The methods of centrifugal elutriation, two-dimensional gel electrophoresis, and dual isotopic labeling were applied to the study and identification of a number of purified yeast proteins. The location of polypeptide spots corresponding to specific proteins was determined on two-dimensional gels. A dual-label method was used to determine the rates of synthesis through the cell cycle of the identified proteins as well as to confirm the results of previous studies from our laboratory on unidentified proteins. The identified proteins, and the more generally defined phosphorylated, heat shock, and heat stroke proteins were found to follow the general pattern of exponential increase in rate of synthesis through the cell cycle. In addition, colorimetric enzyme activity assays were used to examine the catabolic enzyme ca-glucosidase (EC 3.2.1.20). Both the activity and synthesis of a-glucosidase were found to be nonperiodic with respect to the cell cycle. These data contrast with earlier reports of periodicity, which employed induction and selection synchrony to study enzyme expression through the yeast cell cycle.The cell cycles of eucaryotic organisms contain a number of distinct events which take place only once in each cell cycle and are restricted to a defined period. In the budding yeast Saccharomyces cerevisiae, such distinct, one-time events include: spindle pole body duplication and separation, bud emergence, DNA synthesis, nuclear migration, nuclear division, cytokinesis, and cell separation (23). In addition to distinct events, cells undergo an exponential increase in size through the cell cycle (23). In light of the temporal sequence of events through the cell cycle, one might expect to see temporal expression of specific protein products responsible for (or dependent upon) such events.Workers in this laboratory have approached the problem by applying the technique of centrifugal elutriation to the separation of asynchronous, exponentially growing populations of yeast cells into cell cycle specific fractions (14). During elutriation, cells in the separation chamber are acted upon by a centrifugal force which tends to sediment them. This force is opposed by a flow of liquid, and with increasing flow rate, cells of increasing size are washed from the chamber and collected into specific fractions. t Current address:

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucoseor fructose-nutrient medium. The FBP activity in glucoseor fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactoseand ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gluconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.son, the activities of PFK and phosphoglucose isomerase (PGI; D-glucose-6-phosphate-ketolisomerase, EC 5.3.1.9), an enzyme used both in glycolysis and gluconeogenesis, were determined. A preliminary report of this investiga-978 on August 3, 2020 by guest http://jb.asm.org/ Downloaded from gensis. J. Bacteriol. 117:965-970.

No abstract

The intracellular levels of adenosine triphosphate and several glycolytic intermediates were determined in Saccharomyces cerevisiae in relation to the presence of the metabolically antagonistic enzymes phosphofructokinase and fructose-1,6-bisphosphatase. Phosphofructokinase is synthesized constitutively in cells grown in the presence of glucose and fructose-1,6-bisphosphatase derepression occurs upon the exhaustion of glucose from the growth medium. Transcriptional regulation of fructose-1,6-bisphosphatase was suggested based on experiments with wild type cells using 8-hydroxyquinoline, a known inhibitor of nuclear transcription, and with the S. cerevisiae mutant strain A364A (ts-136) blocked in the transport of nuclear RNA at non-permissive temperature. The level of phosphofructokinase was reduced more than 25-fold under conditions of high citrate accumulation in an aconitase-less, glutamate requiring mutant strain, MO-1-9B. There was a rapid decrease in the levels of adenosine triphosphate and fructose-1,6-bisphosphate at the end of log-phase of culture growth when both fructose-1,6-bisphosphatase and phosphofructokinase were present in the cells simultaneously. The changes in the levels of key glycolytic intermediates, but not the changes in adenosine triphosphate, during the simultaneous presence of these two enzymes, can be explained without involving any futile cycling.

Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.