Messenger ribonucleic acid and protein metabolism during sporulation of Saccharomyces cerevisiae

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Abstract
To investigate differences between growing yeasts and those undergoing sporulation, several parameters of mRNA transcription and translation were compared. The general properties of mRNA metabolism were not significantly altered by the starvation conditions accompanying sporulation. The average mRNA half-life, calculated from the kinetics of incorporation of [3H]adenine into polyadenylic acid-containing RNA, was 20 min in both cell populations. Furthermore, 1.3-1.4% of the total RNA was adenylated in both growing and sporulating cells. The proportion of RNA that could be translated in a wheat germ system slowly decreased during sporulation. Within 8 h after the induction of sporulation, isolated RNA stimulated half as much protein synthesis as the equivalent amount of vegetative RNA. There were significant differences in protein synthesis. The percentage of ribosomes in polysomes decreased 3-fold as the cells entered sporulation. This decrease began within 5 min of the initiation of sporulation, and the steady-state pattern was attained within 120 min. The ribosomes were not irreversibly inactivated; they could be reincorporated into polysomes by returning the sporulating cells to growth medium. Though unable to sporulate, strains homozygous for mating type, MAT.alpha./MAT.alpha., showed a similar decrease in the number of polysomes when placed in sporulation medium. The same shift toward monosomes was observed during stationary phase of growth. The redistribution of ribosomes represents a general metabolic response to starvation. Apparently, the loss of polysomes is most likely caused by a decrease in the initiation of translation rather than a severe limitation in the amount of mRNA. The loss of polysomes is not due to the decreased synthesis of a major class of abundant proteins. Of the 400 vegetative proteins resolved by 2-dimensional gel electrophoresis, only 19 were not synthesized by sporulating cells. Approximately 10-20% of the cells in a sporulating culture failed to complete ascus formation. [35S]methionine is incorporated equivalently into cells committed to sporulation and cells that fail to form asci. The proteins synthesized by these 2 populations were indistinguishable on 1-dimensional gels. Proteins labeled by various protocols were compared, including long-term and pulse-labeling during sporulation and prelabeling during vegetative growth before transfer to sporulation medium. The resulting 2-dimensional gel patterns differed significantly. Many spots labeled by the long-term techniques may have arisen by protein processing. Pulse-labeling may produce the most accurate reflection of instantaneous synthesis of proteins.