The study of subcellular components often requires purifying a large amount of a particular component. One might be interested in isolating and purifying for further analysis the components of a complex unit like a ribosome, proteasome, or spliceosome. Or one might want to measure the metabolic activity of organelles such as the mitochondrion or peroxisome. Cell fractionation methods have been developed that allow the researcher to separate relatively pure samples of subcellular components in reasonably active states. Lloyd & Cartledge (1991) and Zinser & Daum (1995) review methods of isolation of yeast subcellular organelles. Additional methods for specific organelles are listed in Walker (1998).
The first step is to rupture the cells and release the contents. A variety of methods are available for Saccharomyces that are similar to those used for other types of cell with variations to accommodate the rigid cell wall and the high level of protease activity found in Saccharomyces. Cells are grown under the appropriate culture conditions, harvested by centrifugation or filtration, and resuspended in a buffered salt solution containing protease inhibitors. A number of mechanical, chemical, or enzymatic methods are available for breaking open the cells and releasing the contents. A French press is used for large-sized samples (several grams of cells). This device forces cells through a small hole under pressure. Cells can also be ruptured by aggressive agitation of a cell suspension with glass beads. This can be done on small samples simply by vortexing the cell-glass bead suspension or by shaking the suspension in any of a variety of devices designed specifically for this purpose. Alternately, the Saccharomyces cell wall may be stripped using glusulase or zymolyase, enzymes that attack the structural components of the cell wall, after which the cells are burst by altering the osmolarity of the cell suspension.
Differential-velocity centrifugation separates subcellular components based on size/ shape and density. The theory is that the larger more dense components will pack at the bottom of a centrifuge tube faster and at lower speeds then smaller less dense ones. Initially, the total cell extract is centrifuged at a low speed for a short time to remove unbroken cells. In the next steps, the speed and time of centrifugation are progressively increased removing some components (packing them at the bottom of the tube as a precipitate) and leaving others in the supernatant at each step. The final supernatant, after the step-wise removal of nuclei, mitochondria, vacuoles, peroxisomes, plasma membrane and vesicles, endoplasmic reticulum, and ribo-somes, is called the soluble fraction and contains soluble proteins and other small molecule components of the cytosol such as tRNAs.
Equilibrium density gradient centrifugation separates subcellular components based only on their density. For this method, one must first prepare a density gradient in a centrifuge tube. A nonionic molecule like sucrose, glycerol, or Ludox is used to vary the density of the buffer solution. The concentration of the molecule is varied, and therefore the density of the solution, and the concentration, is greatest at the bottom of the centrifuge tube and decreases slowly towards the top of the tube. Special devices are available for making these gradients. A step gradient can also be prepared. Here a series of solutions of different concentration (30%, 25%, 20%, etc.) are layered on top of one another with the step with the highest concentration at the bottom.
The cell extract is layered at the top of the gradient and the tube is subjected to centrifugation at high speed for several hours. During this time the different subcellular components move down the tube until they reach the position in the density gradient that corresponds to the density of the component and will remain in this position indefinitely. In a step gradient, the organelle will position itself between two steps. Using a fraction collector, individual small samples are gently removed from the tube starting at the top or bottom in a manner that does not disturb the gradient. The samples are then analyzed by Western analysis, electron microscopy (EM), or biochemical assay to identify the subcellular location of a particular protein. The purified fractions also can be used for other biochemical studies.
Figure 2.1 illustrates the results from a typical equilibrium density gradient separation experiment. Western analysis (see below) was used to identify the fractions containing the protein of interest, Gaplp (the general amino acid permease) in this experiment, and biochemical assays of marker enzymes from the different subcellular compartments were carried out to identify the location of the compartment in the gradient.
The purity of the subcellular fractions is often at issue. Samples obtained by differential-velocity centrifugation are generally not considered to be a highly homogeneous purified product. No matter which method is used it is essential to test the purity. The samples can be observed by EM to determine the presence of contaminating components. Marker enzymes or proteins characteristic of a particular
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