- Email: [email protected]
[7] Characterization of protein prenylation in Saccharomyces cerevisiae
68
PRENYLATION
[7]
tural genes involved in prenyltransferase activity can be easily confirmed with biochemical assays using recombinant enzyme reconstitution. Yeast FTase and GGTase I produced in E. coli are indistinguishable from the native proteins and can be studied without interference from contaminating cellular protein prenyltransferases. Structure-function analysis of the yeast prenyltransferase subunits is also simplified by the rapidity with which mutant enzymes can be analyzed in E. coli and their biological activity characterized in yeast defective for the particular subunit gene. Acknowledgments The authors thank Scott Powers and Douglas Johnson for providing all original DNA clones and yeast strains and Charlie Mantel for assistance with HPLC. We would also like to acknowledge original protocols by Y. Reiss for partial purification of FTase and P. Casey for identification of attached isoprenoid species which were adapted to the study of the yeast prenyltransferases. This study was supported in part by the American Cancer Society.
[7] C h a r a c t e r i z a t i o n
of Protein Prenylation Saccharomyces cerevisiae
By
in
D A V I D A . M I T C H E L L a n d R O B E R T J. D E S C H E N E S
Introduction A growing number of membrane-localized proteins require posttranslational modification of a C-terminal sequence motif called the CaaX box, where C is Cys, a is generally an aliphatic residue, and X is the C-terminal residue. 1-3 The steps in the modification pathway include prenylation of the CaaX box cysteine, proteolytic removal of the -aaX residues, methyl esterification of the newly exposed o~-carboxyl group, and palmitoylation of a second cysteine often found within 1-6 residues of the prenylated cysteine. The importance of these steps has been established by studying the effect of mutating the CaaX b o x , 4-6 isolating mutations in genes encod1 S. Clarke, Annu. Rev. Biochem. 61, 355 (1992). 2 W. R. Schafer, and J. Rine, Annu. Rev. Genet. 26, 209 (1992). 3 A. D. Cox and C. J. Der, Curr. Opin. Cell Biol. 4, 1008 (1992). 4 K. Kato, A. D. Cox, M. M. Hisaka, S. M. Graham, J. E. Buss, and C. J. Der, Proc. Natl. Acad. Sci. U.S.A. 89, 6403 (1992). s R. J. Deschenes and J. R. Broach, Mol. Cell Biol. 7, 2344 (1987). 6 B. M. Willumson, K. Norris, A. G. Papageorge, N. L. Hubbert, and D. R. Lowy, E M B O J. 3, 2581 (1984).
METHODS IN ENZYMOLOGY, VOL. 250
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[7]
PROTEINPRENYLATIONIN YEAST
69
ing the CaaX box processing enzymes, 7's and pharmacological inhibitor studies of the modifying enzymes. 9 Blocking the prenylation step prevents subsequent steps in the modification pathway and results in a cytosolic form of the CaaX box protein. Interestingly, the requirement for prenylation can be overcome by substituting alternative membrane localization signals, although the properties of the altered protein may differ from the normally modified form. 1°'11 In the course of evaluating the sequence requirements for CaaX box processing of yeast Ras2 protein, we found it necessary to devise ways to improve the sensitivity of our assays to study the function of CaaX box mutants and the structure of the fully modified protein. We describe in this chapter two assays for studying protein prenylation in yeast. The first is a convenient plasmid loss assay for rapid evaluation of the activity of Ras2 CaaX box mutants. The second is a sensitive method for identification of the prenyl group that modifies a CaaX box protein in yeast. Direct labeling of prenylated proteins with tritiated isoprenoids or precursors such as mevalonate is not possible in yeast, presumably owing to an inability to transport these compounds into the cell. A method is described that circumvents the problem. The method involves (1) labeling all newly synthesized proteins in vivo with Na235SO4, (2) isolating the protein of interest as a fusion protein with glutathione S-transferase (GST), (3) completely digesting the purified fusion protein with proteases, and (4) high-performance liquid chromatography (HPLC) analysis of the labeled products by comparing the elution position with authentic prenylated cysteine standards. Using the assays, we have shown that the cysteine residue of the Ras2 protein CaaX box can be changed to a serine without complete loss of Ras2 function. The residual activity is attributable to a low level of prenylation of a second cysteine that is normally modified by palmitate. Rapid Qualitative A s s a y of Yeast Ras CaaX Box M u t a n t s B a s e d on Sensitive Plasmid Loss Assay Outline o f Plasmid Loss Assay to Measure Function o f C a a X Mutants
Mutational analysis of the amino acids in and around the CaaX box provides important information concerning the posttranslational modifica7A. Fujiyama, K. Matsumoto, and F. Tamanoi, EMBO J 6, 223 (1987). S. Powers, S. Michaelis, D. Broek, S. Santa Anna, J. Field, I. Herskowitz, and M. Wigler, Cell (Cambridge, Mass.) 47, 413 (1986). 9F. Tamanoi, Trends Biochem. Sci. 18, 349 (1993). 10j. E. Buss, P. A. Solski, J. P. Schaeffer, M. J. MacDonald, and C. J. Der, Science 243, 1600 (1989). 11p. A. Solski, L. A. Quilliom, S. G. Coats, C. J. Der, and J. E. Buss, this volume [33].
70
PRENYLATION
[71
Ras2
NH2.
CYS CYS ILE ILE SER-cooH
Ras2 (C318S)
NH2.
SER (~Y~ ILE ILE SER - cooH
Ras2 (C319S)
NH2
(~Y$ SER ILE ILE SER- COOH
Ras2 (C318S, C319S)
NH2Amino Acids1-317
SER SER ILE ILE ILE SER-cooH 318 319 320 321 322
FIG. 1. Amino acid sequences (residues 318-322) of the C terminus of the Ras2 proteins used in this study. Residues 1-317 of yeast Ras2 are represented by a solid bar.
tions required for the function of the protein. To assess the function of a large number of CaaX box mutations conveniently, we have developed a yeast genetic screen based on the plasmid shuffle procedure. 12 To demonstrate the method, we have analyzed a series of yeast Ras2 CaaX box mutations. Figure 1 shows the C-terminal amino acid sequences of wildtype Ras2 and three CaaX box mutants: Ras2(C318S), Ras2(C319S), and Ras2(C3188, C3198). The Ras2(C3198) and Ras2(C3188, C3198) mutants change the conserved CaaX box Cys to a Ser, which is expected to abolish prenylation completely. Ras2(C318S) substitutes a Ser for the cysteine that is normally palmitoylated. This change has no effect on prenylation and a very minor effect on Ras activity.5 The scheme for carrying out the plasmid loss assay is shown in Fig. 2. The genotype of the parent strain we have constructed is as follows: RJY504: M A T a leu2 lys2 ura3 his3 rasl :: HIS3 ras2A [YCp50RAS1] Note that RJY504 has had the chromosomal R A S 1 and R A S 2 genes deleted, a condition that would normally be lethal to the cell. Cell viability is maintained by the presence of a YCp50-based plasmid expressing the wild-type R A S 1 gene (YCp50RAS1). YCp50RAS1 is marked by the U R A 3 gene, an anxotrophic marker that permits growth on medium that lacks uracil (SC - Ura). SC medium consists of 0.67% yeast nitrogen base (YNB) appropriately supplemented to satisfy the auxotrophic requirements of the strain. Because the viability of the strain requires the presence of YCp50RAS1, RJY504 must remain a uracil prototroph and is therefore sensitive to 5'-fluoroorotic acid (FOA), a drug that kills uracil prototrophs. 13 Media containing FOA consists of 0.67% (w/v) YNB, 0.05 g/liter uracil, 12 R. 8. Sikorski and J. D. Boeke, this series, Vol. 194, p. 302. 13 j. D. Boeke, J. Trueheart, G. Natsoulis, and G. R. Fink, this series, Vol. 154, p. 164.
[7]
71
PROTEIN PRENYLATION IN YEAST
I MATaleu2ura3his3rasl::HIS3ras2A(RJY.g04)"h
CEN41ARSI Ras2allele~ CEN61ARS4~/
J
I SC- Ura - Leu
IC MATaleg2ura3his3rasl::HIS3r~2A 1
1
~
5'-Fluoroorotic Acid (FOAl
Growth ( F O /
I
MATaleu2ura3his3r~;I::HIS3~
Growth (FOA-)
C MATaItm2ura3his3r~sl::HIS3ras2A !
LEU2~[pRS3|5) CEN6/A~ j
J
LF:U2X
~
|pRS31S|
/'
v\
i VCpSO /
/
/
FIG. 2. Schematic diagram of the plasmid loss assay used to measure the function of various mutant Ras2 alleles. The laboratory strain RJY504 is transformed with pRS315 plasmids harboring the Ras2 allele to be tested. Transformants containing both of the Rasexpressing plasmids are selected on synthetic medium plates lacking uracil and leucine (SC - Ura - Leu). To test for the ability of the strain to grow in the absence of the RAS1expressing plasmid, cells are replica-plated to medium containing FOA. Two possible outcomes are shown which depend on the function of the Ras2 allele being tested.
72
PRENYLATION I
2
[7]
3
YEPD
SC-Ura-Leu
5'-FOA
Fie;. 3. Plate assay of the function of Ras2 CaaX box mutants. Approximately 107 cells from the strain RJY504 transformed with either pRS315Ras2 (column 1), pRS315Ras2(C319S) (column 2), or pRS315Ras2(C318S, C319S) (column 3) were grown on synthetic medium lacking uracil and leucine (SC - Ura - Leu). After growth at 30° for 2 days, the cells were transferred to plates of rich medium (YEPD) (top), SC Ura - Leu (middle), or medium containing FOA (bottom). YEPD medium consists of 1% (w/v) yeast extract, 2% (w/v) Bactopeptone, and 2% (w/v) glucose. All plates were incubated at 30° for 2 days. and 0.1% (w/v) F O A . This provides a powerful drug selection that can be used as the basis of the plasmid loss screen (refer to Fig. 2). Ras2 C a a X box m u t a n t genes (generically referred to as Ras2 alleles in Fig. 2) were cloned into a s e c o n d type of yeast plasmid based on the p a r e n t plasmid pRS31514 to create pRS315Ras2, pRS315Ras2(C319S), and pRS315Ras2(C318S, C319S). T h e pRS315 plasmid carries a LEU2 gene conferring leucine p r o t o t r o p h y to a leu2 host strain such as RJY504. E a c h plasmid was t r a n s f o r m e d into R J Y 5 0 4 and selection for the presence of the plasmid assayed on SC - U r a - L e u plates. T o d e t e r m i n e if the Ras2 C a a X box m u t a n t s carried on the pRS315 plasmid were capable of supporting R a s - d e p e n d e n t growth, the new strains were assayed for the ability to grow in the absence of Y C p 5 0 R A S 1 by plating on m e d i u m containing F O A . If Y C p 5 0 R A S 1 is dispensable, they b e c o m e uracil a u x o t r o p h s and are resistant to the drug. T h e function of various Ras2 C a a X m u t a n t s can be rapidly and easily assessed by this c o n v e n i e n t plate assay.
Ability of Ras2(C319S) but Not Ras2(C318S, C319S) to Support Ras-Dependent Growth in Yeast T o illustrate the screen, R J Y 5 0 4 was t r a n s f o r m e d with pRS315Ras2, pRS315Ras2(C319S), or pRS315Ras2(C318S, C319S), and the resulting strains were assayed by the plasmid loss assay. A s seen in Fig. 3, the strain 14R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989).
[7]
PROTEIN PRENYLATION IN YEAST
73
of RJY504 containing pRS315Ras2 was able to grow in the presence of the drug FOA, indicating that expression of Ras2p is sufficient for viability and the YCp50RAS1 plasmid is no longer required. Next, we examined the importance of the CaaX box Cys, by testing whether Ras2(C319S) was able to support Ras-dependent growth. To our surprise, we found that Ras2(C319S)-expressing cells were also able to grow on medium containing FOA, indicating that a RAS gene lacking the CaaX box cysteine was capable of supporting life. However, the cells grew more slowly than cells expressing Ras2(C318S), suggesting that the mutant was not modified as efficiently as the wild type. The strain containing the double mutant, Ras2(C318S, C319S), was unable to grow on FOA (Fig. 3). Because substitution of serines at both positions 318 and 319 leads to a nonfunctional protein, this raises the possibility that Cys-318 can be farnesylated when the CaaX box cysteine (Cys-319) has been changed to Ser. Method for Characterization of Protein Prenylation in Yeast
Principle of Assay It has not been possible to radiolabel prenylated yeast proteins directly because yeast are unable to take up radiolabeled isoprenoids or precursors such as mevalonate and mevalonolactone. We have developed an alternative method to assess these modifications. It involves overexpression of the CaaX box proteins as an easily purified 35S-labeled fusion protein, followed by complete digestion with proteases and analysis of the products by reversed-phase HPLC. A detailed description of each step in the procedure is given below.
Inducible Expression and Affinity Purification of Glutathione S-Transferase Fusion Proteins in Yeast We have described two plasmids for the expression of GST fusion proteins in yeast. 15The plasmids possess several useful features for producing large quantities of a fusion protein. First, they are propagated in yeast as high-copy number plasmids. Second, expression of the GST fusion is under the control of the galactose-inducible upstream activator sequence, UASGA L. The activity of this promoter can be controlled by incubating cells in medium containing glucose (repressing condition) or galactose (inducing condition). The expression level obtained from this system can be further elevated by coexpression of the transcriptional activator, Gal4p, which binds to the UASGAL. A plasmid, pMA210, has been described which ~s D. A. Mitchell, T. K. Marshall, and R. J. Deschenes, Yeast 9, 715 (1993).
74
PRENYLATION
[71
expresses G A L 4 under the control of the ADH1 promoter. 16This promoter is induced by growing cells on a fermentable carbon source such as galactose. The combination of using a high-copy vector with a strong, inducible promoter and coexpression of the transcriptional activator protein for the UAS allows one to express sufficient quantities of protein in yeast for biochemical analysis of rare posttranslational modification events.
Metabolic Labeling of Cells with Na235S04 An efficient method for radiolabeling yeast proteins on Met and Cys residues is to grow cultures in sulfate-free medium containing Na235SO4.17'18 To obtain efficient labeling, it is necessary to deplete intracellular sulfate reserves. 17,18It typically requires 36-48 hr to deplete completely the sulfate pools causing G1 arrest (cells are unbudded). Once cells are arrested, the medium is supplemented with just enough (NH4)2804 to allow the cells to resume growth and 4% galactose to allow the cells to begin overproduction of the desired protein. The amount of (NH4)2SO4 that is added is somewhat strain dependent and should be determined empirically, but the final concentration is generally in the range of 50 /xM. The time needed for the cells to resume growth is also strain dependent, but is usually between 2 and 4 hr. The cells are then harvested by centrifugation at 2500 g for 5 min at 4 ° and washed once with sulfate-free medium to remove residual sulfate. The cells are resuspended in 3 ml of SC - SO4 supplemented with 4% (w/v) galactose and 1 mCi Na235804. The culture is then incubated with gentle shaking at 30 ° for 6-8 hr.
Purification of Fusion Proteins Expressed in Yeast Following induction and Na235SO4 labeling, cells are harvested by centrifugation at 2500 g for 5 min at 4 °, the medium is decanted, and the pellet is washed once in sorbitol buffer (0.3 M sorbitol, 100 m M NaC1, 5 m M MgC12, and 10 m M Tris-HC1, p H 7.4). All subsequent manipulations are performed at 4 °. The resulting pellet is resuspended in 1 ml of sorbitol buffer with protease inhibitors [final concentrations 100 units/ml aprotinin, i m M phenylmethylsulfonyl fluoride (PMSF), 1 m M pepstatin, and 100 tzM leupeptin]. Cells are lysed by eight 30-sec bursts on a vortex mixer in the presence of 1/3 volume of glass beads (425-600/xm, Sigma, St. Louis, MO, G-9268). U n b r o k e n cells are removed by centrifugation at 2500 g for 5 min, 16j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 48, 847 (1987). 17R. J. Deschaies and R. Schekman, J. Cell Biol. 1115,633 (1987). 18j. B. Stimmel, R. J. Deschenes, C. Volker, J. Stock, and S. Clarke, Biochemistry 29, 9651 (1990).
[7]
PROTEIN PRENYLATION IN YEAST
75
and the supernatant is transferred to a clean tube. The beads are washed once with 1 ml sorbitol buffer containing protease inhibitors, spun (2500 g, 5 min), and the supernatants pooled. The membrane fraction is separated from the soluble fraction by centrifugation at 100,000 g for 20 min and the soluble fraction set aside. To purify GST fusion proteins from the membrane fraction, the pellet is solubilized on ice for 20 min in sorbitol buffer with protease inhibitors and containing 1% (w/v) Nonidet P-40 (NP-40), 0.5% (w/v) deoxycholate, and 0.1% (w/v) sodium dodecyl sulfate (SDS). A successful labeling results in approximately 3-4 × 108 total counts/min (cpm) of 35SO4-1abeled material. Particulate material that is not solubilized is removed by centrifugation at 12,000 rpm in a microcentrifuge for 3 rain. Affinity purification of the GST fusion protein is then performed by adding 150 ~1 of a 50% slurry of agarose beads conjugated with glutathione (GSH) (Sigma, G-4510) to the solution of solubilized proteins. The mixture is incubated with gentle shaking for 1 hr at 4°. The beads are then recovered by centrifugation (12,000 rpm, 30 sec) and then washed with 1 ml of sorbitol buffer. Three additional washes are performed to remove unincorporated 35SO4.
Proteolytic Digestion of Fusion Protein and Analysis of Products by Reversed-Phase Chromatography We have previously reported a protease digestion strategy that converts 35SO4-1abeled proteins to the constituent amino acids or prenylated derivatives. 17 The protease cocktail works well for yeast Ras proteins; however, we have not systematically tested it for other prenylated proteins, and the optimal conditions may vary. Ras2-GST fusion proteins bound to GSHagarose beads are resuspended in 500 ~1 of 20 mM sodium phosphate buffer (pH 6.8), containing 7.5 units of pronase E [Streptomyces griseus, Sigma type XIV, 5.8 units/mg of protein (1 unit hydrolyzes casein to produce Folin-Ciocalteau color equivalent to 1 ~mol of tyrosine per minute at pH 7.5 at 37°)], and the mixture is incubated for 40 hr at 30°. At that time, 18 units of leucine aminopeptidase M [porcine kidney microsomes, Sigma type IV-S, 20 units/mg of protein (1 unit hydrolyzes 1 ~mol of /-leucine-p-nitroanilide per minute at pH 7.2 at 37°)] and 42 units of prolidase [porcine kidney, Sigma, 205 units/mg of protein (1 unit hydrolyzes 1 ~mol of Gly-Pro per minute at pH 8.0 at 37°)] are added, and the incubation is continued for another 4 hr at 30°. The beads are then removed by centrifugation (12,000 rpm, 30 sec at room temperature) and the supernatant recovered for analysis. Approximately 2 × 10 6 cpm is injected onto a Ca reversed-phase HPLC column (Vydac, 4.6 × 250 mm) at an initial flow rate of 1 ml/min of a buffer consisting of 24% (v/v) acetonitrile/12%
0.2-
A S-GG-CYS 0.15 -
S-Far-CYS Abs 214 nm
0.1
0.05
0-
12000 -
B 10000 -
8000-
cpm
6000-
4000-
2000-
02000-
C 1600-
1200 -
cpm 800-
400 o
0
' ' 5' ' I0'
0
15
20
~'5 ;0 ;5 40'/5'
Time
50
[7]
PROTEINPRENYLATIONIN YEAST
77
(v/v) 2-propanol/0.1% (v/v) trifluoroacetic acid. A 45-min linear gradient is performed ending with a final buffer composed of 60% acetonitrile/30% 2-propanol/0.1% trifluoroacetic acid. The effluent is monitored at a wavelength of 214 nm and collected as 0.5-ml fractions across the gradient. The entire fraction is mixed with 5 ml of Budget-Solve (RPI) and radioactivity determined by scintillation counting.
Evidence for Ras2(C319S) Prenylation on Cys-318 As an example of this technique, we have examined the posttranslational modification of the protein product of the Ras2(C319S) allele described above. Plasmids expressing GST fusions between Ras2(C318S), Ras2(C319S), and Ras2(C318S, C319S) were constructed and transformed into the yeast strain RJY690 (MATa/a leu2 ura3 his3 [pMA210]). Cells were labeled with Na235SO4 and processed by the method described above. Figure 4A shows the elution position of S-farnesylcysteine (10 nmol) and S-geranylgeranylcysteine (15 nmol). S-Farnesylcysteine was synthesized by established procedures and confirmed by fast atom bombardment ( F A B ) mass spectrometry, t8 S-Geranylgeranylcysteine was kindly provided by Dr. Pat Casey (Duke University). Labeled products derived from digestion of Ras2(C318S) (Fig. 4B) and Ras2(C319S) (Fig. 4C) comigrate with S-farnesylcysteine (Fig. 4B). Although the number of counts recovered from the digestion of purified Ras2(C319S) is less than from the Ras2(C318S) protein with an intact CaaX box, the major peak derived from Ras2(C319S) still corresponds to S-farnesylcysteine. The difference in recovered counts most likely reflects inefficient farnesylation of the Ras2(C319S) protein and not a lower level of expression of the protein. Western blots reveal that similar levels of the two proteins were expressed in the two strains (data not shown). This interpretation is also consistent with the plasmid loss assay. A second minor peak is also observed that elutes 2-3 min later than S-farnesylcysteine (Fig. 4C). The identity of the second peak has not been determined, but its elution position is consistent
FIG. 4. Reversed-phase HPLC analysis of prenylated Ras2 proteins. (A) Elution profile of S-farnesylcysteine(16.5-17.0 min) and S-geranylgeranylcysteine(26.5-27.0rain) standards measured by monitoring absorbance at 214 nm. (B and C) Analysis of approximately 2 × 10 6 cpm of the 35SO4-1abeledmaterial derived from proteolysis of samples prepared from Ras2(C318S) (B: diamonds), Ras2(C319S) (C: circles),or Ras2(C318S, C319S) (C: triangles). A C4 reversed-phase HPLC column (Vydac, 4.6 × 250 ram) was used for the fractionation. The column profile consisted of a 45-min linear gradient (1 ml/min) beginning with a buffer consisting of 24% acetonitrile/12% 2-propanol/0.1% trifluoroacetic acid and ending with a buffer consisting of 60% acetonitrile/30%2-propanol/0.1%trifluoroaceticacid. Fractions were collectedevery 30 sec and the contents of the entire fraction measured by scintillationcounting.
78
PRENYLATION
[71
with S-farnesylcysteine methyl ester. As expected, no peak is observed in this portion of the gradient for Ras2(C318S, C319S). We were intrigued by the ability of Ras2(C319S) to support Ras-dependent growth. As Ras2(C318S, C319S) is not functional, we conclude that the ability of Ras2(C319S) to function relies on the cysteine at position 318, one residue upstream of the normal CaaX box cysteine (position 319). The in vivo assay of prenylation described above revealed that Cys-318 was modified by a farnesyl group. We consider two explanations for this result. The first is that if a nonspecific protease removes the C-terminal residue of Ras2(C319S), a true CaaX box would be formed with the sequence Cys-Ser-Ile-Ile. This sequence could be a substrate for yeast protein farnesyltransferase (FTase). The second possibility is that the yeast FTase, which normally recognizes CaaX sequences, may also be capable of recognizing a cysteine five residues from the end of a protein in vivo. Concluding Remarks The methods described in this chapter provide a means for measuring the prenylation of proteins in yeast and assessing the function of proteins altered in the ability to be posttranslationally modified. The power of the system is in the sensitivity of the plasmid shuffle assay which allows detection of even low levels of activity produced by inefficiently modified proteins. In addition to scoring the function of known CaaX box mutations, the plasmid shuffle assay can be adapted to search for intragenic or second site suppressors that allow unmodified proteins to function. We have also described a method to characterize protein prenylation in yeast. To date, direct measurement of prenylation in yeast has been hampered by the inability to radiolabel cells with [3H]mevalonate or other precursors such as farrlesyl and geranylgeranyl pyrophosphate. The method we describe combines overproduction and radiolabeling the protein of interest as a GST fusion, followed by reversed-phase HPLC to identify the prenyl moiety. Acknowledgments We thank Pat Casey for generously providing the S-geranylgeranylcysteine standard, Lynn Farh for synthesizing the S-farnesylcysteine, and Jun Ma for the Gal4 overproduction plasmid, pMA210. This work was partially supported by grants from the National Institutes of Health {CA50211) and the Pardee Cancer Research Fund to R. J. D.
PRENYLATION
[7]
tural genes involved in prenyltransferase activity can be easily confirmed with biochemical assays using recombinant enzyme reconstitution. Yeast FTase and GGTase I produced in E. coli are indistinguishable from the native proteins and can be studied without interference from contaminating cellular protein prenyltransferases. Structure-function analysis of the yeast prenyltransferase subunits is also simplified by the rapidity with which mutant enzymes can be analyzed in E. coli and their biological activity characterized in yeast defective for the particular subunit gene. Acknowledgments The authors thank Scott Powers and Douglas Johnson for providing all original DNA clones and yeast strains and Charlie Mantel for assistance with HPLC. We would also like to acknowledge original protocols by Y. Reiss for partial purification of FTase and P. Casey for identification of attached isoprenoid species which were adapted to the study of the yeast prenyltransferases. This study was supported in part by the American Cancer Society.
[7] C h a r a c t e r i z a t i o n
of Protein Prenylation Saccharomyces cerevisiae
By
in
D A V I D A . M I T C H E L L a n d R O B E R T J. D E S C H E N E S
Introduction A growing number of membrane-localized proteins require posttranslational modification of a C-terminal sequence motif called the CaaX box, where C is Cys, a is generally an aliphatic residue, and X is the C-terminal residue. 1-3 The steps in the modification pathway include prenylation of the CaaX box cysteine, proteolytic removal of the -aaX residues, methyl esterification of the newly exposed o~-carboxyl group, and palmitoylation of a second cysteine often found within 1-6 residues of the prenylated cysteine. The importance of these steps has been established by studying the effect of mutating the CaaX b o x , 4-6 isolating mutations in genes encod1 S. Clarke, Annu. Rev. Biochem. 61, 355 (1992). 2 W. R. Schafer, and J. Rine, Annu. Rev. Genet. 26, 209 (1992). 3 A. D. Cox and C. J. Der, Curr. Opin. Cell Biol. 4, 1008 (1992). 4 K. Kato, A. D. Cox, M. M. Hisaka, S. M. Graham, J. E. Buss, and C. J. Der, Proc. Natl. Acad. Sci. U.S.A. 89, 6403 (1992). s R. J. Deschenes and J. R. Broach, Mol. Cell Biol. 7, 2344 (1987). 6 B. M. Willumson, K. Norris, A. G. Papageorge, N. L. Hubbert, and D. R. Lowy, E M B O J. 3, 2581 (1984).
METHODS IN ENZYMOLOGY, VOL. 250
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[7]
PROTEINPRENYLATIONIN YEAST
69
ing the CaaX box processing enzymes, 7's and pharmacological inhibitor studies of the modifying enzymes. 9 Blocking the prenylation step prevents subsequent steps in the modification pathway and results in a cytosolic form of the CaaX box protein. Interestingly, the requirement for prenylation can be overcome by substituting alternative membrane localization signals, although the properties of the altered protein may differ from the normally modified form. 1°'11 In the course of evaluating the sequence requirements for CaaX box processing of yeast Ras2 protein, we found it necessary to devise ways to improve the sensitivity of our assays to study the function of CaaX box mutants and the structure of the fully modified protein. We describe in this chapter two assays for studying protein prenylation in yeast. The first is a convenient plasmid loss assay for rapid evaluation of the activity of Ras2 CaaX box mutants. The second is a sensitive method for identification of the prenyl group that modifies a CaaX box protein in yeast. Direct labeling of prenylated proteins with tritiated isoprenoids or precursors such as mevalonate is not possible in yeast, presumably owing to an inability to transport these compounds into the cell. A method is described that circumvents the problem. The method involves (1) labeling all newly synthesized proteins in vivo with Na235SO4, (2) isolating the protein of interest as a fusion protein with glutathione S-transferase (GST), (3) completely digesting the purified fusion protein with proteases, and (4) high-performance liquid chromatography (HPLC) analysis of the labeled products by comparing the elution position with authentic prenylated cysteine standards. Using the assays, we have shown that the cysteine residue of the Ras2 protein CaaX box can be changed to a serine without complete loss of Ras2 function. The residual activity is attributable to a low level of prenylation of a second cysteine that is normally modified by palmitate. Rapid Qualitative A s s a y of Yeast Ras CaaX Box M u t a n t s B a s e d on Sensitive Plasmid Loss Assay Outline o f Plasmid Loss Assay to Measure Function o f C a a X Mutants
Mutational analysis of the amino acids in and around the CaaX box provides important information concerning the posttranslational modifica7A. Fujiyama, K. Matsumoto, and F. Tamanoi, EMBO J 6, 223 (1987). S. Powers, S. Michaelis, D. Broek, S. Santa Anna, J. Field, I. Herskowitz, and M. Wigler, Cell (Cambridge, Mass.) 47, 413 (1986). 9F. Tamanoi, Trends Biochem. Sci. 18, 349 (1993). 10j. E. Buss, P. A. Solski, J. P. Schaeffer, M. J. MacDonald, and C. J. Der, Science 243, 1600 (1989). 11p. A. Solski, L. A. Quilliom, S. G. Coats, C. J. Der, and J. E. Buss, this volume [33].
70
PRENYLATION
[71
Ras2
NH2.
CYS CYS ILE ILE SER-cooH
Ras2 (C318S)
NH2.
SER (~Y~ ILE ILE SER - cooH
Ras2 (C319S)
NH2
(~Y$ SER ILE ILE SER- COOH
Ras2 (C318S, C319S)
NH2Amino Acids1-317
SER SER ILE ILE ILE SER-cooH 318 319 320 321 322
FIG. 1. Amino acid sequences (residues 318-322) of the C terminus of the Ras2 proteins used in this study. Residues 1-317 of yeast Ras2 are represented by a solid bar.
tions required for the function of the protein. To assess the function of a large number of CaaX box mutations conveniently, we have developed a yeast genetic screen based on the plasmid shuffle procedure. 12 To demonstrate the method, we have analyzed a series of yeast Ras2 CaaX box mutations. Figure 1 shows the C-terminal amino acid sequences of wildtype Ras2 and three CaaX box mutants: Ras2(C318S), Ras2(C319S), and Ras2(C3188, C3198). The Ras2(C3198) and Ras2(C3188, C3198) mutants change the conserved CaaX box Cys to a Ser, which is expected to abolish prenylation completely. Ras2(C318S) substitutes a Ser for the cysteine that is normally palmitoylated. This change has no effect on prenylation and a very minor effect on Ras activity.5 The scheme for carrying out the plasmid loss assay is shown in Fig. 2. The genotype of the parent strain we have constructed is as follows: RJY504: M A T a leu2 lys2 ura3 his3 rasl :: HIS3 ras2A [YCp50RAS1] Note that RJY504 has had the chromosomal R A S 1 and R A S 2 genes deleted, a condition that would normally be lethal to the cell. Cell viability is maintained by the presence of a YCp50-based plasmid expressing the wild-type R A S 1 gene (YCp50RAS1). YCp50RAS1 is marked by the U R A 3 gene, an anxotrophic marker that permits growth on medium that lacks uracil (SC - Ura). SC medium consists of 0.67% yeast nitrogen base (YNB) appropriately supplemented to satisfy the auxotrophic requirements of the strain. Because the viability of the strain requires the presence of YCp50RAS1, RJY504 must remain a uracil prototroph and is therefore sensitive to 5'-fluoroorotic acid (FOA), a drug that kills uracil prototrophs. 13 Media containing FOA consists of 0.67% (w/v) YNB, 0.05 g/liter uracil, 12 R. 8. Sikorski and J. D. Boeke, this series, Vol. 194, p. 302. 13 j. D. Boeke, J. Trueheart, G. Natsoulis, and G. R. Fink, this series, Vol. 154, p. 164.
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PROTEIN PRENYLATION IN YEAST
I MATaleu2ura3his3rasl::HIS3ras2A(RJY.g04)"h
CEN41ARSI Ras2allele~ CEN61ARS4~/
J
I SC- Ura - Leu
IC MATaleg2ura3his3rasl::HIS3r~2A 1
1
~
5'-Fluoroorotic Acid (FOAl
Growth ( F O /
I
MATaleu2ura3his3r~;I::HIS3~
Growth (FOA-)
C MATaItm2ura3his3r~sl::HIS3ras2A !
LEU2~[pRS3|5) CEN6/A~ j
J
LF:U2X
~
|pRS31S|
/'
v\
i VCpSO /
/
/
FIG. 2. Schematic diagram of the plasmid loss assay used to measure the function of various mutant Ras2 alleles. The laboratory strain RJY504 is transformed with pRS315 plasmids harboring the Ras2 allele to be tested. Transformants containing both of the Rasexpressing plasmids are selected on synthetic medium plates lacking uracil and leucine (SC - Ura - Leu). To test for the ability of the strain to grow in the absence of the RAS1expressing plasmid, cells are replica-plated to medium containing FOA. Two possible outcomes are shown which depend on the function of the Ras2 allele being tested.
72
PRENYLATION I
2
[7]
3
YEPD
SC-Ura-Leu
5'-FOA
Fie;. 3. Plate assay of the function of Ras2 CaaX box mutants. Approximately 107 cells from the strain RJY504 transformed with either pRS315Ras2 (column 1), pRS315Ras2(C319S) (column 2), or pRS315Ras2(C318S, C319S) (column 3) were grown on synthetic medium lacking uracil and leucine (SC - Ura - Leu). After growth at 30° for 2 days, the cells were transferred to plates of rich medium (YEPD) (top), SC Ura - Leu (middle), or medium containing FOA (bottom). YEPD medium consists of 1% (w/v) yeast extract, 2% (w/v) Bactopeptone, and 2% (w/v) glucose. All plates were incubated at 30° for 2 days. and 0.1% (w/v) F O A . This provides a powerful drug selection that can be used as the basis of the plasmid loss screen (refer to Fig. 2). Ras2 C a a X box m u t a n t genes (generically referred to as Ras2 alleles in Fig. 2) were cloned into a s e c o n d type of yeast plasmid based on the p a r e n t plasmid pRS31514 to create pRS315Ras2, pRS315Ras2(C319S), and pRS315Ras2(C318S, C319S). T h e pRS315 plasmid carries a LEU2 gene conferring leucine p r o t o t r o p h y to a leu2 host strain such as RJY504. E a c h plasmid was t r a n s f o r m e d into R J Y 5 0 4 and selection for the presence of the plasmid assayed on SC - U r a - L e u plates. T o d e t e r m i n e if the Ras2 C a a X box m u t a n t s carried on the pRS315 plasmid were capable of supporting R a s - d e p e n d e n t growth, the new strains were assayed for the ability to grow in the absence of Y C p 5 0 R A S 1 by plating on m e d i u m containing F O A . If Y C p 5 0 R A S 1 is dispensable, they b e c o m e uracil a u x o t r o p h s and are resistant to the drug. T h e function of various Ras2 C a a X m u t a n t s can be rapidly and easily assessed by this c o n v e n i e n t plate assay.
Ability of Ras2(C319S) but Not Ras2(C318S, C319S) to Support Ras-Dependent Growth in Yeast T o illustrate the screen, R J Y 5 0 4 was t r a n s f o r m e d with pRS315Ras2, pRS315Ras2(C319S), or pRS315Ras2(C318S, C319S), and the resulting strains were assayed by the plasmid loss assay. A s seen in Fig. 3, the strain 14R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989).
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PROTEIN PRENYLATION IN YEAST
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of RJY504 containing pRS315Ras2 was able to grow in the presence of the drug FOA, indicating that expression of Ras2p is sufficient for viability and the YCp50RAS1 plasmid is no longer required. Next, we examined the importance of the CaaX box Cys, by testing whether Ras2(C319S) was able to support Ras-dependent growth. To our surprise, we found that Ras2(C319S)-expressing cells were also able to grow on medium containing FOA, indicating that a RAS gene lacking the CaaX box cysteine was capable of supporting life. However, the cells grew more slowly than cells expressing Ras2(C318S), suggesting that the mutant was not modified as efficiently as the wild type. The strain containing the double mutant, Ras2(C318S, C319S), was unable to grow on FOA (Fig. 3). Because substitution of serines at both positions 318 and 319 leads to a nonfunctional protein, this raises the possibility that Cys-318 can be farnesylated when the CaaX box cysteine (Cys-319) has been changed to Ser. Method for Characterization of Protein Prenylation in Yeast
Principle of Assay It has not been possible to radiolabel prenylated yeast proteins directly because yeast are unable to take up radiolabeled isoprenoids or precursors such as mevalonate and mevalonolactone. We have developed an alternative method to assess these modifications. It involves overexpression of the CaaX box proteins as an easily purified 35S-labeled fusion protein, followed by complete digestion with proteases and analysis of the products by reversed-phase HPLC. A detailed description of each step in the procedure is given below.
Inducible Expression and Affinity Purification of Glutathione S-Transferase Fusion Proteins in Yeast We have described two plasmids for the expression of GST fusion proteins in yeast. 15The plasmids possess several useful features for producing large quantities of a fusion protein. First, they are propagated in yeast as high-copy number plasmids. Second, expression of the GST fusion is under the control of the galactose-inducible upstream activator sequence, UASGA L. The activity of this promoter can be controlled by incubating cells in medium containing glucose (repressing condition) or galactose (inducing condition). The expression level obtained from this system can be further elevated by coexpression of the transcriptional activator, Gal4p, which binds to the UASGAL. A plasmid, pMA210, has been described which ~s D. A. Mitchell, T. K. Marshall, and R. J. Deschenes, Yeast 9, 715 (1993).
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PRENYLATION
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expresses G A L 4 under the control of the ADH1 promoter. 16This promoter is induced by growing cells on a fermentable carbon source such as galactose. The combination of using a high-copy vector with a strong, inducible promoter and coexpression of the transcriptional activator protein for the UAS allows one to express sufficient quantities of protein in yeast for biochemical analysis of rare posttranslational modification events.
Metabolic Labeling of Cells with Na235S04 An efficient method for radiolabeling yeast proteins on Met and Cys residues is to grow cultures in sulfate-free medium containing Na235SO4.17'18 To obtain efficient labeling, it is necessary to deplete intracellular sulfate reserves. 17,18It typically requires 36-48 hr to deplete completely the sulfate pools causing G1 arrest (cells are unbudded). Once cells are arrested, the medium is supplemented with just enough (NH4)2804 to allow the cells to resume growth and 4% galactose to allow the cells to begin overproduction of the desired protein. The amount of (NH4)2SO4 that is added is somewhat strain dependent and should be determined empirically, but the final concentration is generally in the range of 50 /xM. The time needed for the cells to resume growth is also strain dependent, but is usually between 2 and 4 hr. The cells are then harvested by centrifugation at 2500 g for 5 min at 4 ° and washed once with sulfate-free medium to remove residual sulfate. The cells are resuspended in 3 ml of SC - SO4 supplemented with 4% (w/v) galactose and 1 mCi Na235804. The culture is then incubated with gentle shaking at 30 ° for 6-8 hr.
Purification of Fusion Proteins Expressed in Yeast Following induction and Na235SO4 labeling, cells are harvested by centrifugation at 2500 g for 5 min at 4 °, the medium is decanted, and the pellet is washed once in sorbitol buffer (0.3 M sorbitol, 100 m M NaC1, 5 m M MgC12, and 10 m M Tris-HC1, p H 7.4). All subsequent manipulations are performed at 4 °. The resulting pellet is resuspended in 1 ml of sorbitol buffer with protease inhibitors [final concentrations 100 units/ml aprotinin, i m M phenylmethylsulfonyl fluoride (PMSF), 1 m M pepstatin, and 100 tzM leupeptin]. Cells are lysed by eight 30-sec bursts on a vortex mixer in the presence of 1/3 volume of glass beads (425-600/xm, Sigma, St. Louis, MO, G-9268). U n b r o k e n cells are removed by centrifugation at 2500 g for 5 min, 16j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 48, 847 (1987). 17R. J. Deschaies and R. Schekman, J. Cell Biol. 1115,633 (1987). 18j. B. Stimmel, R. J. Deschenes, C. Volker, J. Stock, and S. Clarke, Biochemistry 29, 9651 (1990).
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PROTEIN PRENYLATION IN YEAST
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and the supernatant is transferred to a clean tube. The beads are washed once with 1 ml sorbitol buffer containing protease inhibitors, spun (2500 g, 5 min), and the supernatants pooled. The membrane fraction is separated from the soluble fraction by centrifugation at 100,000 g for 20 min and the soluble fraction set aside. To purify GST fusion proteins from the membrane fraction, the pellet is solubilized on ice for 20 min in sorbitol buffer with protease inhibitors and containing 1% (w/v) Nonidet P-40 (NP-40), 0.5% (w/v) deoxycholate, and 0.1% (w/v) sodium dodecyl sulfate (SDS). A successful labeling results in approximately 3-4 × 108 total counts/min (cpm) of 35SO4-1abeled material. Particulate material that is not solubilized is removed by centrifugation at 12,000 rpm in a microcentrifuge for 3 rain. Affinity purification of the GST fusion protein is then performed by adding 150 ~1 of a 50% slurry of agarose beads conjugated with glutathione (GSH) (Sigma, G-4510) to the solution of solubilized proteins. The mixture is incubated with gentle shaking for 1 hr at 4°. The beads are then recovered by centrifugation (12,000 rpm, 30 sec) and then washed with 1 ml of sorbitol buffer. Three additional washes are performed to remove unincorporated 35SO4.
Proteolytic Digestion of Fusion Protein and Analysis of Products by Reversed-Phase Chromatography We have previously reported a protease digestion strategy that converts 35SO4-1abeled proteins to the constituent amino acids or prenylated derivatives. 17 The protease cocktail works well for yeast Ras proteins; however, we have not systematically tested it for other prenylated proteins, and the optimal conditions may vary. Ras2-GST fusion proteins bound to GSHagarose beads are resuspended in 500 ~1 of 20 mM sodium phosphate buffer (pH 6.8), containing 7.5 units of pronase E [Streptomyces griseus, Sigma type XIV, 5.8 units/mg of protein (1 unit hydrolyzes casein to produce Folin-Ciocalteau color equivalent to 1 ~mol of tyrosine per minute at pH 7.5 at 37°)], and the mixture is incubated for 40 hr at 30°. At that time, 18 units of leucine aminopeptidase M [porcine kidney microsomes, Sigma type IV-S, 20 units/mg of protein (1 unit hydrolyzes 1 ~mol of /-leucine-p-nitroanilide per minute at pH 7.2 at 37°)] and 42 units of prolidase [porcine kidney, Sigma, 205 units/mg of protein (1 unit hydrolyzes 1 ~mol of Gly-Pro per minute at pH 8.0 at 37°)] are added, and the incubation is continued for another 4 hr at 30°. The beads are then removed by centrifugation (12,000 rpm, 30 sec at room temperature) and the supernatant recovered for analysis. Approximately 2 × 10 6 cpm is injected onto a Ca reversed-phase HPLC column (Vydac, 4.6 × 250 mm) at an initial flow rate of 1 ml/min of a buffer consisting of 24% (v/v) acetonitrile/12%
0.2-
A S-GG-CYS 0.15 -
S-Far-CYS Abs 214 nm
0.1
0.05
0-
12000 -
B 10000 -
8000-
cpm
6000-
4000-
2000-
02000-
C 1600-
1200 -
cpm 800-
400 o
0
' ' 5' ' I0'
0
15
20
~'5 ;0 ;5 40'/5'
Time
50
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PROTEINPRENYLATIONIN YEAST
77
(v/v) 2-propanol/0.1% (v/v) trifluoroacetic acid. A 45-min linear gradient is performed ending with a final buffer composed of 60% acetonitrile/30% 2-propanol/0.1% trifluoroacetic acid. The effluent is monitored at a wavelength of 214 nm and collected as 0.5-ml fractions across the gradient. The entire fraction is mixed with 5 ml of Budget-Solve (RPI) and radioactivity determined by scintillation counting.
Evidence for Ras2(C319S) Prenylation on Cys-318 As an example of this technique, we have examined the posttranslational modification of the protein product of the Ras2(C319S) allele described above. Plasmids expressing GST fusions between Ras2(C318S), Ras2(C319S), and Ras2(C318S, C319S) were constructed and transformed into the yeast strain RJY690 (MATa/a leu2 ura3 his3 [pMA210]). Cells were labeled with Na235SO4 and processed by the method described above. Figure 4A shows the elution position of S-farnesylcysteine (10 nmol) and S-geranylgeranylcysteine (15 nmol). S-Farnesylcysteine was synthesized by established procedures and confirmed by fast atom bombardment ( F A B ) mass spectrometry, t8 S-Geranylgeranylcysteine was kindly provided by Dr. Pat Casey (Duke University). Labeled products derived from digestion of Ras2(C318S) (Fig. 4B) and Ras2(C319S) (Fig. 4C) comigrate with S-farnesylcysteine (Fig. 4B). Although the number of counts recovered from the digestion of purified Ras2(C319S) is less than from the Ras2(C318S) protein with an intact CaaX box, the major peak derived from Ras2(C319S) still corresponds to S-farnesylcysteine. The difference in recovered counts most likely reflects inefficient farnesylation of the Ras2(C319S) protein and not a lower level of expression of the protein. Western blots reveal that similar levels of the two proteins were expressed in the two strains (data not shown). This interpretation is also consistent with the plasmid loss assay. A second minor peak is also observed that elutes 2-3 min later than S-farnesylcysteine (Fig. 4C). The identity of the second peak has not been determined, but its elution position is consistent
FIG. 4. Reversed-phase HPLC analysis of prenylated Ras2 proteins. (A) Elution profile of S-farnesylcysteine(16.5-17.0 min) and S-geranylgeranylcysteine(26.5-27.0rain) standards measured by monitoring absorbance at 214 nm. (B and C) Analysis of approximately 2 × 10 6 cpm of the 35SO4-1abeledmaterial derived from proteolysis of samples prepared from Ras2(C318S) (B: diamonds), Ras2(C319S) (C: circles),or Ras2(C318S, C319S) (C: triangles). A C4 reversed-phase HPLC column (Vydac, 4.6 × 250 ram) was used for the fractionation. The column profile consisted of a 45-min linear gradient (1 ml/min) beginning with a buffer consisting of 24% acetonitrile/12% 2-propanol/0.1% trifluoroacetic acid and ending with a buffer consisting of 60% acetonitrile/30%2-propanol/0.1%trifluoroaceticacid. Fractions were collectedevery 30 sec and the contents of the entire fraction measured by scintillationcounting.
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PRENYLATION
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with S-farnesylcysteine methyl ester. As expected, no peak is observed in this portion of the gradient for Ras2(C318S, C319S). We were intrigued by the ability of Ras2(C319S) to support Ras-dependent growth. As Ras2(C318S, C319S) is not functional, we conclude that the ability of Ras2(C319S) to function relies on the cysteine at position 318, one residue upstream of the normal CaaX box cysteine (position 319). The in vivo assay of prenylation described above revealed that Cys-318 was modified by a farnesyl group. We consider two explanations for this result. The first is that if a nonspecific protease removes the C-terminal residue of Ras2(C319S), a true CaaX box would be formed with the sequence Cys-Ser-Ile-Ile. This sequence could be a substrate for yeast protein farnesyltransferase (FTase). The second possibility is that the yeast FTase, which normally recognizes CaaX sequences, may also be capable of recognizing a cysteine five residues from the end of a protein in vivo. Concluding Remarks The methods described in this chapter provide a means for measuring the prenylation of proteins in yeast and assessing the function of proteins altered in the ability to be posttranslationally modified. The power of the system is in the sensitivity of the plasmid shuffle assay which allows detection of even low levels of activity produced by inefficiently modified proteins. In addition to scoring the function of known CaaX box mutations, the plasmid shuffle assay can be adapted to search for intragenic or second site suppressors that allow unmodified proteins to function. We have also described a method to characterize protein prenylation in yeast. To date, direct measurement of prenylation in yeast has been hampered by the inability to radiolabel cells with [3H]mevalonate or other precursors such as farrlesyl and geranylgeranyl pyrophosphate. The method we describe combines overproduction and radiolabeling the protein of interest as a GST fusion, followed by reversed-phase HPLC to identify the prenyl moiety. Acknowledgments We thank Pat Casey for generously providing the S-geranylgeranylcysteine standard, Lynn Farh for synthesizing the S-farnesylcysteine, and Jun Ma for the Gal4 overproduction plasmid, pMA210. This work was partially supported by grants from the National Institutes of Health {CA50211) and the Pardee Cancer Research Fund to R. J. D.