[31] Pulse labeling of yeast cells and spheroplasts

Publisher Summary This chapter focuses on the pulse labeling of yeast cells and spheroplasts. Pulse-labeling experiments are technically simple however several variables must be carefully controlled. These variables are discussed in this chapter. It is always useful to know the amino acid composition of the polypeptide to be studied. The specific radioactivity of the completed polypeptide depends not only on the specific radioactivity of the amino acid pool in the cell, but also on the abundance of that amino acid in the polypeptide chain. To utilize as much of the added radioactive tracer as possible, pulse-labeling should be performed under conditions where protein synthesis is rapid. One important factor is to transfer the cells or spheroplasts from growth medium to labeling medium rather quickly; only 3–4 minutes are necessary for centrifugation and resuspension. The labeling medium contains no amino acids, however small amounts may be carried over from the growth medium of spheroplast buffer because the cells and spheroplasts are not washed before suspending in labeling buffer. These small amounts do not significantly compete with [ 35 S]methionine for incorporation into protein, however may provide pools of other amino acids necessary to sustain protein synthesis.