Vol.

172,

No.

November

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1990

Pages

TOTAL

IN VITRO

MATURATION

a-FACTOR

Stevan

Marcus’,

1 Department

of Microbiology Biology, University of Chemistry,

October

THE

LIPOPEPTIDE

Guy A. Caldwelll,

2 Department

Received

OF

SACCHAROMYCES

MATING

Chu-Biao

and Program of Tennessee,

1310-1316

CEREVISIAE

PHEROMONE

Xue *, Fred

Naide?,

in Cellular, Knoxville,

Molecular, and Developmental Tennessee 37996

and Jeffrey

College of Staten Island, City University Staten Island, New York 10301

M. Becker’

of New York,

1, 1990

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is posttranslationally modified in a manner analogous to that of the fasproto-oncogene product. A consensus Cterminal amino acid sequence, CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAtWDPRI gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs. ‘-’ 199” ncademlc Press, 1l.C.

Sexual

conjugation

the reciprocal which

action

is secreted

In contrast, post-translational

a-factor

amino

acids,

CAAX

results

lysates

were

include

used

catalyses

capable CAAX

This

assay

of blocking

via the yeast

(2), is processed

amino

contained

acid sequence,

in the primary

have

important

in activity

which prove

shown

that

of both

useful

any of the steps

S. cerevisiae

total maturation

in screening involved

1310

is essential as removal

gene

that

to a-factor

for

A is aliphatic

sequence,

for normal of the rabbit

The

Ras

isoprenyl

reticulocyte

S. cerevisiae

cell

(7, 10) is a component

Ras and a-factor

for potential

(1).

contains

peptides.

of the -AAX

(9). In addition,

(4).

and detects

anti-Ras-related

in the post-translational

sequences.

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in urg fr,rm reserved.

lamins

which

(C is cysteine,

et al. demonstrated

of the RAMDPR7

pathway

pathway

proteolysis

bioactivity,

of nuclear

the product

and o-factor,

secretory

of each of these

that isoprenylation

(8). Vorburger

demonstrates

sulfur,

upon

(3, 4, 5, 6). Essential

CAAX

precursors

for a-factor

the isoprenylatjon

product

is dependent

by a cells,

by an alternative

(5, 6). Studies

the isoprenylation

may

is secreted

of the cysteine

to show

the first in vitro assay

which occurs

by the ras proto-oncogene

is likewise

cells

a-factor,

isoprenylation

decrease

and Q haploid

and secretion

is a C-terminal

of catalyzing

recently

which

shared

acid),

esterification

capable

this pathway.

and Ras

steps

in a marked

were

the activity

agents

events

cerevisiaea

termed

maturation peptide

and Ras maturation

methyl

moiety

present

processing

(5, 7). lsoprenylation

extracts

pheromones

a-factor

and X is any amino

and carboxy

Saccharomyces

an isoprenylated

processing

function

of peptide

by CLcells.

a-factor,

proper

between

modification

In this report,

of we

intermediates cancer

therapeutic

of the a-factor

in

Vol.

172,

Materials

No.

3, 1990

and

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Methods

Preoaration of veast extracts and strains used S. cerevisiae cultures grown at 30 “C to mid-log phase in YEPD (1% yeast extract, 2% peptone, and 2% dextrose) were harvested by centrifugation, washed once with sterile distilled H,O, and resuspended to 0.3 g/ml (wet weight/volume) in ice-cold lysis buffer (7.5 mM KH2P04, 42.5 mM K2HP04, 2 mM EDTA, 20 mM P-mercaptoethanol, and 20 mM MgC12, pH 7.6). Cells were lysed using glass beads (0.45-0.50 mm, 6. Braun, Germany) by voriexing for 60 set intervals, with placement in ice for 2 min between each interval. Unbroken cells were removed by centrifugation for 10 min. at lOOxg, and the supernatant was centrifuged for 45 min at 20,OOOxg. The resulting supernatanfs (cell extracts) were diluted to 2.5 mg protein/ml, after estimation of protein concentration (Bio-Rad Protein Assay Concentrate), and stored at -70 “C until use. Cell extracts were prepared from the following strains: X21801A (MATa SUCZ ma/ me/ gal2 CUPl) (Yeast Genetics Stock Center [YGSC]), SM1267 (MATa rar@his3 ura3 trp1 a&8 canl) (from Susan Michaelis), and SM1287 (isogenic to SM1267, but transformed with the episomal plasmid pYPG1, which contains the RAM/W/?7 gene, thus allowing for overexpression of this gene) (also a gift from Susan Michaelis). $vnthesis of a-factorJzzursorg The various a-factor analogs used in this study are shown in Fig. 1. The a-factor (NH2YllKGVFWDPAC[S-farnesyl].OCH$ and a-factor pentadecapeptide (NH,-YIIKGVFWDPA-CVIA-COOH) were synthesized as previously reported (11, 12, 13). All other analogs were synthesized using either solution-phase peptide synthesis or a combination of solid-phase and solution-phase peptide synthesis. Ail peptides were purified to greater than 98% homogeneity using reversed phase HPLC and were characterized using amino acid analysis, 400 MHz ‘H-NMR spectroscopy and Fast Atom Bombardment mass spectrometry. Details of the synthesis and characterization of these compounds will be published separately. a-Faclpr maturation w The standard a-factor maturation assay was a modification of the assay described by Fieiss et al. (14) and consisted of the following components: 50 pl assay buffer (50 mM Tris-Cl, pH 7.5, 50 PM ZnCI2, 20 mM KCI, 1 mM DTT) containing 1 mg/ml a-factor precursor, 2 pl [3H]farnesyl pyrophosphate (pH]FPP) solution (from New England Nuclear, 10 Ci/ml, 0.0125 PM/ml), and 10 PI cell extract (50 pg protein). Reaction mixtures were incubated at 30 “C for 5 hr, then stopped by adding 62 pl of glacial acetic acid and placing in dry ice. Assays were analyzed by HPLC (Beckman Instruments, System Gold computer software) using the following methodology: column: DuPont Zorbax Protein Plus C, Reversed Phase; flow rate: 1.5 ml/min; gradient: 15% acetonitrile (ACN) from 0 to 10 min, 15 to 80% ACN from 10 to 75 min (l%/min). One min fractions were collected, then counted by liquid scintillation (Beckman LS 7000). Site-directed mutaaenesis of fhe Mfal uene The MFal gene (15, a gift from Jeremy Thorner) was originally subcloned as a 1.6 kb BanMl gene fragment into the polylinker of phagemid vector pTZ1W (Bio-Rad) using standard recombinant DNA techniques (16). This construct was used for the generation of single-stranded template DNA and subsequent in vitro mutagenesis following a modification of the method of Kunkel et al. (17, 18). The mutagenic primer used to generate a Cys ‘2->Ser12 substitution was a 21 mer: 5’-GACCCAGCATCTGTTATTGCT-3’. Putative mutants were screened directly by dideoxy DNA seqencing to identify desired clones. The vectors containing the mutated MFa7 and wild type MFa7 genes, respectively, were subjected to further mutagenesis to allow for subcloning into yeast-E. co/i shuttle vector pRS129 (19, Figure 3A). S. cefevisiae strain SM1229 (see Figure 5) was transformed with the constructs pGCGa1003 (wild type MFaIf or pGCGai004 {Cys 12->Ser12 mutatron) and transformants tested for the ability to produce a-factor in the presence of galactose, as determlned by the halo assay (Figure 38).

Results

and

Discussion

Because

several post-translational

modification

steps are shared by Ras proteins

and the

S. cerevisiae a-factor, we set out to develop an assay whereby these processes could be studied in vitro. We synthesized

potential

a-factor precursors,

as well as standards necessary for the identification by 1311

Vol.

172,

No.

3, 1990

HPLC

of a-factor

extracts

were

precursors which

and

(Figure

2A and

from

esterification,

28,

respectively),

mature

a-factor

(Figure

suggested

that

omission peak

of the a-factor (Figure

2D).

pyrophosphate factor that

proteolysis

This

of the

precursor,

peptide

indicates

that

To provide

we added

if the 42 min peak

was

indeed

of this

conversion

of the 42 min RT peak

a-factor

15mer.

15mer

Schafer

processing

product

assay.

to mature

et al. (4) reported

was

is not

in this

precursor, in Figure

farnesylation

Since

the same

we believe

of a 15mer

that

including product

of SAM

in their

peak

assay,

by using

cell

methyl

also The

the 42 min

of [3H]farnesyl

was

a farnesylated

a-

We expected

would

of SAM

42 min RT peak this

The

assay.

to the assay.

addition

= 42 min) 15mer.

maturation

in this

products,

3A, addition

peptide

also detected.

detectable

(SAM)

cell

and carboxy

as 15mer

a degradation

a-factor

assay,

were

that the 42 min RT peak

As shown a-factor.

[RT]

(detectable

products

sequence

peak

15mer

time

2C, RT = 48 min),

S-adenosyl-L-methionine

a-factor.

is used

a-factor

the same

evidence

S. cerevisiae and

(retention

in no a-factor-related

42 min

a farnesylated

to mature

precursor

favor

further

did allow

for the

is obtained

when

represents

farnesylated

but did not observe

additional

to a-factor. To provide

used

the

Plmer

B, and C; RT = 51 min),

resulted

additional

product

that

et al. (4), to be farnesylated

Figure

produced precursor

nonradiolabeled

processing

the

N-terminal

precursor

([3H]FPP).

2A,

apparently

the a-factor

nonmethylated strain,

COMMUNICATIONS

1). We found

a major

farnesylated,

The fact that 21 mer maturation

both

by Schafer

RAhVDPR7 overexpressing

the

producing

of the

obtained

RESEARCH

(Figure

producing

results

producing

BIOPHYSICAL

intermediates

isoprenylation

on similar

of proteolysis,

prepared

AND

biosynthetic

of catalyzing

based

steps

extract

potential

capable

we believe,

additional

BIOCHEMICAL

substantiating

evidence

S-[methyl-3H]adenosyl-L-methionine Only

a single

radiolabeled

that we were ([3H]SAM)

product

(RT

indeed

detecting

total

and nonradiolabeled

= 51 min)

was

produced

maturation

FPP in our a-factor

maturation

under

conditions

these

a-factor NH2-Tyr-lle-lle-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys-COOCH3 A Nonmethylated

3

3

3

3

3

3

a-Factor.

NH2-Tyr-lle-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys-C00H L -722-T a-Factor

2lmer-

a-Factor

15mer

a-factor

12mer:

Fiqure

1. Structures

of

a-factor

and various 1312

a-factor

analogs

of a-factor,

used In this study

assay

we

Vol.

172,

No.

BIOCHEMICAL

3, 1390

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

nonmethylated a-laclor (RT = 48 min)

50000-

D

4000030000

_

20000

-

1000001 30

35

40

45

50

Fraction Number

tSC

55

(min)

Figure 2. In vitro maturation of a-factor. a-Factor maturation assays were performed, then analyzed by HPLC and liquid scintillation, as described in the Materials and Methods section. A) Maturation of a-factor 21 mer by X2180-1A (wild type) cell extract. B) Maturation of a-factor 15mer by X2180-IA cell extract. C) Maturation of a-factor 2lmer by SM1287 (RAM/DPRl overexpressing) cell extract. D) Assay without a-factor precursor. The retention times (FIT) indicated in panel A were determined using synthetic peptide standards.

(Figure

38).

factor,

strongly

not processed

Thus,

this

peak

suggesting (not

shown),

42 and 48 min, respectively),

is farnesylated,

the formation and since

carboxy

methyl

of the mature only

is methylated,

mature

a-factor,

we have

esterified,

pheromone.

and

coelutes

the a-factor

but not the isoprenylated

the first direct

1313

Since

biochemical

evidence

with 12mer

synthetic (Figure

precursors

a1) is

(RT’s

for the order

= of

Vol.

172, No. 3, 1990

BIOCHEMICAL

a-factor CAAX

maturation

nonmethylated,

farnesylated

AND BlOPHYSlCAL

steps: 1) isoprenylation,

2) proteolysis

of the -AAX sequence,

for the isoprenylation

gene product is a component

/?AM/DPRI

(SM1287)

RAM/DPR7

much greater isoprenyl transferase activity than extracts prepared from wild type (X2180-1A)

cells. An accumulationof nonmethylated

had

(Figure 4)

a-factor (RT = 48 min)) was also apparently detectable when

overexpressing

l?AM/DPR7

of the

of a-factor and Ras. Our assay provides strong evidence to

support this conclusion. Extracts prepared from a strain which overexpresses

extracts from the

producing

a-factor, then 3) carboxy methyl esterification.

A recent report (4) provided evidence that the activity responsible

RESEARCH COMMUNICATIONS

strain were used. Extracts from ram/dprl mutants, however,

contained no detectable isoprenyl transferase activity under our assay conditions. As stated above, an unmodified 12mer a-factor peptide (Figure 1) lacking the -AAX sequence was not processed to an a-factor-related

peptide, thus substantiating

sequence is essential for protein isoprenylation requirement

of the CAAX

and MFa2

(M&37

a plasmid

50000

sequence for proper a-factor maturation. S. cerevisiae

mfa7A

mfa2A

mutants

are the a-factor structural genes), which do not produce a-factor, were transformed with

containing an a-factor gene with a Cys t*-,Serl*

galactose-inducible

previous results showing that the -AAX

(4, 5, 6). We have also obtained in viva evidence for the

GAL

7

mutation expressed

promoter (Figure 5A). These transformants

under control of the

did not produce a-factor, in contrast

A

40000 30000 20000 10000 2 0 6

50000

80000 5 a 0

60000 40000

0

0

3

30

36

40

Fraction

45

Number

50

0

55

0

4

(min)

30

35

40

Fraction

Figure 3. In vitro methylation of a-factor. a-Factor maturation assays were performed using a-factor 21 mer and X2180-IA cell extract. A) Effect of addition of S-adenosyl-L-methionine (SAM) (to 160 ug/ml) to the standard maturation assay. B) a-Factor maturation assay using 13H]SAM. Nonradiolabeled farnesyl pyrophosphate (to 320 pg/ml) was added to the reaction. Figure 4. Evidence that the S. cerevisiae RAM/DPRi gene product is required for isoprenyl transferase activity. a-Factor maturation assays were performed using a-factor 21mer. Open circles represent maturation by SM1287 (RAAUDPR7 overproducing) cell extract. Closed circles represent maturation by X2180-IA (wild type) cell extract. Extract from a ram/dprl mutant (SM1267) lacked isoprenyl transferase activity (open triangles).

1314

45

Number

50

(min)

55

I

Vol.

172,

No.

BIOCHEMICAL

3, 1990

A

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

B

CENIARS

Figure 5. In viva evidence for the requirement of the CAAX sequence for a-factor maturation. A) Overexpression vector pGCGal003. Constructed by the ligation of the 1 kb BarnHI-EcoRI MFaf gene fragment into vector pRS129 (19). pRS129 is a derivative of plasmid pRSS56 with the GAL1110 promoter region subcloned into the Kpnl site of the polylinker. The MFa7 gene was manipulated by site-directed mutagenesis to create a novel EcoRl site upstream from the MFaf translational start and downstream from its own promoter sequence. This change enabled the ligation of the Mfal gene fragment into pRS129 for galactose-inducible control of expression. Additional attributes of plasmid pGCGa1003 include: Amp’. ampicillin resistance for selection in E. co/i; ori, E. co/i origin of replication; CENIARS, for stability and replication in yeast; TRPl , selectable marker for yeast; fl[+], origin of replication of bacteriophage 11 for production of ssDNA for use in mutagenesis and sequencing; IacZ, gene encoding P-galactosidase. Note: phagemid pGCGa1004 is identical to pGCGa1003, with the exception that the MFaf DNA encodes a mutation to Ser12 in place of the wild type Cys’*. B) Results of site-directed mutagenesis of the MFal gene. A lawn of strain RC757 (MATa ~~12-1 me his6 met1 can7 cyhq cells was spread onto YPGal plates (1% yeast extract, 2% peptone, 2% galactose, 0.05% dextrose, 2% agar). The following S. cerevisiae strains were patched onto the lawns and incubated at 30 ‘C for 24 hr: A, SMI 058 (MA Ja trpf leu2 ura3 hi.54 canf); B, X2180-1B (MATa); C, SM1229 (isogenic to SM1058, but mfafA::LEU2 mfa23::URAS); D, SM1229 (pRS129): E, SM1229 (pGCGa1003 [wild type MFa7)); and F, SM1229 (pGCGa1004[Cys12 ->Ser12]). to transformants that

overexpressing

a synthetic

a-factor

Saccharomyces secretion

15mer

cerevisiaecell

of a-factor

the in vivo effect

eukaryotic

proteins (7)

and

(3). Therefore,

isoprenylation

of activated

strong

evidence

growth,

might

isoprenylation significance been

is now

report,

we have

doses

considered methyl

presented

of intermediates

inhibitors

which

might

block

of Cys t2-,Sert2

modification

has been

(8).

Studies

shown

or abolish

its ability drugs

target

an important allows

effective

for

demonstrates

This

common against

1315

do not Ras

has not yet been

may

to a-factor Ras-related

for normal which

prove

cells.

therapeutic

of a-factor useful

In addition, affect

cell

(3, 20).

Thus,

agents.

The

(21).

in vitroand for the

and Ras maturation, cancers.

Ras

although

of this protein

of

prevent

adversely proteins

established,

role in the activity

assay

to a number

mutations

cancer

for the total maturation

pathway.

any one of the steps

that

for anti-Ras-related

to Ras function

which

directly

to transform

of activated

have

by

is required

common

that

potential

may

not isoprenylated

to be essential

suggest

reducing

the first assay

potentially

substitution

of cholesterol

esterification

was

demonstrated

that isoprenylation

the transforming

of the maturation

of drugs

alteration

results

production.

decrease

a possible

that this modification

detection

for the identification

that

Previous

shown

modification

activity

58).

it has been

post-translational

this

a-factor

in inhibiting

of carboxy

speculated

on a-factor

an important

(Figure

a Cys 12->Ser12

this site-specific

Ras significantly

suggests be useful

gene

(4). Moreover,

(5, 6). Furthermore, maximal

MFa7

containing

farnesylation

is clearly

for

type

precursor extracts

of blocking

lsoprenylation

function

the wild

it has In this for the

screening thus,

allowing

of

Vol.

172, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Acknowledgments We wish to thank Susan Michaelis for S. cerevisiae strains, Bill Michaud and Phil Hieter for vector pRS129, Munira A. Basrai for helpful discussions and suggestions, and Hui-Fen Lu and Angus Dawe for technical assistance. This work was supported by Public Health Service grants GM-22086 and GM-22087 from the National Institutes of Health. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Fuller, R.S., Sterne, R.E., and Thorner, J. (1988) Ann. Rev. Physiol. 50, 345-362. Anderegg, R.J., Betz, R., Carr, S.A., Crabb, J.W., and Duntze, W. (1988). J. Biol. Chem. 263, 1823618240. Schafer, W.R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H., and Rine, J. (1989) Science 245, 379. 385. Schafer, W.R., Trueblood, C.E., Yang, C.-C., Mayer, M.P., Rosenberg, S., Poulter, C.D., Kim, S-H., and Rine, J. (1990) Science 249, 1153-1139. Glomset, J.A., Gelb, M.H., and Farnsworth, C.C. (1990) TIES 15, 139-142. Goldstein, J.L., and Brown, MS. (1990) Nature 343, 425-430. Powers, S., Michaelis, S., Broek, D., Santa Anna-A., S., Field, J., Herskowitz, I., and Wigler, M. (1986) Cell 47, 413-422. Marcus, S., Caldwell, G.A., Xue, C-B., Naider, F., and Becker, J.M. manuscripf in preparation. Vorburger, K., Kitten, G.T., and Nigg, E.A. (1989) EMBO J. 13, 4007-4013. Goodman, L.E., Perou, CM., Fujiyama, A., and Tamanoi, F. (1988) Yeast 4,271-281. Becker, J.M., Marcus, S., Kundu, B., Shenbagamurthi, P., and Naider, F. (1987) Mol. Cell. Biol. 7, 4122-4124. Xue, C.-B., Caldwell, G.A., Becker, J.M., and Naider, F. (1989) Biochem. Biophys. Res. Comm. 162, 253-257. Xue, C.B., Ewenson, A., Becker, J.M., and Naider, F. (1990) Intern. J. Peptide Res. 33, (in press). Reiss, Y., Goldstein, J.L., Seabra, M.C., Casey, P.J., and Brown, M.S. (1990) Cell 62,81-88. Brake, A., Brenner, C., Najarian, R., Laybourne, P., Merryweather, J. (1985) In Protein Transport and Secretion (M.J. Gething, Ed.) p. 103-108. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Kunkel, T.A., Roberts, J.D., and Zakour, R.A. (1987) Meth. Enzymol. 154, 367-382. McCleary, J.A., Whitney, F., and Geisselsoder, J. (1989) Biotechiques 7, 282-289. Sikorski, R.S., and Hieter, P. (1989) Genetics 122, 19-27. Finegold, AA., Schafer, W.R., Rine, J., Whiteway, M., Tamanoi, F. (1990) Science 249, 165-169. Broach, J.R., and Deschenes, R.J. (1990) Adv. Cancer Res. 54, 79-139.

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