IDENTIFICATION OF COMPOUNDS MODULATIONS PROTEIN/CELL MEMBRANE ASSOCIATION

Abstract: A method for identifying compounds which modulate protein/cell membrane association which method comprises contacting a test compound with a cell, having (i) a cell membrane, (ii) a heterologous protein comprising a reporter sequence and a recognition sequence for cell membrane association, (iii) a reporter system which is acted upon by the reporter sequence such that there is a measurable change in cell phenotype upon modulation of protein/cell membrane association by the test compound, and detecting any change in cell phenotype.

Description: IDENTIFICATION OF COMPOUNDS MODULATING PROTEIN/CELL MEMBRANE ASSOCIATION The present invention relates to a method for identifying compounds wnich modulate protein/ceil emnrane association. The method provides an m v vo assay for inhibitors of protein/cell metnorane association wherein modulation of the association leads to a detectable change in cell phenotype. The invention also relates to heterologous proteins, nucleic acid sequences encoding these, corresponding DNA constructs, and recomninant cells, all for use m the aoove method. Numerous proteins are modified by the covalent addition of lipids \ ) . The hydropnob c side-cnams of the lipids serve to anchor the proteins m selected cellular memoranes. We refer to this process as membrane association. Inhibition of membrane association of certain i pid-modifled proteins is a potential target for therapeutic intervention, since membrane association is usually critical for full biological activity of these proteins. By way of example, inhibition of the memnrane association of ras proteins has been proposed as a method for the selective inhibition of oncogenic forms of ras (2) . Additionally the activity of oncogenic src variants is dependent upon their memnrane association througn N-myπstoylation (3) . Current proceαures for identifying inhibitors of lipid modification and membrane association involve screening compounds, such as peptides, against purified samples of individual enzymes involved m the lipid modification process (8) . This is time-consuming and expensive since it involves the purification and pernaϋs cloning of these enzymes. Furthermore, not all steps involved m tne memnrane association process may be amenable to study m this wav. For examole, the proposed interaction of lipid-modif ed proteins with memnrane components (9) may be difficult to reproduce in vitro. An alternative approacn was reported by Fmegold e_t a . (10) . Activation of rhe pneromone response pathway of Saccharomvces S cerevisiae) leads to growth arrest. The activity cf tne tr.ercracπs response patnway is dependent upon tne integrity of tne g sununit of the pheromone receptor-associated G protein. This sunun t is isoprenylated. Thus, inmbition of isoprenylation reverses growth arrest in a iX_ cerevisiae strain m wnich tne pheromone response pathway is active. This provides a growth/no growtn assay for inhibitors of isoprenylation. However, this assay is not specific to isoprenylation, since compounds inhibiting the pneromone response pathway at any point will be active. It has been reported elsewhere that ammo acid sequences around the site of lipid modification m some proteins act as memnrane association signals (5,6) . Hancock et al (5) have demonstrated -nat fusion of ras membrane association signals to protein A relocates protein A to the inner surface of the plasmamembrane. Similarly Pellman et al ^ (6) showed that fusion of β-globm to a src memnrane association signal relocated that fusion to the plasmamembrane. In both cases neither of the fusions conferred a readily measurable phenotype on the cell and the location of the fusion protein could only be determined by time-consuming and labour intensive immunological methods. We now provide an in vivo assay for inhibitors of protein/cell membrane association wnerem modulation of association leads to a detectable change m cell phenotype. In a first aspect of the present invention we provide a method for identifying compounds which modulate protem/cell memnrane association which method comprises contacting a test compound witn a ceil, having (I) a cell membrane, (n) a heterologous protein comprising a reporter sequence and a recognition sequence for ceil memnrane association, (m) a reporter system wnich is acted upon ny the reporter sequence such that there is a measuranle change m ceil pnenotype upon modulation of protein/ceil memnrane association ny tne test coπvDound, and detecting any cnange m cell pnenotype. The method may ne useα to laentifv mnibitors or ennar.cers of ar.v nioiogicai process wnicn leacs α rectly or mαirectly to ""oαuiation of protein/cell memnrane association. A particular aαvantage of the methoα is that it enables compounds to be screeneα for their ability to inhibit membrane association without the need to c-one or purify any of the enzymatic species involved in memnrane association. The method will detect, in addition to direct inhibitors of memnrane association, mnibitors of any lipid modification process and mnibitors of lipid biosynthesis The method may be used screens, such as rapid throughput screens, for compounds which are potential mnibitors/ennancers of biochemical processes resulting m modulation of protem/cell membrane association. Such screens may for example involve large collections of chemical compounds, natural products and/or broths . Alternatively such screens may involve -ntracellularly expressed protein libraries. Since active compounds confer a gain function (activation of the reporter system) upon a test cell, the method is very sensitive; a small level of reporter activity is easily observed if the background is zero. Any convenient cell may be used where modulation of protem/cell memnrane association such as lipid modification or • nemnrane insertion can be linked via a reporter system to a phenotypic cnange Examples of convenient cells include yeasts such as Ξ erevisiae, Schizosaccharomvces pomne, Kluvermvces lactis and Pichia nastoris, bacteria such as Escheπschia coli, plant cells such as tonacco and maize, and animal cells. We include cells which naturally contain the machinery required for membrane association of proteins, and cells where such machinery has been introduced, for example by genetic engineering, and cells where the natural machinery has been aitereα by addition, alteration (for example by mutation) replacement or deletion of some component The invention also relates to a cell for use m tne above metnoα and having ) a ceil memnrane, (11) a heterologous protein co orismg a reporter sequence ana a recognition sequence for cell "emorane association, (111) a reporter system wnicn is acteα upon by tne rεnorter sequence such that there is a measuranle cnange ceil nneno t vne U D O Π modulation of protem/cell membrane association ny a test compound. The ceil memnrane used in the above method may be any convenient intracellular ceil memnrane, including the nuclear memnrane, organelle membranes and the plasmamembrane. The memnrane with which the heterologous fusion protein is associated need not be the same membrane to which proteins which naturally contain tne membrane association signal are targeted; the presence of other signals m the fusion protein may alter the targeting. For example, the presence of a nuclear localisation signal m combination with a membrane localisation signal may locate the fusion protein to tne nuclear memnrane, whereas m the absence of the nuclear localisation signal the protein would be localised to other cell membranes. The heterologous protein used the above method comprises a reporter sequence and a recognition sequence for cell membrane association. It conveniently comprises a fusion of the reporter sequence and the recognition sequence. The reporter sequence is conveniently a transcriptional activator and preferably comprises a DNA binding domain and a transcription activation domain. The DNA binding domain may be sunstituted for by a protein domain that interacts with a protein already bound to a promoter; m eit.iεr case the function of this part of the protein is to enanle the protein to bind to a specific promoter when the fusion protein is not membrane-associated. Such transcriptional activator/recognition sequence fusions can confer a simple pnenotype on cells containing an appropriate reporter gene m that the gene is inactive wnen tne activator is memnrane-associated; but when that membrane association is inhibited the activator fusion can move to the reporter gene, bind to its promoter, and activate transcription of the reporter In essence, the memnrane association signal enables the activator to ne sequestered away from the reporter gene The above heterologous proteins together rfitn nucleic acid sequences encoding these, form further and independent aspects of the invention. The transcr ntionai activator conveniently comprises a DNA ninαmg oomam from a euκaryotιc protein such as GAL4, or a pro aryotic protein sucn as tne repressor protein LexA. These DNA binding domains are well characterised and known to be capable of tolerating fusion a variety of activation domains (21, and refs. therem) . Other DNA binding domains, such as the zmc binding domains, homeodomams, and basic domains (see ref 23) may also be used this method. The transcriptional activation domain may be natural or synthetic. Suitable activation domains of the 'acidic' class are described by Ptashne (ref 21) ; these acidic domains have the useful property of being able to function in many eukaryotic species. It may be convenient for the trancπptional activator to contain a nuclear localisation signal. The activator must be able to tolerate fusion of c imaeric signals without destroying these functions. The membrane association signal may, for example be a signal for farnesylat on, geranylgeranyiation, palmitoylation, myristoylation or some other lipid modification. Additionally the signal may ne a novel sequence created, for example from of all or part of two or more different signals. The heterologous protein containing the membrane association signal is encoded on an expression construct. This construct contains a promoter to drive expression of the fusion protein together with appropriate nucleic acid sequences to allow efficient RNA processing and translation of the mRNA. The promoter may be constitutive or regulated. The reporter system conveniently comprises a promoter / reporter gene acted on by a transcriptional activator as described above The promoter of the reporter gene will contain o nαmg sites for the transcriptional activator and s preferably essentially inactive the ansence of bound activator, thus maximising tne response upon modulation of memnrane association. The promoter may be a naturally occurring DNA sequence, or it may be a syntnetic sequence, or it may be a comnmation of the two. A number of factors w ll influence tne difference netween the inactive and acti-e states of the reporter gene These include the affinity of tne rranscrmt onai activator ror ts n αmg sites the promoter, tne number of those binding sites, and strengtn of the activation domain of the activator. The reporter gene conveniently confers an reaαily measurable phenotype upon the cell. The reporter gene may conveniently comprise the coding sequence of a enzyme sucn as E___ coii β-gaiactosidase, firefly luciferase, or choramphenicol acetyl transferase, m which the phenotype conferred is an enzyme activity which may easily be measured, often ny use of a colour change. Alternatively the reporter gene may be a gene essential for the growth of the cell, for example a gene encoding an essential metabolic enzyme. In this case, activation of the gene will allow cell growth. Alternatively tne reporter gene may encode an enzyme which metanoiises a toxic substrate. In the presence of this susbstrate, activation of the reporter gene will again allow growth of the cell. Examples of other convenient promoter/reporter gene combinations will be apparent to the person of ordinary skill. If required the reporter system may incorporate an amplif cation step. By way of example, the transcriptional activator released from membrane association activates transcription of a gene encoding another transcription factor, preferably one comprising a strong transcriptional activation domain and a different DNA binding specificity. This second transcription factor then activates transcription of a reporter gene such as those described above. The effect of an inhibitory agent on memnrane association can, this way, ne amplified into a larger response from a reporter system. The invention also relates to DNA constructs wnich enanle the heterologous protein comprising a reporter sequence and a recognition sequence for cell membrane association optionally together .ith tne reporter system to be introduced/transfected into tne host cell. The DNA constructs contain sequences to allow maintenance of the constructs as episomai plasmids, or to enable integration of the constructs into tr.e genome of the nost ceil. The constructs will, n general , also ne snuttle vectors wnicn can ne propagated Ξ_ cell to allc^ tne generation of sufficient quantities cf material. In a further aspect of tne invention at least two distinct fusion protein/reporter gene systems are introduced into the same ceil . This allows simultaneous screening for the ability of compounds to modulate either or both of the relevant processes . In such a poiyfunctional assay, distinct reporter genes and promoters are conveniently used. The method of the invention is particularly applicable to lipid-modifled proteins which are anchored to intracellular membranes and to the inner face of the plasmamembrane. It therefore encompasses such modifications as palmitoylation, isoprenylation and N-myristoylation nut is not directly applicable to, for example, giycolipid modifications wnicn ancnor proteins to the extracellular face of the plasmamembrane. N-myπstoylation is the cotranslational addition of myπstate to the ammo terminal glycine residue of selected proteins (3) . These proteins include the a subunit of some heterotπmeric G proteins, cAMP-dependent protein kmase, src, myristoyiated protein -tmase C substrate ( ARCKS) protein and numerous retroviral coat proteins . The sequence motif which confers N-myristoylaticn is localised to the first ten ammo acids of the protein. It has neen demonstrated that an N terminal peptide from p60src can direct myristoylation and plasmamembrane association when fused to heterologous proteins (6) . Considerable conservation of the N-myristoylation machinery has occurred evolution, such that the numan N-myπstoyi transferase, which shares 44% homology with the S . cerevis ae enzyme, will complement a deficiency in the yeast enzyme Isoprenylation may ne conveniently subdivided into three types (reviewed m ref. 4) (1) Farnesylation. This is the attachment of farnesyl (15 rarncn cnam) groups to cysteme residues near the carboxy terminus o: irotems . The target seσuence for farnesylation is CAAX (wnere C s cysteme, A is an aliphatic ammo acid, and X, the C-term nai residue, is any ammo acid except ieucme) . Following attacnment of the farnesyl group to the cysteme residue of this sequence, target proteins are further modified by a proteoiytic cleavage wnich removes the last three residues, and methylation of the new carboxy terminus. Additionally, palmitoylation of cyste es close to the farnesylated cysteme occurs m some proteins. Farnesylated proteins include g subunits of some heterotr meπc G proteins, ras proteins, and the nuclear larnms. The farnesyl group is donated by farnesyl pyrophosphate, an intermediate on the sterol biosynthetic pathway. The enzymology of farnesylation and its associated modifications appears to have been conserved m eukaryotes . These processes occur m both mammalian cells and m the budding yeast S_;_ cerevisiae. Of particular interest are the ras proteins. Recently, the sequence requirements for stable plasmamembrane association of ras proteins have been determined (5) . In addition to the carboxy terminal tetrapeptide which signals farnesylation, an adjacent sequence is required. In c-Ha-ras, this adjacent sequence includes a cysteme which becomes palmitoylated subsequent to the initial farnesylation. For c-Ki-ras, the adjacent sequence contains a set of positively cnarged ammo acids which, though they do not necome modified, are necessary for membrane association. It has neen demonstrated that it is possible to fuse the last eleven ammo acids of a ras protein to a cytosolic protein and confer membrane association upon that protein (5) . Thus this region of ras proteins defines a signal for membrane association. (2) Geranylgeranyiation at CAAL sequences. The addition of geranyigeranyl (C-20 chain; groups to the cysteme residues of proteins terminating m CAAX, where X is ieucme, s very similar to farnesylation. As with farnesylation, proteoiytic cleavage and metnyiation steps follow tne initial modification. The enzyme wnicn performs this type of geranylgeranyiation is geranyigeranyl transferase I. This enzyme contains a sununit whicn s common to farnesvi transferase. Proteins wnicn undergo tn s modification mciuαe several ras-related G proteins sucn as racl, rac2 and ralA an d the g summits of some neterotr meric G proteins. The similarity of th s form of geranylgeranyiation to farnesylation suggests that the carnoxy terminal regions of these proteins will, like the comparable regions of ras proteins, constitute distinct membrane association signals capable of tolerating fusion to heterologous proteins. (3) Geranylgeranyiation at CC and CAC sequences. Geranylgeranyiation of cysteme residues in these carboxy terminal sequences is mediated by a different enzyme from that above. Furthermore, the signal for geranylgeranyiation appears to be distributed throughout the target protein (see ref. 24) . Thus, for tnis particular form of isoprenylation, tnere may not be a distinct memnrane association sequence wnich can be transferred to a neterologous protein. The method of the present invention is conveniently used to f nd inhibitors of famesylation-dependent and geranylgeranylation-dependent membrane association. In addition to direct mhinition of these processes, it will be apparent from Figure 4 that lnninitors of sterol biosynthetic pathway, such as inhibitors of HMG-CoA reductase, w ll inhibit these membrane association processes by depleting the substrates used m these reactions. Inhibitors of sterol biosynthesis have application m the management of cholesterol levels. Inhibitors of farnesylation are potential antitumour agents against tumours containing an oncogenic form of a ras gene and may have application m modulating signalling pathways involving farnesylated Gg proteins . Inhibitors of geranylgeranyiation -lay nave application modulating signalling pathways mediated by geranvigeranylated Gg proteins Inhibitors of myristoylation have potential application as modulators of signal transduction pathways dependent upon a myristoyiated Gα protein, as inhibitors of src-mediated cell transformation and as inhibitors of retroviral coat assemnly Inhibitors of memnrane association processes may also be ~"tcto:: c aσents In another aspect of tne present invention we provide a method for identifying memnrane association signals We have s.io n that membrane association can be used to confer a simple phenotype upon a cell. This phenotype can ne used to screen libraries of peptide sequences to find those which function as memnrane association signals. In another aspect of the invention, we provide a met od to generate mutations m memnrane association processes. Mutagenesis of a ceil containing the heterologous protein/reporter gene system may generate mutants m which the fusion protein can no longer be localised to the membrane. These mutants may be distinguished ny the cnange cell phenotype consequent upon reporter gene activation. Compounds whicn modulate protem/cell memnrane association can act as reporter gene mducers. In a further feature of the invention we use this to provide cells wnere inhibitors of protem/cell membrane association are used to switch on genes of interest. Therefore a further aspect of the invention we provide a cell, navmg (1) a cell membrane, (11) a heterologous protein comprising a transcription activator and a recognition sequence for cell memnrane association, (m) a promoter/gene transcription system wnich is acted upon by the transcription activator such that gene transcription is activated by a compound which inhibits association of the neterologous protein and the ceil memnrane. In this aspect of the invention the gene which is transcπned is conveniently a gene which is used for some other purpose other than to monitor the membrane attachment of the activator. For example the gene may encooe a protein whose production in n gh quantities is desirable nut the constant expression of wnicn _s detrimental to the cell. Multicelluiar organisms sucn as plants or animals may be engineered to contain cells containing tne components of tr. s switch. Application of the inducing compound can then ne used to induce expression of a αesireα gene m some or ail of the cells cf tne crσanism. The applications cf sucn a system are varied and include for example controlling the fertility and yield of crop plants ano, m animal cells, inducing the expression of heterologous proteins cr increasing the levels of endogenously expressed proteins. Additionally, if the expressed gene encodes for example a toxin gene, the system can ne used to anlate certain cells at a desired time. The -se of a tissue-specific promoter, or some other form of regulated promoter to drive expression of the fusion protein enanles the mducible gene regulatory system to be established m a desired subset of cells. The invention will now be illustrated but not limited by reference to the following Figures and Examples wherein: Figure 1 illustrates the method cf the invention. The dounle solid lines denated by A represent a membrane. The protein denoted by B consists of; a DNA binding domain (open oval) , a transcription activation domain (shaded oval) and a membrane association signal (open box) . The zigzag denoted by C represents lipid moieties anchoring the protein to the membrane. The reporter gene is represented by D and E. D denotes binding sites for the transciptional activator within the promoter of the gene. Ξ denotes tne coding region of the reporter gene. Situation I represents the situation the tester cell m tne ansence of any inhibitors of membrane association. The activator s lipid-modifled and sequestered m a memnrane. The reporter gene is therefore inactive since no activator is hound to he binding sites m tne promoter. Situation II represents the situation m the presence of an inhibitor of lipid modification. In the ansence of lipid modifications tne activator is no longer membrane-associated and is free to migrate to the reporter gene and aind to the promoter of that gene This results activation of reporter gene transcription, d eno t ed cv tne _arger arrow The figure snows now inhibitors cf -^emnrane association whicn act directly cr indirectly upon np α modification may be detected. This system may be able to detect agents wnich inhibit memnrane association by some mechanism other t h an inhibiting lipid modification; for example, if membrane association of a lipid-modifled protein requires a specific interaction netween t h at protein and a component of the target membrane, the inhibition of that interaction may be detectable. -Ficruge 3 illustrates the ctruoturo of the activators and reporter gcnea uaod. Figure 2A shows the structure of the GAL4-based activators Galll2FS and Galll2NF. The DNA binding domain consists of ammo acids 1-147 of GA 4 (ref. 17) . The activation domain consists of am o acids 1-108 of the activation domain B112 (ref. 15) . The C-termmal ten ammo acid residues of Galll2FS are identical to the C-termmal ten ammo acid residues of the S. cerevisiae RAS2 protein (ref. 18) . The C-termmal ten am o acid residues of Galll2NF are identical to those of Galll2FS except for the exchange of an alanme for a cysteme four ammo acids from the carboxy terminus. The primary structure of each activator consists of 268 ammo acids. In the yeast expression vectors YCpGalll2FS and YCpGalll2NF the expression of each activator is driven by the S_^_ cerevisiae ADH1 promoter. In the mammalian expression vectors pECE-FS and PECE-NF the expression of eacn activator is driven by the SV40 promoter/enhancer. Figure 2B shows the schematic structure of the reporter genes used in yeast and mammalian cells. Both are based on the E.coli lacZ gene which encodes the enzyme β-galactosidase. The yeast reporter gene GALl-lacZ is integrated into the S cerevisiae genome. The promoter is the S. cerevisiae GAL1 promoter which contains four binding sites for GAL4 idenoted by shaded boxes) . The mammalian reporter gene G5SGal is supplied as a plasmid by transfecticn. The promoter is a synthetic promoter consisting of the human β-glonm minimal promoter (open ncx) and 5 consensus GAL4 binding sites The arrows above eacn construct indicate tne start cf transcription. Figure 3 onowo a grann of the QOOG reoponαe to iovαatαcin of Figure 3A shows a dose response cf reporter gene activity in YT6: :171 containing YCpGalll2FS measured using ONPG as the substrate tne β-galactosidase assay. Yeast cultures were incubated overnight m tne presence of various concentrations of lovastatm. Growth was measured as the increase in optical density at 595nm over an 18 hour period m a microtitre plate. Reporter gene activity is the rate of increase in optical density at 420nm after addition of reaction buffer OD change in 120 minutes) to the cultures. The figure snows that at intermediate concentrations of lovastatm, reporter gene transcription s induced. Figure 3B shows a dose response of reporter gene activity m YT6: :171 containing YCpGalll2FS measured using CPRG as the substrate tne β-galactosidase assay. Activity is given as the change m absornance at 570nm over a period of 1 hour. Each point is the average of 20 readings,- the error bars are standard deviations. The nasal level of OD570 change n the ansence of lovastatm was 0.25 -mits Figure 4 outlines the biochemical pathway involved m sterol oiosynthesis eukaryotic cells. The substrates for geranylgeranyiation and farnesylation of proteins are derived from the intermediate farnesyl pyrophosphate. Hence inhibition of this pathway at the level of HMG-CoA reductase by agents such as lovastatm can lea d to inhibition of protein modification. This can suppress the effects of activating mutations ras (see ref. 16 ) Figure 5 shows the effect of lovastatm on reporter gene ac t ivi t y in COS-1 cells. Reporter gene activity was measured by tne CPRG-based β-gaiactosidase assay. Lovastatm (1 uM) is anbreviated as In. ^ se t cf transfect cns was peroformed using various comnmaticns cf t ne repor t er piasmid pG5βGal (labelled as G5 ) , the activator c ^ aspids oECΞ-FS (labelled as FS) and pECΞ-NF ( laneiled as NF ) and lovastatm (labelled as Ln) . Lovatstat was added at lμM to tne indicated transfections . Each transfection was performed m duplicate; the shaded parts of the nars represent the averages cf these duplicates and the unshaded parts of the bars represent the standard deviations. The vertical axis represents the change in OD 570 over 1.5 hours. The reaction was linear during this period, so these values are directly proportional to enzyme activity. The efficiency of the tranfections, as measured by a growth hormone assay, was roughtly equal. Figure 6 shows a Western blot of COS-1 cells transfected with activator constructs . The lanes are as follows : 1. lOul high molecular weight rainbow markers (from Amersham: molecular weights 14,300-200,000) . 2. 25μg total protein extract from COS-1 cells transfected with pECE-FS plus pRSVhGH. 3. 25μg total protein extract from COS-1 cells transfected with pECE-FS plus pRSVhGH and incubated m the presence of lμM Lovastatm. 4. 25ug total protein extract from COS-1 cells transfected with pECE-NF plus pRSVhGH. 5. 25ug total protein extract from COS-1 cells transfected with pECE-NF plus pRSVhGH and grown m the presence of lμM Lovastatm. 6. 25μg total protein extract from COS-1 cells transfected with pRSVhGH alone. 25ug total protein extract from COS-1 cells transfected with pRSVhGH alone and incubated the presence of lμM Lovastatm. Ξ. lD α .1 low molecular weight rainbow markers (from Amersnam: molecular weights 2,350-46,000) . Example 1 Inhibition of membrane association of a transcriptional activator in the yeast S_;_ cerevisiae by the H G-CoA reductase inhibitor, lovastatin: - In this example we show (I) that fusion of a membrane association signal to a transcriptional activator causes that protein to become inactive, presumably through lipid modification and consequent membrane association and (ii) that inhibition of this presumed lipid modification renders the protein active again. The yeast S_ cerevisiae was used as a host organism. We useo the E_;_ coli β-galactosidase gene under control of the S. cerevisiae GAL1 promoter as a reporter gene. The GAL1 promoter contains binding sites for the transcriptional activator GAL4. Since GAL4 has been deleted from this strain, the reporter gene is inactive. We transformed plasmids expressing ch maeric GAL4-based activators into this strain. We found that fusion of a membrane-association signal from a ras protein to the carboxy terminus of the activator resulted inhibition of reporter gene activity, presumably due to farnesylation and membrane association of the activator. This inhibition could be reversed by the application of lovastatin, an HMG-CoA reductase inhibitor, which would be expected to inhibit farnesylation by inhibition of the sterol biosynthetic pathway. Materials and methods ϊ'east strains and plasmids : The host S_ j . cerevisiae strain used was YT6 : :171 (11) , whicn contains a GALl-lacZ reporter gene integrated at the URA3 locus . The p asmid YCpGalll2FS, wnich encodes a fusion protein consisting of the DNA binding domain of GAL4 (ammo acids 1-147) , the activation domain ΞL1Z and the last ten ammo acids cf the S_ cerevisiae RAS2 protein, ,as constructed as follows - The Hmo I -EcoR I fragment frcm pSG424 (12) encoding GAL (1-147) was ligated into the Hmd III and ΞcoR I sites of pBluescπpt SK+ (Stratagene) to create pSK147. The polylmker of pSK147 was modified by replacing the sequence between the Pst I and Not I sites with the following annealed oligonucleotides: GCCCGGGGTCGACACTAGTTAACTAG and ACGACGGGCCCCAGCTGTGATCAATTGATCGCCGG. This created the plasmid PSK147BX. The H d III-Not I fragment of this plasmid was ligated into the Hmd III-Not I fragment of the plasmid pADNS (13) to create pADGall47. The polylmker of the yeast single copy vector pRS315 (14) was replaced by cleaving the plasmid with Xho I and Sac I and inserting the following annealed oligonucleotides : TCGACGGATCCGAGCT and GCCTAGGC. Into the Bam HI site of the resultant plasmid, pRS315XS, the Bam HI fragment of pADGall47 was inserted such that the SacI site is 5' to the ADH1 promoter and the Kpn I site is 3 ' to the ADH1 terminator, creating the plasmid YCpl5ADH147. A 320bp Eco Rl fragment of plasmid pB112 (15) containing amino acids 1-108 of the acidic activation region B112 was ligated into the Eco RI site of pSK147 m the orientation maintaining the integrity of the reading frame to create pBXG112. The H d III-Pst I fragment of pBXG112 was used to replace the Hmd III- Pst I fragment of YCpl5ADH147 to create YCpl5G112. YCpl5G112 is a centromeπc expression plasmid wnich expresses the activator GAL(1-147) -B112 from the yeast ADH1 promoter. YCpGalll2FS contains a memnrane association signal . This was created by inserting the following annealed oligonucleotides between the Pst I and Sal I sites: ATCTGGTTCTGGTGGTTGTTGTATTATTTCTTAAG and ACGTTAGACCAAGACCACCAACAACATAATAAAGAATTCAGCT. YCpGalll2NF contains a mutated membrane association signal created by inserting the following annealed oligonucleotides between the Pst I and Sal I sites : ATCTGGTTCTGGTGGTTGTGCTATTATTTCTTAAG and ACGTTAGACCAAGACCACCAACACGATAATAAAGAATTCAGCT. Plasmids YCpl5G112, YCpGalll2FS and YCpGalll2NF were transformed nto YT6::17l by the lithium acetate method (19) Some of the clones obtained wnicn contained YCpGalll2FS exhibited instability. when they were restreaked and transferred to X-Gal indicator plates, these unstanle clones gave rise to some blue colonies . Other clones were apparently stanle since thev σave rise to oniv wnite colonies. One of tne clones that gave only white colonies was picked and restreaked twice more to confirm tnat it was stable This clone, designated clone 19, was used β-galactosidase assays. β-galactosidase assay: Yeast cells containing plasmids were grown m synthetic complete media lacking Ieucme. For reporter gene assays, stationary cultures were diluted 1:50 and grown overnight in 200μl aliσuots m 96 well microtitre dishes, β-galactosidase activity was measured by a modification of the method of Ausabel et al (25) . Equal volumes of lOxZ buffer and 4mg/ml ONPG (o-Nitrophenyl-β-D-Galactopyranoside, from Sigma) m 1M phoshate pH7 were mixed. SDS was added to a final concentration of 0.1%. 50μl of this cocktail was added to each well of the microtitre plate and the rate of appearance of product measured at 420nm using a platereader. The optical density at 595nm was taken as a measure of growth. Similar experiments were performed using lOmM CPRG (chloropnenolred-β-D-galactopyranoside, from Boehr ger Mannheim) m place of 4mg/ml ONPG. For this substrate, changes m optical density /ere measured at 570nm. Results and Discussion: YCpGalll2FS expresses the protein shown n Fig. 2. This protein consists of a DNA binding domain which will bind to GAL4 sites, an activation domain, and the last ten residues of the yeast AS2 protein (ΞGSGGCCIIS) By analogy with the experiments cf HancocK et a_ (5) , this carboxy terminal sequence from RAΞ2 should confer farnesylation, palmitoylation and membrane association upon the protein. In YT6: .17l cells transformed with this construct there was a very low level of reporter gene activity, less than 1% of the activity onserveo witn tne parental activator Galll2 (15) . Evidently tne fusion cf tne carnoxv terminal sequence of RAS2 inactivates tne GAL4 fusion, presumably ny causing it to necome attached to a memnrane so that it is unable to activate transcription of the reporter gene. Inhibition of HMG-CoA reductase leads to inhibition of the sterol biosynthetic pathway and thus to the inhibition of formation of farnesyl pyrophosphate, the intermediate of this pathway used by farnesyl transferase. Application of lovastat to mammalian cells leads to an accumulation of nonisoprenyiated substrate proteins (20) . Furthermore, lovastatm will revert the pnenotype of activated ras mutations n Xenopus oocytes (16) and will rescue lethal yeast mutations dependent upon isoprenylation for their activity (10) We reasoned that if the inactivity of Galll2FS was due to farnesylation-dependent membrane association, then addition of lovastatm should lead to inhibition of farnesylation and memnrane association of Galll2FS and consequently to activation of the reporter gene (see Figure 4) . Yeast cultures were incubated overnight m the presence of lovastatm (sodium salt, from Merck and Co.) . We observed two effects of lovastatin upon YT6::17l containing YCpGalll2FS (see Figure 3A) . Firstly, the lovastatm inhibited growth at concentrations of around lmM. This growth inhibition is attributanle to cytotoxicity resulting from inhibition of sterol biosynthesis. Secondly, at lower concentrations of lovastatm, the predicted induction of reporter gene activity was onserved. The optimum level for reporter gene stimulation was about O.lmM. The maximal level of induction of reporter gene was roughly equivalent to the level observed with the parental activator Galll2. Lovastatin had no effect on the reporter gene m the parental strain YT6: :171; the reporter gene was inactive at all lovastatm concentrations tested. Thus lovastatm reverses the effect of fusion of the carboxy terminus of RAS2 to a transcriptional activator. We presume that this is cue to inhibition of the sterol biosynthetic pathway and consequent depletion of isoprenyl substrates required for farnesylation. A more precise dose-response cf reporter gene activation YT6 1"1 containing YCpl5Galil2FS to lovastatm concentrations ir. tr.e range lOOnM to lOOuM was onta eo using tne CPRG form cf the -galactosιdase assay. This dose-response is diplayed m Figure 3B. A continuous reproducible increase m reporter gene induction s observed as the lovastatin concentration is increased. To obtain further evidence that the RAS2-derived sequence m Galll2FS is acting as a farnesylation signal, we also constructed a vector which expresses the activator Galll2NF. Galll2NF is identical to Galll2FS except that the cysteme four residues from the carboxy terminus has been replaced w th an alanme. This alteration to the carboxy terminal sequence should destroy the farnesylation signal. Unlike Galll2FS, we found that this activator was as strong as the parental activator Galll . Increasing concentrations of lovastat cid not induce reporter gene expression activity, but rather caused tne level of reporter gene activity to decrease in a manner that closely paralleled the inhibition of growth. These results suggest that, as expected. Galll2NF is neither farnesylated nor membrane associated. Conclusions : We have shown that fusion of a farnesylation-dependent membrane association signal to a transcriptional activator results m inhibition of the ability of this protein to activate transcription. We have shown that lovastatin can be used to restore the activating anility of this protein. Thus we have used lovastat to induce expression of a reporter gene. We believe that the mechanism of th s induction is via inhibition of farnesylation-dependent membrane association of a chimaeπc transcriptional activator. The S . cerevisiae yeast strain described containing the activator Galll2FS readily allows a simple assay for compounds capable of inhibiting farnesylation and farnesylation-dependent membrane association. Using tne fi-gaiactosidase assay described, this assay is amenable to use m nign throughput screens to identify sucn compounds, though we advise tnat if such compounds are also cytotoxic, they may not be detected by sucn a screen Example 2 Inhibition of membrane association of a transcriptional activator in COS-1 cells by the HMG-CoA reductase inhibitor lovastatin: - In this example we show that, in a transient transfection in mammalian cells, (i) fusion of a membrane association signal to a transcriptional activator causes that protein to become inactive, (ii) that this chimaeric transcriptional activator displays a mobility in gels consistent with lipid modification and (iii) that application of the HMG-CoA reductase inhibitor lovastatin restores the correct mobility of the protein and restores its ability to activate transcription. COS-l cells were used as an experimental system. Mammalian cells do not contain any DNA binding proteins with the specificity of the S . cerevisiae protein GAL4. However, GAL4 derivatives which function as transcriptional activators in S . cerevisiae will function in mammalian cells if GAL4 binding sites are placed near the start site of gene transcription (21) . To study the function in mammalian cells of the chimaeric activators generated in Example 1, we transferred the genes encoding Galll2FS and Galll2NF into mammalian expression vectors. These constructs were cotransfected into COS-l cells with a reporter gene consisting of a promoter containing five GAL4 binding sites driving transcription of a β-galactosidase reporter gene. Materials and methods: Plasmids The mammalian expression vector pECE72 was created by cloning the insert of pECE (26) as a Pvu II- Bam HI fragment into the backbone c: the pUC-based vector pSP72 (Promega) cleaved with PvuII and Bgl II. The vector pECΞ72 contains the SV40 late region enhancer/promoter followed by a poiylinker and the ΞV40 poly(A) addition site. DNA cloned into the polylmκer of this plasmid is expressed at high levels _n mammalian cells, particularly cells such as the monkey line COS-l wnicn amplify the plasmid by replication. Plasmid pECE-FS was CDtamed by cloning the 0.6kb Hmd Ill-Spe I fragment of YCpl5G112FS into pECE72 cleaved with Hmd III and Xba I. pECE-FS expresses the chimaeric transcription factor Galll2FS from the SV40 promoter/enhancer. Plasmid pECE-NF was obtained by cloning the 0.6kb H d III- Spe I fragment of YCpl5G112NF into pECE72 cleaved with H d III and Xba I. pECE-NF expresses the chimaeric transcriptional activator Galll2NF from the SV40 promoter/enhancer. The reporter gene plasmid pG5βGal was constructed m the following way. A 0.lkb DNA fragment containing 5 consensus GAL4 binding sites was obtained by PCR from the CAT-based reporter plasmid pG5EC (27) using the following oligonucleotides: CCATGCTTAAGCGCCAAGC and ATACCCTCTAGAGTCGAC This fragment was cleaved with Afl II and Xba I and cloned into the plasmid pVIP-GAL cleaved with Spe I and Afl II. pVIP-GAL. pVIP-GAL (M. Needham/Zeneca Pharmaceuticals) was derived from pGSE1417 (28) and pSVGal (Pharmacia) and is a mammalian expression plasmid containing the E_ ; _ coli β-galactosidase gene driven by a minimal β-globm promoter. The GAL4 sites were inserted immediately upstream of this promoter to make pGSGal. Cell culture and transfection COΞ-1 cells were maintained DMEM plus 10% foetal calf serum supplemented witn 50 μg / ml penicillin/streptomycin and 2 mM -giutamme. Calcium chloride transfections were performed essentially as desribed (25) For each individual transfection, tne 6 procedure was as follows One day prior to transrection 1.5x10 ceils were seeded into 75 cm " flas and incubated overnight at 37 C. One hour prior to transfection fresh media was added. Calcium chloride precipitates were made with 60μg per plate of the test plasmids together with 15μg of pRSVhGH (M. Needham/ Zeneca Pharmaceuticals) as a control for levels of transfection. The precipitate was added to the cells and cells incubated for seven hours at 37 C, followed by a 60 second 15% glycerol shock. Fresn media was added and the cells were allowed to recover for 24 hours . After 24 hours fresh media was added to the flasks. Some flasks also received lμM Lovastatin (Merc and Co.) . After a further 24 hours the cells were trypsmised, washed twice m phosphate buffered saline and the pellet finally resuspended m 300ul of water. For β-galactosidase assays, 1.25x10 cells were diluted to 200ul and dispensed into a microtitre well. 50μl of 5xZ nuffer (see Example 1) and CPRG (to a final concentration of ImM) and SDS (to a final concentration of 0.1% were added to start the reaction. The rate of reaction was measured as the rate of change m OD570. For Western blots, cell extracts were prepared by freeze-thawmg the transfected cells three times. Protein concentration estimated using a protein assay kit (Pierce) . Growth hormone assays were performed using a Hybritech kit. Aliqouts of cell extract were electrophoresed througn 10% polyacrylamide/SDS gels and electrcnlotted onto nitrocellulose filters . GAL4 derivatives were detected using a 1:500 dilution of GAL4 polyclonal antibody (M. Ptashne, Harvard) and visualised using 1:1000 dilution of a goat anti-rabbit alkaline phosphatase antibody, detected using a NBT/BCIP k t (from Eiorad) . Results We first examined the transcriptional activity cf the proteins Galll2FS and Galll2NF m mammalian cells. COΞ-1 cells were cotransfected with the reporter gene plasmid pG5βGal and the expression constructs encoding Galll2FS and Galll2NF. The transfectε: ceils were incubated m tne presence cr ansence of lovastatin. After 24 ncurs tne cells were assaved for s-σaiactcsicase activity. Tne results are displayed m Figure 5. COS-l cells contain an endogenous 3-σalactosιdase activity in the ansence of the reporter gene (lanes 1 and 2) . Transfection of the reporter gene increases this activity 1.5-2 fold (lanes 3 and 4) , snowing that the reporter gene plasmid pGSβGal is transcribed at a basal level m the absence of any GAL4-based activators. Lovastatm does not increase this basal level; instead it reduces it slightly. Cotransfection of the plasmid expressing Galll2NF with the reporter gene resulted m a high level of reporter gene activity (lane 5) . This is consistent with the results obtained in Example 1, where Galll2NF was demonstrated to be a strong transcriptional activator. This activator is predicted to be ncn-lipid modified and, consistent with this, lovastatm does not affect its activity (lane 6) . Cotransfection of the plasmid containing Galll2FS with the reporter gene resulted m a small increase m reporter gene activity compared to reporter gene alone (compare lanes 3 and 7) . However, incubation of this transfection with lovastatm resulted m a substantial induction of reporter gene activity (compare lanes 7 and 8) . Subtracting respectively the basal levels due to reporter gene alone (lanes 3 and 4) , this amounts to an induction of the activity of Galll2FS by lovastatin of 5.8 fold. Farnesylation and palmitoylation of ras proteins leads to an increase in their electrophoretic mobility m SDS-polyacrylamide gels (ref. 29) . We used Western blotting to analyse the mobility of Galll2NF and Galll2FS in the presence and absence of lovastatin. Plasmids pECE-NF and pECE-FS were transfected into COS-l cells. After incubation m tne presence or absence of lovastatm, protein was prepared from the transfected cells and electrophoresed through an SDS-polyacrylamide gel . This was blotted onto a nitrocellulose membrane and probed with antι-GAL4 antibodies. Figure 6 shows the resulting Western blot. Lanes 6 and 7 are control lanes which snow the result of mocK transfecticns . No proteins are detected cy Gal4 antibodies m either lane. Lanes 4 and 5 show Galll2NF with and without lovastat . GaillCFS and Galll2NF are predicted to be about 31kdal; a band of approxima t e size 33kdal is detected lanes 4 and 5. Lovastatin has no effec t cn tne monility cf this nand, implying that Galll2NF s not modified by any intermediate on the sterol biosyntnetic pathway. By contrast, lanes 2 and 3 show Galll2FS in the ansence and presence of lovastatm respectively. Galll2FS migrates ahead of Galll2NF, despite containing the same number of ammo acids m its primary structure The large mobility difference is consistent with lipid modification (29) . In the presence of lovastatm, Galll2FS migrates with the same mobility as Galll2NF. These results imply that Galll2FS is modified by an intermediate on the sterol biosynthetic pathway, and that lovastatm inhibits this modification. Discussion In this example we show that the activator Galll2FS behaves m the same fashion m a mammalian cell line (COS-l) as in S cerevisiae. Galll2FS has a low activity which can be induced by lovastatin. The control activator Galll2NF is strongly active and its activity is unaffected by lovastatin, again as observed m S. cerevisiae. The mobilities of the proteins as examined by Western blotting are consistent with Galll2FS being lipid-modifled. These results demonstrate that activation of reporter gene expression correlates with restoration of normal electropnoretic mobility With reference to tne wor of Hancock et: al . (5) we conclude that activation of reporter gene expression results from inhibition of farnesylation. In ras proteins, farnesylation is a requirement for subsequent palmitoylation, proteoiytic cleavage and methylation. Therefore the inhibition of farnesylation would result m complete inhibition of lipid modif cation. These modifications are required for memnrane association of ras proteins. A chimaeric protein m which these modifications are inhibited should not be associated with a memnrane. Therefore tnese results imply that the lovastatm-induced activation of reporter gene expression we observe m noth mammalian (COS-l) and yeast tS cerevisiae) cells is due to the production of a chimaeric activator //nich is not memnrane associated and is therefore anle to nmd to and activate a reporter gene. We note tr.at tne man nacκσround of 1-σaiactcsιoase activity CCS-1 ceils and the transient nature of the assay described would not favour tne use of mammalian ceils as an assay system to detect novel inhibitors of memnrane association processes. However, it would be possible to construct stanle cell lines containing integrated copies cf both Galll2FS and a GAL4-responsive reporter gene. Such a cell line could be conveniently used high througnput screening. References : 1. Chow et al. , (1992) Curr. Opinion Cell Biol. 61, 355-386. 2. Gibbs (1991) , Cell 65, 1-4. Towler et_ al . , (1988) Ann. Rev. Biochem. 57, 69-99. Clarke, (1992) Ann. Rev. Biochem. 61, 355-386. Hancock et al. , (1991) EMBO J. 10, 4033-4039. Pellman et al. , (1985) Nature 314, 374-377. Duronio et al . , (1992) Proc. Natl. Acad. Sci. USA 89, 4129-4133. Brown et al. , (1992) Proc. Natl. Acad. Sci. USA 89, 8313-8316. Resh and Ling, (1990) Nature 346, 84-86. 10. F egoid et. al . , (1990) Science 249, 165-169. 11. Himmeifarb et al . , (1990) Cell 63, 1299-1309. 12. Sadowski and Ptashne, (1989) Nuc. Acids Res. 17, 7539. 13. Coliceili et al . , (1989) Proc. Natl. Acad. Sci. USA. 86, 3599-3603. 14. Ξikorski and Hieter, (1989) Genetics 122, 19-27. 15. Ruden et al . , (1991) Nature 350, 250-252. 16. Schafer et al. , (1989) Science 380, 379-385. 17. Laughon and Gesteland (1984) Mol. Cell Biol. 4, 260-267. 18. Powers et al. , (1984) Cell 36, 607-612. 19. Ito et al. , (1983) J. Bactenol. 153, 163-168. 20. Repko and Maltese (1989) J. Biol. Chem. 264, 9945-9 21. Ptashne (1988) Nature 335, 683-689. 22. Lenardc and Baltimore (1989) Cell 58, 227-229. 23. Harrison (1991) Nature 353, 715-719. Seabra et al . , (1992) Cell 70, 1049-1057. :. Ausacei et <________, (1990) Current Protocols Molecular Biology . " ileyj ;. Ξll o al. , (1986) Cell 45, "21-731. 27. Sadowski et al . , (1992) Gene 118, 13"-141. 28. Needham et al . , (1992) Nuc. Acids Res. 20, 997-1003 29. Hancock et al . , (1989) Cell 57, 1167-1177. SEQUENCE LISTING GENERAL INFORMATION (i) APPLICANT: ZENECA Limited (ii) TITLE OF INVENTION: PEPTIDES (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Group Patents Services Department (B) STREET: Bessemer Road (C) CITY: Welwyn Garden City (D) STATE: Hertfordshire (E) COUNTRY: United Kingdom (F) ZIP: GB-AL7 1HD (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: dISKETTE, 3.5 INCH, 1.44 Mb storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS 3.20 (D) SOFTWARE: ASCII from WPS-PLUS (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER (B) FILING DATE: :vii) PRIOR APPLICATION DATA: (A) APPLICATION NO. 9226065.2 (B) FILING DATE: 14-Dec-1992 (2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GCCCGGGGTC GACACTAGTT AACTAG 26 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:2 : ACGACGGGCC CCAGCTGTGA TCAATTGATC GCCGG 35 \ 2 ) INFORMATION FOR SEQ ID NO:3 : ii) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TCGACGGATC CGAGCT 16 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : ^TCTGGTTCT GGTGGTTGTT GTATTATTTC TTAAG 35 (2) INFORMATION FOR SEQ ID NO:5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : ACGTTAGACC AAGACCACCA ACAACATAAT AAAGAATTCA GCT 43 (2) INFORMATION FOR SEQ ID NO: 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : ACGTTAGACC AAGACCACCA ACACGATAAT AAAGAATTCA GCT 43 ,2) INFORMATION FOR SEQ ID NO: 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : ATCTGGTTCT GGTGGTTGTG CTATTATTTC TTAAG 35 (2) INFORMATION FOR SEQ ID NO:8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (x ) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : CCATGCTTAA GCGCCAAGC 19 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : ATACCCTCTA GAGTCGAC 18

Claims: Claims : 1. A method for identifying compounds wnicn modulate protem/cell memnrane association wnicn method comprises contacting a test compound with a ceil, navmg (1) a cell memnrane, (n) a heterologous protein comprising a reporter sequence and a recognition sequence for cell membrane association, (m) a reporter system which is acted upon by the reporter sequence such that tnere is a measurable cnange m cell pnenotype upon modulation of protem/cell membrane association by the test compound, and detecting any change m cell pnenotype . 2 A method as claimed n claim 1 wherein the recognition sequence for membrane association is a signal for farnesylation, geranylgeranyiation or myristoylation, including endopeptidase cleavage, carboxy terminus methylation or palmitoylation; or any combination thereof. 3 Use of the method as claimed n claim 1 for the identif cation of inhibitors of farnesylation-, geranylgeranyiation-, or myristoylation dependent events. 4 Use of the method as claimed claim 1 for the identification of inhibitors of lipid modification. 5 Use of the method as claimed in claim 1 for the identification of inhibitors of lipid biosynthesis. 6 Use of the method as claimed m claim 1 for the identif cation of inninitors of memnrane association of ras proteins se of the method as claimed m claim 1 to screen a collection cf chemical compounds, natural products and/or nroths for nimtors cf protem/cell memnrane association. Ξ -. heterologous protein comprising a reporter seσuence ano a recognition sequence for cell memnrane association. 9. A protein as claimed in claim 8 wherein the reporter sequence comprises a transcriptional activator. 10. A protein as claimed m claim 8 or claim 9 wherein the recognition sequence for membrane association is a signal for farnesylation, geranylgeranyiation or myristoylation,- including endopeptidase cleavage, carboxy terminus methylation or palmitoylation; or any combination thereof. 11. A DNA construct for expression of a protein as claimed n any one of claims 8-10. 12. A vector comprising a DNA construct as claimed claim 11. 13. A cell having (i) a cell membrane, (ii) a heterologous protein as claimed in any one of claims 8-10 and (m) a reporter system which is acted upon by the reporter sequence such that there is a measurable change in cell phenotype upon modulation of protem/cell memnrane association by a test compound. 14. A cell as claimed in claim 13 when dependent on claim 9 wherein the reporter system comprises a promoter/gene transcription system which is acted upon by the transcription activator such that gene transcription is activated by a compound which inhibits association of the heterologous protein and the cell membrane. 15. A polyfunctional cell having a cell membrane and at least two heterologous protein/reporter systems as stated m claim 13 or claim 14. 16. A cell, having d) a cell memnrane, (ii) a heterologous protein co m prising a transcription activator and a recognition seσuence for cell memnrane association, ,111) a promoter/gene transcription system wnicn is acted upon n tne transcription activator such that gene transcription s activated by a compound nich inhibits association of the heterologous protein and the cell -emnrane. 1". A method for inducing gene expression which method comprises applying a known inhibitor of cell memnrane association to a cell as claimed m claim 16. 13. A method for the identification of recognition sequences for cell membrane association which method comprises constructing a population of cells, eacn having d) a cell membrane, (ii) a neterologous protein comprising a reporter sequence and a putative recognition sequence for cell membrane association, (m) a reporter system which is acted upon by the reporter sequence such that there is a measuranle change m cell phenotype upon modulation of protem/cell memnrane association by the test compound, and detecting those cells vnere no change n cell phenotype has occurred. 19. A method for identifying a mutant cell defective n protem/cell membrane association which method comprises treating one cr more cells as claimed in any one of claims 13-16 with one or more "utagenic agents and detecting a cell wnich is mutated by reference to a cnange m cell phenotype. 23. A reporter system for use m the method of claim 1 which includes a heterologous protein comprising a transcriptional activator and a recognition sequence for cell membrane association, and wherein m tne absence of cell membrane association the transcriptional activator activates transcription of a gene encoding a further transcriptional activator, which further transcriptional activator activates transcription of a reporter gene, expression of whicn leads to a measurable change m cell phenotype. 21 Yeast strain Saccnaromyces cerevisiae YT6: :171 comprising tne plasmid YCol5Galll2FS

Inventors: BROAD PETER MICHAEL, BROAD, PETER, MICHAEL
Applicants: BROAD PETER MICHAEL, ZENECA LTD, BROAD, PETER, MICHAEL, ZENECA LIMITED
Application year: 1993
Main classification: C12N15
IPC codes: C07K14, C07K19, C12N1, C12N15, G01N33