Proteins comprise about half of the dry weight of most cells and include the many structural proteins, catalysts, receptors and signaling proteins responsible for cell function. To understand cell function, scientists often want to analyze the protein composition of cells. Protein analysis begins with the preparation of a cell extract, ideally under conditions that minimize protein degradation. Preparing good cell extracts is something of an art, and many factors need to be considered during the design of an extraction protocol.
In this course, we will be analyzing protein function in yeast. An average haploid yeast cell contains ~6 pg protein (Sherman, 2002). Although yeast cells have many advantages for genetic studies, they are notoriously difficult to use for biochemical studies. Nonetheless, scientists have been able to develop procedures for extracting yeast proteins that circumvent these experimental barriers.
The first consideration in designing an extraction procedure is the compartmentalization of cells. All cells are surrounded by a plasma membrane and eukaryotic cells contain additional membranes that surround organelles. Fungal cells also have cellulose-based cell walls that protect the cells against mechanical and osmotic forces. Cell extraction procedures begin with the disruption of the plasma membrane and cell wall by mechanical and/or chemical treatments. Mechanical disruption of yeast cells must be fairly vigorous because their cell walls are very tough. Mechanical methods commonly used to disrupt yeast include sonication, high pressure, and “beating” with glass beads. These vigorous treatments run the risk of damaging proteins because of the heat and foaming generated during the processes.
Chemical treatments offer a gentler alternative to mechanical disruption for preparing extracts. Chemical extraction procedures frequently include detergents that solubilize membrane lipids, thereby allowing proteins to diffuse out of the cell. Most detergents do not discriminate between intracellular and plasma membranes, so a detergent extract usually contains proteins from multiple organelles as well as cytoplasmic proteins. In this experiment, we will use sodium dodecyl sulfate (SDS) as the detergent. SDS is a denaturing detergent that unfolds protein structures by breaking the thousands of weak bonds that normally stabilize protein structures. The proteins are converted to random coils coated along their lengths by negatively charged SDS molecules.
When preparing extracts, care must be taken to protect proteins from degradation by cellular proteases. Cells contain proteases with many different specificities that are responsible for normal turnover of proteins in cells. Cell disruption often releases proteases from compartments such as lysosomes, providing them access to cytoplasmic proteins. Yeast are notoriously rich in proteases. In an intact yeast cell, many of these proteases are located in the yeast vacuole, which is analogous to the mammalian lysosome. The protocol that we will use in this course (Amberg
et al., 2005) uses a combination of heat and SDS to rapidly destroy the yeast proteases. Cell suspensions are immediately plunged into a boiling water bath after the addition of SDS. Extracts prepared by this method are suitable for electrophoresis and western blot analysis.