A MUTANT OF YEAST APPARENTLY DEFECTIVE IN THE INITIATION OF PROTEIN SYNTHESIS* BT LELAND H. HARTWELLt AND CALVIN S. MCLAUGHLIN DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA (IRVINE) Communicated by Edward A. Steinhaus, December 4, 1968 Abstract.-A temperature-sensitive mutant of yeast, ts-187, which is apparently unable to initiate the synthesis of new polypeptide chains after a short incubation at the restrictive temperature, is described. The existence of this mutant demonstrates that in eucaryotic cells, as in procaryotic cells, there are processes unique to the initiation of polypeptide chains. Introduction.-Our understanding of macromolecule synthesis and its regulation in eucaryotic organisms would undoubtedly benefit from a series of mutants conditionally defective in specific steps of macromolecule synthesis, if such were available. We have, accordingly, isolated and characterized a series of 400 temperature-sensitive mutants of yeast unable to grow at 36 even on highly enriched medium.1 Some of these mutants exhibit a rapid inhibition of protein synthesis following a shift from the permissive to the restrictive temperature. An investigation of polyribosome content and polysaccharide synthesis in the mutants defective in protein synthesis allowed a grouping of these mutants into four distinct classes.2 Three mutants grouped together in class IA exhibit a loss of polyribosomes at the restrictive temperature but retain the capacity for polysaccharide synthesis. It was argued that this behavior was consistent with a defect either in the initiation of protein synthesis or in messenger RNA synthesis. We wish to report further studies which indicate that one of these three mutants, ts-187, displays properties that strongly suggest a defect in the initiation of protein synthesis; a second, ts-171, does not complement with ts-187 and is probably defective in the same gene function. Materials and Methods.-Yeast spheroplasts were prepared, resuspended to a concentration of about 1 X 107 cells/ml (calculated from the number of whole cells used in their preparation), and incubated for 3 hr at 210C in YM-5 medium containing 0.4 M MgSO4. This was done prior to each experiment. Temperature shifts were achieved by the addition of an equal volume of medium at 600C to a culture of cells at 210C while they were being swirled, unless otherwise indicated; this procedure brought the flask and its contents to 370C in a matter of seconds. The source of materials and the complete description of experimental techniques have been described in previous publications as indicated: the origin of the parental strain and its genetic constitution, the origin of the mutants, the composition of YM-5 medium, and the techniques used to determine the amount of radioactivity incorporated into macromolecules;' the preparation of yeast spheroplasts and their growth;3 and the analysis of polyribosomes by sucrose gradient centrifugation.2 Results.-Protein and RNA synthesis in mutant ts-187: Mutant ts-187, isolated for its ability to form colonies on enriched medium at 230C but not at 360C, originated from strain A364A by mutagenesis with N-methyl-N'-nitro-Nnitrosoiuanidine.'t When cultures of ts-187 growing at 230C with a doubling 468
VOL. 62, 1969 BIOCHEMISTRY: HARTWELL AND McLAUGHLIN 469 time of about three hours are shifted to 360C, no significant increase in the protein or RNA content of the culture occurs over a period of six hours; DNA increases about 35% over the first three hours.' An examination of the kinetics of protein synthesis in cultures of ts-187 reveals that protein synthesis is very quickly inhibited after a shift to the restrictive temperature. A culture shifted from 21 to 370C synthesizes protein slightly faster than a culture remaining at 21 C for a period of 1.5 minutes and then protein synthesis is abruptly inhibited (Fig. 1). The kinetics of RNA synthesis, FIG. 1.-Kinetics of protein syn- - 21 thesis at 21 and 370C in spheroplast tsls7s cultures of mutant ts -187. Sphero- 6 plasts of mutant ts-187 growing at 210C were concentrated tenfold by centrifugation and preincubated at 210C for 15 min. To one half of the x culture an equal volume of media at E 4 210C containing 0.4 MAc/ml 14C-isoleucine was added. The other half received the same labeled medium prewarmed to 600C so that the tem- 2 perature was immediately raised to 37 37. 1-ml samples were removed at various times and analyzed for the amount of radioactivity incorporated 0 5 10 into protein. MINUTES examined under the same experimental conditions, are quite different. In the culture shifted to 370C, the rate of RNA synthesis is accelerated for at least ten minutes, relative to the culture at 210C, and gradually decreases after this time (Fig. 2). These results suggest that the primary defect of this mutant resides in the protein synthetic system and that the eventual cessation of RNA synthesis may be a stringent response to the prior cessation of protein synthesis.4 Polyribosomes in mutant ts-187: A previous examination of polyribosome content in a series of mutants at the restrictive temperature revealed that after one hour at 370C, all the polyribosomes of mutant ts-187 were converted to monoribosomes.2 A detailed investigation of polyribosome breakdown in mutant ts-187 has been carried out. Sucrose gradients of cytoplasmic extracts prepared from cultures of mutant ts-187 growing at 210C exhibit 90 per cent of the ribosomes present as polyribosomes (Fig. 3). After a five-minute incubation at 370C, essentially all of the polyribosomes are converted to monoribosomes. However, the addition of 100 gg/ml of cycloheximide at the time of the shift from 21 to 370C prevents the conversion of polyribosomes to monoribosomes (Fig. 3). Cycloheximide is known to inhibit the incorporation of amino acids from aminoacyl-trna into the growing polypeptide chain.6 Apparently, continued translation of messenger RNA into polypeptide at the restrictive temperature is a necessary prerequisite for polyribosome breakdown. Cytoplasmic extracts were prepared from mutant ts-187 after a variety of incubation times at 370C to establish the kinetics of polyribosome decay (Fig. 4). Approximately 90 per cent of the ribosomes are present as polyribosomes until
XSmin 470 BIOCHEMISTRY: HARTWELL AND MCLAUGHLIN PRoc. N. A. S. ts 187S 2.5JI 2. -, F ;* f.omin at37 S--.5minat37+ at37 + cyclohex _e a 0(4 Ni ci ~'2 Lo. 0.5 ' 0 0 10 20 FRACTION 30 0 10 20 30 MINUTE FIG. 2.-Kinetics of RNA synthesis at 21 and 370C in spheroplasts of mutant ts-187. Spheroplasts of mutant ts-187 growing at 210C were concentrated tenfold by centrifugation and preincubated at 210C for 15 min in YM-5 medium containing only 2 Mg/ml adenine. To one half of the culture an equal volume of medium lacking carrier adenine at 210C containing 0.2,uc/ml 14C-adenine (45 mc/mm) was added. The other half received the same labeled medium warmed to 600C so that the temperature was immediately raised to 370C. 1-ml samples were removed at various times and analyzed for the amount of radioactivity incorporated into RNA. FIG. 3.-Polyribosome patterns from spheroplasts of mutant ts-187 at 370C in the presence and absence of cycloheximide. Spheroplasts of mutant ts-187 growing at 210C were shifted to 370C. One 40-ml aliquot was collected immediately after the shift; a second 40-ml aliquot was collected after incubation at 370C for 5 min; a third 40-ml aliquot received 40 Mg/ml of cycloheximide at the time of the temperature shift and it also was collected after 5 min at 370C. The cell extracts were layered on 10-60% w/v sucrose gradients and centrifuged at 24,000 rpm in a Spinco SW25.1 head at 50C for 3 hr. The gradients were collected by pumping out the top of the tube so that the top of the gradient is fraction 1. The 80S monoribosome peak is at fraction 8, and the polyribosomes are in fractions 12 through 30. one minute after the shift to 37. The decay of polyribosomes to monoribosomes begins at one minute and is essentially complete by two minutes. In order to ensure that the preceding results were a true reflection of intracellular events and not an artifact of extract preparation, a mixing experiment was performed. A large aliquot of cells (unlabeled) which had been incubated for 90 seconds at 370C was mixed with a small aliquot of cells (labeled with 14Cadenine) which had been incubated for 60 seconds at 370C. From the previous results, we expect that in the former most of the polyribosomes will break down while in the latter most of the polyribosomes will stay intact. A cytoplasmic extract was prepared from the mixture and analyzed on a sucrose gradient. Ninety per cent of the ribosomal OD260 was present as monoribosomes, while 85 per cent of the radioactivity sedimented in the polyribosome region. Thus the addition of a large amount of extract in which polyribosome breakdown had occurred did not influence the sedimentation characteristics of polyribosomes
VOL. 62, 1969 BIOCHEMISTRY: HARTWELL AND McLAUGHLIN 471 FIG. 4.-The percentage of the ribosomes which are present as polyri- bosomes in spheroplasts of mutant ts-187 as a function of time at 370C. Spheroplasts of mutant ts-187 grow- 60 ing at 210C were shifted to 370C by -I the addition of medium warmed to a Rt187s 600C. At various times after the L shift, the cultures were collected and 40 extracts were prepared and analyzed for polyribosome content, as indicated 20 in the legend to Fig. 3. The data have 5 20 been corrected for a small absorption at A 260 m1a by the sucrose solution. 00 1 2 3 4 5 15' 30'6 MINUTES AT 37* from a small amount of extract. Polyribosome breakdown evidently occurs in vivo and not during or after extract preparation. The prevention of polyribosome breakdown by cycloheximide indicated a requirement for polypeptide synthesis before breakdown could occur. It is reasonable to expect that this requirement reflects the need for a ribosome to finish the translation of a messenger RNA molecule into a polypeptide chain before it can disengage the messenger RNA and become a monoribosome. If this idea is correct, we would expect the nascent protein chains associated with polyribosomes to be released from the ribosome upon polyribosome breakdown. This expectation was tested in the following experiment. Spheroplasts of mutant ts-187 growing at 21'C were pulse-labeled with a mixture of "4C-amino acids, shifted to 370C, and sampled at short intervals for polyribosome analysis (Fig. 5). In the sample removed 45 seconds after the shift to 370C, polyribosome breakdown had not begun and a significant amount of radioactivity had been incorporated into nascent protein chains on polyribosomes. In the samples removed at 90 and 135 seconds, polyribosome breakdown was evident, but the specific activity of the polyribosomes which remain was increasing due to an increase in the specific activity of the amino acid pool. It is evident that the radioactivity present in the polyribosomes is not transferred to the monoribosomes upon polyribosome breakdown; the specific activity of the monoribosome peak at 135 seconds was threefold less than that of the polyribosome region at 45 seconds and tenfold less than the polyribosome region at 135 seconds. We conclude that nascent protein chains are released from the ribosomes when polyribosomes are converted to monoribosomes. Time required to synthesize a polypeptide chain: Polyribosome breakdown occurs in mutant ts-187 over a time period of approximately one minute. If this breakdown is a result of a cessation of polypeptide chain initiation, then the time period required for the synthesis of a complete polypeptide chain on yeast polyribosomes cannot be longer than about one minute. In order to determine the time required for polypeptide chain synthesis, spheroplasts of the parent strain, A364A, were pulse-labeled with a mixture of "4C-amino acids, and samples
472 BIOCHEMISTRY: HARTWELL AND McLAUGHLIN PRoc. N. A. S3. LS O.S~~~~~~~~~~~~ I I I0 1 20 2 E FIG. 5. Pulse-labeled nascent protein chains on the polyribosomes of mutant ts-187 at various times after the shift to VI C. Spheroplasts of mutant ts-187 growing at 21 C were -concentrated tenfold by centrifugation and incubated for 15 min at 21 C. Reconstituted 14Cprotein hydrolysate was added at a concentration of 5 pe/ml and the culture was incubated at 21IC for an additional 30 see. At this time, the culture was brought to VI C by being swirled in a 600C water bath for 15 see and then placed in a 37 C water bath. At 45, 90, and 135 see, after the culture was placed at 37 C, 6-ml aliquots were collected into 30 ml of cold medium containing- cycloheximide at a concentration of 10-3 M; extracts were prepared and analyzed int1-60% w/vsucrose gradients. were withdrawn from the culture at various times for polyribosome analysis..cytoplasmic extracts, prepared after two, four, and six minutes of "IC-amino acid incorporation, were analyzed on sucrose gradients. The radioactivity in the polyribosomes continued to increase over this time period (Table 1); however, the rate of 14C-amino acid incorporation into protein is constant by four minutes (see Fig. 1) and we would therefore expect the specific activity of the nascent polypeptide chains on polyribosomes to become constant. Evidently, part of the radioactive protein associated with polyribosomes is contributed by the entry of newly synthesized ribosome structural proteins into polyribosomes.,to determine the fraction of the radioactivity actually present in nascent polypeptide chains, polyribosomes from each gradient were collected and recentrifuged on sucrose gradients under conditions which dissociate polyribosomes into ribosomale subunits. A fraction of the radioactivity sedimented with the ribosomal subunits in this experiment and this fraction increased with the time of incubation. We assume that only the radioactivity at the top of the subunit gradients free from the ribosomal subunits is truly nascent polypeptide chains in the process of synthesis. The total radioactivity in the polyribosome gradients, the radioactivity associated with polyribosomes, and the percentage of the polyribosomae-associated radioactivity which is truly nascent polypeptide are recorded in Table 1. If we assume that, on the average, each nascent polypeptide chain is. half completed, then the radioactivity present in nascent polypeptide chains must be multiplied by two in order to obtain the amount of radio-
VOL. VBIOCHEMISTRY: 62, 1969 HARTWELL AND McLAUGHLIN 473 TABLE 1. Radioactivity in pulse-labeled nascent polypeptide chains associated with polyribosomes. -~~Radioactivity (cpm) Labeling Total in Amount in time polyribosome polyribosome Per cent* Cpm nascent X 2 (min) gradient region nascent (cpm per round) 2 43,421 9790 49.5 9690 4 96,212 15,028 45.5 13,676 6 147,623 23,318 38.5 18,048 * Determined by dissociating polyribosomes into ribosomal subunits. activity which would be associated with polyribosomes if each ribosome contained a completed polypeptide chain. This amount of radioactivity is equivalent to one round of synthesis. The number of rounds of synthesis which have occurred between two time intervals is the increase in total radioactivity over this time period divided by the average amount of radioactivity equivalent to one round of synthesis. The number of rounds of synthesis which have occurred during the time periods two to four minutes and two to six minutes are accordingly 4.51 and 7.51, respectively. The number of minutes per round during the same two intervals are therefore 0.445 and 0.532, respectively. We conclude that an average polypeptide chain is synthesized in about 0.5 minute on yeast polyribosomes at 370C. This time period is quite consistent with the assumption that polyribosome breakdown in mutant ts-187 which takes about one minute is a result of the cessation of polypeptide chain initiation, especially when one considers that some polypeptide chains will be longer than the average. Discussion.-A lesion in any one of a number of cellular processes would be expected to lead to a cessation of protein synthesis. It is important to note, therefore, that not only are the properties of mutant ts-187 consistent with the hypothesis of a defect in the initiation of polypeptide chain formation, but they are inconsistent with the hypothesis of defects in energy metabolism, membrane function, RNA synthesis, or polypeptide chain elongation. Thus, the ability of this mutant to synthesize DNA' and incorporate 14C-glucose into macromolecules at the restrictive temperature argue against the possibility of a defect either in energy metabolism or in membrane function. Several facts argue against the possibility that mutant ts-187 is defective in the synthesis of messenger RNA. First, the kinetics of RNA synthesis at the restrictive temperature indicate that it is not inhibited until several minutes after the inhibition of protein synthesis. Second, other evidence indicates that the half life of messenger RNA in yeast at 370C is about 20 minutes;6 if the primary defect in this mutant were its inability to synthesize messenger RNA, we would expect polyribosomes to decay in mutant ts-187 with a half life of 20 minutes, whereas, in fact, they decay with a half life of 0.5 minute. Third, a study of RNA synthesis in the presence of cycloheximide demonstrates that mutant ts-187 is capable of synthesizing messenger RNA as well as the parental strain at 37o.6 Finally, the breakdown of polyribosomes to monoribosomes at the restrictive temperature in mutant ts-187 is inconsistent with the hypothesis of a defect in the process of polypeptide chain elongation. Conditions which inhibit polypeptide chain elongation, such as cycloheximide addition, amino
474 BIOCHEMISTRY: HARTWELL AND McLAUGHLIN PRoc. N. A. S. acid starvation, or a defect in an aminoacyl-trna synthetase, result in a retention of polyribosomes.2 The experimental results strongly indicate that mutant ts-187 is defective in the initiation of polypeptide chains. The defective reaction might be any reaction which intervenes between the completion of one polypeptide chain and the beginning of a second. The kinetics of protein synthesis and the behavior of polyribosomes suggests that the defect does not manifest itself for the first minute at the restrictive temperature. It may be that the temperaturesensitive protein requires one minute for complete inactivation or it may be that there is enough of the product of the temperature-sensitive enzyme present in the cell to support polypeptide chain initiation for a period of one minute. Between one and two minutes after the shift to the restrictive temperature, protein synthesis is severely inhibited. Several facts supply evidence that this inhibition is due to a cessation of polypeptide chain initiation. During the same time interval, polyribosomes are converted to monoribosomes; nascent polypeptide chains are released from ribosomes upon polyribosome breakdown, and an inhibition of polypeptide chain elongation by cycloheximide prevents polyribosome breakdown. Furthermore, the time period required for the conversion of polyribosomes to monoribosomes agrees with the time required to synthesize a polypeptide chain. The existence of this mutant implies that there is at least one protein which functions in the initiation of polypeptide chains in a eucaryotic organism that is distinct from those factors which function in the elongation of polypeptide chains. In a procaryotic organism, Escherichia coli, several factors are thought to function uniquely in the process of polypeptide chain initiation. One of these factors is the enzyme methionyl-trna transformylase, which catalyzes the formylation of one species of methionyl-trna;7 and two other factors participate in the binding of N-formylmethionyl-tRNA to the ribosome.8-10 * This work was supported by USPH grants GM 13304 and CA10628 and National Science Foundation grants GB4828 and GB8028. t Department of Genetics, University of Washington, Seattle, Washington 98105. Tetrad analysis indicated that the temperature-sensitive lesion in t Note added in proof: this mutant segregates as a single nuclear gene. I Hartwell, L. H., J. Baderiol., 93, 1662 (1967). 2 Hartwell, L. H., and C. S. McLaughlin, J. Bacteriol., 96, 1664 (1968). s Hutchison, H. T., and L. H. Hartwell, J. Bacteriol., 94, 1697 (1967). 4 Stent, G. S., and S. Brenner, these PROCEEDINGS, 47, 2005 (1961). 5 Siegel, M. R., and H. D. Sisler, Biochim. Biophy8. Acta, 87, 83 (1964). 6 Hartwell, L. H., H. T. Hutchison, and C. S. McLaughlin, in preparation. 7 Clark, B. F. C., and K. Marcker, Nature, 211, 378 (1966). 8 Salas, M., M. B. Hille, J. A. Last, A. J. Wahba, and S. Ochoa, these PROCEEDINGS, 57, 387 (1967). ' Lucas-Lenard, J., and F. Lipmann, these PROCEEDINGS, 10 57, 1050 (1967). Revel, M., M. Herzberg, A. Becarevic, and F. Gros, J. Mol. Biol., 33, 231 (1968).