their different amino acid sequence, was unexpected to many students. At the end of this computer session, each student received a copy of the article ''Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates'' [20] to be prepared for the next three laboratory experiments. In preparation for the next laboratory sessions, the instructor transformed vma6D cells that lacked the gene VMA6 [6] with mutagenized pRS316-VMA6 plasmids separately.…”

“…The reading assignment [20] helped to focus students' attention, who described the article as ''very helpful and clear.'' The Week 8 to 10 experiments used the same protocols described in the article to study expression of subunit d and other V-ATPase subunits in the mutant strains made during this laboratory course.…”

Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

“…SDS-PAGE is a simple, rapid technique that is readily adaptable to the undergraduate laboratory. Applications that have been used in undergraduate biochemistry laboratory curricula include assessment of protein purity (10), determination of subunit molecular mass (11,12), determination of subunit stoichiometry (13), investigation of the presence of intermolecular disulfide bonds (14), and Western blotting (15)(16)(17). However, some care is required to set up an experiment to obtain accurate results and to teach students skills that they can use in a professional setting.…”

Preparation, Purification, and Secondary Structure Determination of Bacillus circulans Xylanase. A Modular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry

“…The first was to move away from labs focused on single techniques in the context of different proteins, to an integrated lab with a single protein that allows students to learn techniques that culminate in a discoveryoriented project. Aspects of this basic approach have been documented in this Journal (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”