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Changes in ribosomes of artichoke (Helianthus tuberosus L.) tuber cells following excision and aging of tissue slices in water were studied using biochemical techniques. During the first 2 h of aging total rRNA dropped 28% and then remained constant for a subsequent 46 h. Since ribosome synthesis occurs through at least the first 24 h of aging, turnover of ribosomes must take place in this period. Cells of the dormant tuber gave essentially no membrane-bound (mb) ribosomes. On aging, the mb ribosome fraction rose and reached a maximum of 25% of total ribosomes at 24 h. Density gradient analysis showed that the ribosomes of dormant cells were present largely as monosomes. After 4 h aging a significant number of ribosomes in both free and mb populations sedimented as polysomes and the number of polysomes in both populations increased to a maximum at 24 h. The direct polysome analysis was confirmed by estimates of synthetically "active" ribosomes obtained using 0.8 M KCl to isolate monosomes carrying nascent polypeptides. This approach showed that while unaged cells had only 13% of total ribosomes active, on aging the active fraction rose to about 68% at 24 h. Both free and mb populations showed the same percentage of ribosomes active at all times studied. [(3)H]uridine showed significant incorporation into ribosomes during three periods studied; 2-4h, 12-14h, 22-24h. At the two latter periods the specific activity of the free ribosomes was greater than that of the mb ribosomes. Uridine was incorporated into both active and inactive ribosomes of both populations, judged by KCl fractionation, with the inactive fraction having greater specific activity in both cases. These differences in labelling possibly result from relatively slow mixing of different ribosome populations. Uptake of soluble [(3)H]uridine into the tissue increased 4-fold between 4 h and 14 h accounting at least in part for greater overall specific activity of ribosomes at later aging times.

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The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.

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