[30] Labeling of RNA and phosphoproteins in Saccharomyces cerevisiae

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control for the experiment? Since a chimeric mRNA will utiliTe the same promoter as one of its parental mRNAs, the two mRNAs are presumably transcribed at approximately the same rate. To a first approximation, differences i n the steady-state levels should, therefore, be consistent with differences in the decay rates. Acknowledgments Most of the methods described here were developed with the support of a grant (GM27757) to A J . and a postdoctoral fellowship (GM11479) to R.P. from the National Institutes of Health. We thank David Munroe, Janet Donahue, and Laura Steel for their advice and assistance.

[30] Labeling of RNA and Phosphoproteins Saccharomyces cerev/siae

in

ByJONATHAN R. WARNER

The labeling of macromolecules in vivo is an important part of many types of experiments. In some cases it is necessary only to incorporate as much radioactivity as possible. In other cases it is necessary to incorporate radioactivity under controlled conditions, so that one can, for instance, compare the transcriptional or translational activity of cells under different experimental conditions. In yet other cases it is important to be able to provide a pulse of label followed by a chase with nonradioactive precursors in order to follow the fate of a particular species of macromolecule. We describe methods available for such labeling and potential pitfalls that may be encountered. Methods for preparation of RNA ([27] and [28]) and protein ([35]) are described elsewhere in this volume. [s2p]Phosphate: RNA, Protein Most media contain inorganic phosphate that competes with added for uptake into the cell. Rubin t described a medium, adapted from one used for Escherichia coil, in which the inorganic phosphate is removed by precipitation with Mg2+ at high pH. The cells grow rather well on the organic phosphate present, but they avidly take up inorganic 32po4. The recipe is as follows: Dissolve 10 g yeast extract (Difco, Detroit, MI) and 32po 4

' G. M. Rubin, J. BioL Chem. 248, 3860 (1973).

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20 g peptone (Difco) in 920 ml water. With vigorous stirring, add 10 ml of 1 M MgSO4 followed by l0 ml concentrated NH4OH. Allow the phosphate salts to precipitate at room temperature for 30 rain. Filter twice through a Biichner funnel fitted with Whatman 3 MM paper. Adjust to pH 5.8 with concentrated HCI. Autoclave 25 min. Add 40 ml sterile 50% dextrose. Grow cells to log phase in ihis medium. We generally label with 150 /,Ci/ml of carrier-free a2po4 for periods from 5 to 60 rain. To our knowledge, no one has pei'formed careful experiments to determine the uptake of phosphate from this medium under different experimental conditions. Its use is primarily to prepare highly radioactive RNA, DNA, or protein molecules for further analysis. Because the [32P]phosphate is rapidly converted to polyphosphates, it is not feasible to "chase" under these conditions except over a long period of time. Furthermore, the polyphosphates confound attempts to measure the incorporation of 32p into macromolecules by the usual precipitation with trichloroacetic acid. Nevertheless, one can expect several million counts per minute per milliliter of culture incorporated into RNA, and several thousand into any given phosphoprotein. Organic Precursors: General Considerations It is frequently useful to label macromolecules with their immediate precursors, uracil or adenine in the case of nucleic acids, or amino acids in the case of proteins. In our experience it is always more effective to label a cell prototrophic for the precursor rather than auxotrophic. In the latter case, one must grow the cells in the presence of unlabeled precursor to reach the experimental condition. That precursor will compete with the labeled precursor unless it is removed. To do so, however, the cells must be shifted from the growth medium to a starvation medium. This shocks them in at least 3 ways, by the manipulations of centrifugation or filtration, by the new "unconditioned" medium, and by starvation for the required nutrient. Although controls for such perturbations arc possible, they are tedious and may be unconvincing. In our hands2 cells prototrophic for uracil, adenine, or any of several amino acids, for example, lcucine, lysine, or methionine, incorporate added radioactive precursors avidly when growing in synthetic medium (SC), containing, per liter, 6.7 g yeast nitrogen base (without amino acids) (Difco), 20 g glucose, 50 nag required amino acids, and 20 nag required purincs or pyfimidincs. In some cases addition of 10 g succinic acid and 6 g of NaOH (per liter) improves growth by buffering the medium. 2 j. R. Warner, J. Biol. Chem. 246, 447 (1971).

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Labeling of RNA Bases or Nucleosides as Precursors. ~4C- or 3H-labeled uracil is a useful precursor for the labeling of nucleic acids. Alternatively, labeled uridine, substantially less expensive, can be employed. Adenine can be used as well, though in our hands the signal-to-noise ratio is poorer. For either precursor, there are three potential drawbacks.

1. Uracil, for example, is incorporated into RNA as UTP and CTP. Therefore, a number of enzymatic reactions must ensue between the uptake of the uracil into the cell and its utilization for transcription. Although the incorporation of radioactive uracil sometimes appears linear from an early time, this is in fact a delusion arising from the countervailing effects of two nonlinear events: (a) the convex labeling profile of unstable RNAs, namely, the fact that unstable RNAs are synthesized and degraded [the amount of radioactivity in unstable RNAs after a short pulse is out of proportion to their representation in bulk RNA; for example, poly(A)+ m R N A makes up 3-5% of total RNA but as much as 3096 of a 2-rain pulse], and (b) the approach to m a x i m u m value of the specific activities of UTP and CTP, which can take up to 15 rain. 3 These considerations may be of little import in most instances, but if one is interested in the rate of synthesis of a molecule under different experimental conditions, one must be careful about the equilibration of the labeled precursor with the nucleoside tfiphosphate precursor pools. In one example, 4 it was found that starvation for an amino acid effectively prevented the uptake of exogenous uracil into the precursor pools that were maintained by the reut'dization of nucleotides resulting from the turnover of RNA within the cell. 2. If one desires to measure carefully the flow of precursor into an RNA species, as, for example, when measuring the approach to equilibrium labeling of an unstable species, it is necessary to measure continuously the specific activity of both the U T P and CTP pools. Alternatively, the U T P pool can be measured and the specific activity of the UMP in RNA determined. A method for doing so is detailed in Ref. 3. 3. Once the uracil has been taken into the cell and phosphorylated, it rarely leaves the cell. Therefore, a pulse-chase can be carried out only over a considerable length of time. Realistically, chase times of less than 10-15 rain are impractical. In practice) if one labels a culture at l0 T cells/ml in synthetic medium lacking uracil for 5 min at 23 ° with 200 gCi/ml of [3H]uracil (19 Ci/ mmol), the yield is about 105 cpm//~g RNA. The yield of RNA is generally 3c. H. Kim and J. R. Warner,J. Mol. Biol. 165, 79 (1983). 4 R. W. Shulman, C. E. Sripati,and J. R. Warner, J. Biol. Chem. 252, 1344(1976).

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20 #g/107 cells. Therefore, 1 ml of culture will yield 2 X 107 cpm of [3H]RNA. The radioactivity found in RNA is generally proportional to the amount of radioactivity added to the culture. Methionine as a Source of Labeled Methyl Groups in RNA. One of the most useful ways to label many RNAs, especially ribosomal RNA, is through the methyl group of methionine. [methyl-3H]Methionine is convetted readily to S-adenosylmethionine (SAM), which donates the 3Hmethyl in the methylation of nucleic acids. Many of the problems described above are not present when methionine is used as a precursor. In our experience, labeling a l-ml culture of 107 cells in the minimal medium described above with 100gCi of [methyl-3H]methionine for 3-5 rain yields several thousand counts per minute incorporated into ribosomal RNA precursors. 5 Similar protocols have been useful in studying the mTG cap at the 5' end of mRNAs 6 and its metabolism. A useful feature of labeling RNA with methionine is that the uptake of radioactivity into the SAM precursor pool is far less sensitive to environmental perturbation than is that of nucleotides.4 Furthermore, the pools of SAM are rapidly saturated (a half-time of less than 2 rain) and even more rapidly chased (a half-time of about 0.5 rain). 7 The actual measurement of the specific activity of the SAM pool is rather simple if the cells have been prelabeled with [~4C]adenine. (See Ref. 7 for details.) Methionine can therefore be particularly useful for pulse-chase experiments, for example, to study the processing of ribosomal precursor RNA. A l-ml culture in methionine-free medium is labeled with 60#Ci of [methyl-3H]methionine for 1-5 rain. If needed, cold methionine can be added to 0.5 mg/ml in order to chase the label. RNA is prepared from the cells (see [27] in this volume), and the RNA from 0.2-0.4 ml of culture is fractionated on a 1.5% agarose denaturing gel, containing 2.2 M formaldehyde and 10 m M sodium phosphate. To detect the RNA by autoradiography, the gel is soaked in EN3HANCE (NEN-Du Pont, Boston, MA), dried, and exposed to X-ray film at - 80 ° without an intensifying screen,s A more quantitative analysis is possible if the cells have been uniformly labeled with a purine or pyrimidine precursor to assist in bookkeeping.5 We generally use 0.05 aCi [~4C]uracil, diluted with 10#g cold uracil, per milliliter. In this case the gel must be sliced, solubilized, and analyzed in a scintillation counter. 5 The one potential drawback of the labeling of RNA with methyl groups is uncertainty over the degree of methylation of RNA in a given experi5 S. A. Udem and J. R. Warner, d. Mol. Biol. 65, 227 (1972). 6 C. E. Sripati, Y. Groner, and J. R. Warner, J. Biol. Chem. 251, 2898 (1976). 7j. R. Warner, S. A. Morgan, and R. W. Shulman, ./. Bacterioi. 125, 887 (1976). s M. R. Underwood and H. M. Fried, EMBO J'. 9, 91 (1990).

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IN YEAST

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mental condition. This can usually be assessed by analysis of an alkali digest of the RNA, since the bulk of the methylation in ribosomal RNA is at the Y-hydroxyl, to yield an alkali-resistant dinucleotide. The dinucleotides can be readily separated from mononucleotides on a DEAE-Sepharose column in 7 M urea (see Ref. 4 for details).

Run-On Transcription: Labeling with UTP Although there is much interest in the regulation of transcription of genes in yeast, transcription is rarely measured. Rather, one measures the concentration of mRNA or its products. This is largely due to the problems of incorporating sul~cient radioactivity and of correcting for pool specific activity. As an alternative, investigators working with mammalian cells have developed the procedure of run-on transcription. Nuclei are prepared and incubated with radioactive nucleoside tdphosphates. RNA polymerase molecules which were actively transcribing in the cells continue transcribing for a few hundred nucleotides. The radioactive RNA produced appears to be a faithful representation of the transcription that was going on in the cell.9.1o An analogous method was developed for Saccharomyces cerevisiae by Jerome and Jaehning. 1~ However, to avoid the dit~culty and duration of the procedure for preparing yeast nuclei, we have modified the approach by using a detergent to permeabilize the membrane of whole cells, with cell walls remaining intact? 2 The rigid cell wall protects the permeab'~zed cells during the procedure, which takes only a few minutes. These will now incorporate nucleoside triphosphates for a brief period. A useful feature is that the cells are also permeable to a-amanitin. RNA can then be prepared from the permeabilized cells for analysis, for example, by slot-blot hybridization to cloned gene fragments. The procedure is as follows? 2 Pour a culture containing about 3 X l0 T cells onto one-half volume of crushed ice. Do all subsequent steps on ice until the incubation. Centrifuge for 5 min at 5000 rpm; pour off supernatant. Suspend cells in 5 ml of TMN (10 mMTris, pH 7.4, 100 m M NaCl, 5 mM MgCI2) at 0°. Centrifuge for 5 rain at 5000 rpm; aspirate supernatant. Suspend cells in 0.95 ml of cold water. Add 50 #1 of 10% (w/w) N-lauroylsarcosine (sodium salt) [Sigma (St. Louis, MO) L-5125]. Leave on ice for 15 rain. Transfer to an Eppendorf tube. Spin for 2 rain in the cold. Aspirate superuatant. 9 j. Weber, W. Jdinek, and J. E. DamelL Jr., Cell (Cambridge, Mass.) 10, 611 (1977). ~oG. S. McKnight and R. D. Palmiter, J. Biol. Chem. 254, 9050 (1979). '~ J. F. Jerome and J. A. Jaehnln~ Mol. Cell. Biol. 6, 1633 (1986). 12 E. A. Effort and J. R. Warner, Moi. Ceil. Biol. 6, 2089 (1986).

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]

Suspend permeabilized cells in 120 #1 of reaction mix: Component

Final concentration

Tris, pH 7.9 KCI MgC12 MnCI2 Dithiothreitol ATP GTP CTP Phosphocreatine* Creatine phosphokinase* [c~-32p]UTP(800 Ci/mmol)

50 mM 100 ram 5 mM I mM 2 mM 500 mM 250 mM 250 mM 10 mM 12 ng//d 1#Ci/#l

*These are not essential but seem to increase incorporation slightly.

Incubate at 25 ° for 5-10 min. (The reaction is usually finished in 35 rain.) Transfer 2/tl into cold 5% trichloroacetie acid (TCA) to determine incorporation. Add 1 ml of cold TMN containing 50 BM UTP. Spin, then aspirate and carefully discard the radioactive supematant. Suspend cells and prepare RNA. (See [27] and [28] in this volume.) Under the best conditions we get about 1 epm/eell. Frequently the yield is only one-third of that. Acknowledgments Research in the author's laboratory is supported by grants from the National Institutes of Health (GM25532 and CA13330) and the American Cancer Society(MV-323S).

[31 ] Tackling the Protease Problem in

Saccharomyces cerevisiae By ELIZASETHW. JONES Introduction The yeast Saccharomyces cerevisiae contains a large number of proteases that are located in various compartments (cytosol, vacuole, mitochondria, endoplasmic reticulum, and Golgi complex, at least) and membranes (vacuole, endoplasmic reticulum, Golgi complex, and plasma, at least) o f the cell. These include endoproteinases, carboxypeptidases, antiMETHODS IN ENZYMOLOGY, VOL. 194

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