LETTERS TO NATURE
`
`
`
`14. Adamezewski, M., Kohler, G. & Lamers, M. ©. Eur. J, Immun, 24, 617-626 (1991).
`15. Lu, ¥.J., Johnsen, G. B., Gordon, J. & MacLennan,|. C. M. Immun. Today 13, 17-21 (1992).
`16. Maeda, K. ef ai. J.
`Immun, 248, 2340-2347 (1992),
`i7. Heyman, 8., Tianmin, |. & Gustavsson, 5, Eur, J. immun, 23, 1739-1742 (1993),
`18. Lebrun, P. & Spiegelberg, H. L. J. immun. 139, 1459-1465 (1987)
`19. Kopf, M, et al, Nature 362, 245-247 (1993).
`20. Keegan, A. D., Snapper, C.M., Van Dusen, RL, Paul, W.E, & Conrad, BD. H. J Immun, 142,
`3868-3874 (1989).
`21. Luo, H., Hofstetter, H., Banchereau, J, & Delespesse, G. J. Immriun, £48, 2122-2129 (1991),
`ACKNOWLEDGEMENTS. We thank H. Mittelstaat, E. Stratling, L Egersdortt, C. Westphal and
`A. Fehrenbach for technical assistance; B. Ledermann for the 66 ES ce!
`line; L, Nitschke and
`22. Sherr, E.. Macy. E.. Kimata, H., Gilly, M, & Saxon, AJ Immun, 142, 481-489 (1989)
`M. Kopf for help with gene targeting; D. Conrad for FeerZa cDNA; and F. McConnell, B. Imhot,
`23. Saxon, A.. Kurbe-Leamer, M., Beble, K.. Max, EE. & Zhang. A.J. Immun. 147, 4000-4006.
`{1992}.
`P. Shaw, R. Carsetti, M. Simon and D. H, Katz for critical reading of the manuscript. MLR. isa
`24, Street. N. E. et al. J. Immun. 144, 1629-1639 (1990).
`recipient of a Special Fellow Award from the Leukemia Society of America, This work was
`25. Campbell, K. A, Lees, A, Finkelman, F.D. & Conrad,
`BD. H. Eur.
`supported in part by @ grant from the NIH to D. H. Katz. The Basel Institute for Immunology
`(1992).
`was founded and is supported by Hoffman-La Roche, Basel, Switzerland,
`
`
`J.
`
`immun, 22, 2107-27112
`
`26. Sinclair, N. R. StC. Immun. Today 4, 35-36 (1983).
`27. Nitschke, L, Kopf, M. & Lamers, M. C. BioTechniques 14, 914-916 (1993)
`28. Ayane, M. P., Neilsen. P. & Kohler, G. Nuciew Acids Res. 17, 6722 (1990).
`29. Rao, M.. Lee, WT. & Conrad, 0. HJ. Immun. 138, 1845-1851 (19871
`
`A mammalian protein targeted
`by Gi-arresting rapamycin-
`receptor complex
`Eric J. Brown, Mark W. Albers, Tae Bum Shin,
`Kazuo Ichikawa, Curtis T. Keith, William S. Lane*
`& Stuart L. Schreiber?
`
`complexes interfere with distinct signalling pathways'*. FKBP12—
`rapamy'cin inhibits progression through theGl phase of the cell
`cycle in osteosarcoma’, liver’ and T cells*” as well as in yeast®,
`and interferes with mitogenic signalling pathways that are involved
`in G1 progression’"'", namely with activation of the protein p70°™"
`(refs 5, 11-13) and cyclin-dependentkinases’'*'*. Here weisolate
`a mammalian FKBP-rapamycin-associated protein (FRAP) whose
`binding to structural variants of rapamycin complexed to FKBP12
`correlates with the ability of these ligands to inhibit cell-cycle
`progression. Peptide sequences from purified bovine FRAP were
`used to isolate a human cDNA clonethat is highly related to the
`Department of Chemistry, Howard Hughes Medical Institute and
`DRRI/TORI and DRR2/TOR2gene products from Saccharo-
`“Harvard Microchemistry Facility, Harvard University,
`12 Oxford Street, Cambridge, Massachusetts 02138, USA
`myces cerevisiae™'”"*, Although it has not been previously demon-
`strated that either of the DRR/TOR gene products can bind the
`*To whom correspondence should be addressed
`FKBP-rapamycin complex directly'™'’,
`these yeast genes have
`been genetically linked to a rapamycin-sensitive pathway and are
`thought to encodelipid kinases'”=",
`
`THE structurally related natural products rapamycin and FK506
`bind to the sameintracellular receptor, FKBP12, yet the resulting
`we
`ae ve
`gk ae ok
`SSKKS
`
`@
`
`M, (K)
`
`oma
`158 —>
`
`16
`66 >
`
`_—_
`
`FRAP
`
`500 nM GFK-ligand
`5% gel
`
`b
`
`RBL
`
`Jurkat
`
`Pt bade
`AAAfe
`s
`&
`
`Ir
`M.(K)
`
`200 —>
`
`=
`
`—
`
`<— FRAP
`
`5% gel
`
`116 —>
`
`a 3
`
`45—
`3
`
`—
`
`enqpaeer <—cre
`=sss=
`
`500 nM GFK-ligand
`12.5% gel
`
`7
`66 >
`45
`<— cK
`31>Leeeeeeeee]
`E
`<—FRAP
`S8sengagaes
`12.5%gel
`14—>
`==—=—=——_ ==
`ra
`200 — .
`2.5 uM GFK-ligand
`,
`5% gel
`16 —>
`
`FIG, 1 Identification of FRAP protein in three mammalian cell lines. a,
`GFK alone or individual GFK-ligand complexes were added to MG-63
`cell lysates (2 x 10” cells per condition)to a final concentration of either
`500 nM or 2.5 pM and the mixtures incubated for 10 minat4 C. Fusion
`protein complexes were recovered by glutathione-affinity chromato-
`graphy, and the proteins detected by silver staining after 5% SOS—
`PAGE. Because of compression, FRAP is not resolved by 12.5% SDS-
`PAGE, so both 5%and 12.5%gels are shown. The amount of FRAP that
`was retained by affinity chromatography saturated at concentrations of
`GFK-Rap greater than 500 nM in these experiments and in others using
`concentrations of GFK-Rap ranging fram 100 nM to SuM (data not
`shown). b, GFK alone or individual GFK-ligand complexes were added
`to a final concentration of 500 nM to lysates prepared from either
`2 10° Jurkat T lymphocytes or 10° rat basophilic leukaemia (RBL)cells
`per condition. Lysates were treated as in a. FABP12, but not FABP13 or
`FKBP25 (ref. 23) is able to mediate the actions of rapamycin in S.
`756
`
`cerevisiae. In addition, we found that YFK188 (ref. 24), an FABP12 null
`strain, could be complemented with GFK (P. K. Martin, B. Gladstone,
`G. Weiss, D. T. Hung, S.L.S., in preparation). Thus the GST appendage
`of the fusion protein does not preclude binding of the biologically rel-
`evant target to the GFK-rapamycin complex in yeast.
`METHODS. MG-63,Jurkat and RBLcells were grown in media containing
`10% FBS and lysed at 4 °C in PINT buffer (150 mM NaCl, 50 mM Tris-
`HCl, pH 7.5. 2mM EDTA, 2mM EGTA, 25 mM NaF, 100 pM NasVO.,
`25 mM 2-glycerophosphate, 0.2 mM PMSF, 1 ug mi-* leupeptin, 1 pe
`mi
`* pepstatin A and 2 mM DTT) containing 0.5%Triton X-100. Lysates
`wereclarified by centrifugation at 25,000g, and the Triton X-100 in the
`supernatant was diluted to 0.33% by adding 0.5 vol PINT buffer. GFK
`prebound to stoichiometric quantities of FA506, keto- iso- or unmodified
`fapamycin was added to lysates as described. Each condition was then
`passed through a 250-u! glutathione-Sepharose column, which was
`washed with PINT buffer containing 0.5 M NaCl and 0.3%Triton X-100,
`NATURE - VOL 369 - 30 JUNE 1994
`
`© 1994 Nature Publishing Group
`
`West-Ward Pharm.
`Exhibit 1006
`Page 001
`
`West-Ward Pharm.
`Exhibit 1006
`Page 001
`
`

`

`LETTERS TO NATURE
`
`M,(K)
`
`eos
`
` “
`
`50
`SRNEETRAKA
`MLGTGPAAAT
`AKELQHYVTM
`TAATTSSNVS VLQQFASGLE
`100
`qT
`ASLIGVEGGN
`KGGILAT
`$1
`ELREMSQEES
`150
`TAEYVEFEVK
`GRLAMAGDTF
`lol
`ATRIGRFANY
`LRNLLPSNDP VVMEMASKAT
`200
`FONIFVAVWD
`NEGRRHAAVL VLRELAISVP
`RALEWLGADR
`is.
`TFFFQQVQPF
`250
`AEKGFDETLA
`aoL
`PRQATREGAV
`PQWYRHTFEE
`AALRACLILT TQREPKEMQK
`300
`RLREEMEEIT
`asl
`KEKGMNRDOR
`QOOLVHDKYC
`IHGALLILNE LVRISSMEGE
`350
`GLMGFGTSPS
`&|
`301
`SNAL
`VGLLGYSSHQ
`4090
`TILNLLPRLA
`351
`CCROLMEEKF
`PAKSTLVESR
`RNSKNSLIQM
`DQVCGAVLKC
`a5
`SVAVRSEFKY
`401
`LSCVKKEKER
`AFRPSAFTOT
`TAAFQALGLL
`QYLQDTHNHY
`500
`TVFTCISMLA
`YLPRVLODIIR
`451
`AALPPKDFAH
`RAMGPGIQOD
`KRQKAMQV DA
`550600
`KMLSLVLMHK
`501
`VGLSPALTAV
`IKELLEPMLA
`LKKDIQDGLL
`LYOLSRQIPQ
`SFEFEGHSLT
`SITLALRTLG
`TTLPEASDVG
`551
`PLRHPGMPKG
`LABQLASPGL
`650
`TPSIHLISGH
`EAARTCSRLL
`601
`LNSEHKEIRM
`AHVVSQTAVQ
`QFVRHCADHF
`700
`TRYCVLASLO
`651
`VWGITDPOPD
`WVADVLSKLL
`ENLQALFVAL
`ERFOAHLAQA
`750
`AICTVGRLSS
`LEHSGIGRIK
`MNPAFVMPFL
`TOL
`RKMLIQILTE
`NOQVFEIREL
`600
`YMEPILKALI
`VSNAPRLIRP
`NPGVINNVLA
`LKLKOPDPOP
`751
`EQSARMLGHL
`850
`gor
`LEMRKWYDEL
`FITIMOMLQD
`SSLLAKROVA
`LWTLGQLVAS
`TIGELAQVSG
`900
`e521
`RREATRVLGL
`LGALOPYKHK
`TGYVVEPYAK
`YPTLLEVLLN
`FLETEQNQGT
`850
`901
`TSEMLVNMGN
`LPLDEFYPAV
`
`YNIGMIDOSRDASAYSLSESKSSQDSSOYS
`1000
`951
`SMVALMRIFR
`SLGLKCVQFL
`POVMPTFLNY
`DQSLSHHHTMH VVQALTFIFK
`116 —>
`1050
`1001
`MDELVTLMRE
`IRVCDGATRE
`FAWMNTSIQS
`FLFQQLGMLY SFVKSHIRPY
`1100
`1051
`VFMHONSPGR
`IVSIKLLAAT
`VALGGEFRLY LPQLIPHMLR
`TIILLIEQIV
`1150
`1101
`LHLLLPPIVK LFOAPEAPLP
`SRKAALETVD
`‘QLFGANLDDY
`RLTESLDETD
`1200
`FIPMYNKVLV
`1151
`VFQLGKKYOI
`YASRIIHPIV
`RILDOSPELR STAMDTLSSL
`FIG. 2 Purification of FRAP from bovine brain and cDNA cloning of
`1250
`1201
`RHRINHQRYD
`WLICRIVKGYTLADEEEOPLTYOHBMLRSGOGDALASGPY
`1300
`1251
`LEWLARLSLE LLKDSSSPSL
`human FRAP. a, Fivefold-enriched bovine FRAP (S-column eluate; see
`ETGPMKKLHYSTINLOKAWG AARRVSKDOW
`1350
`WPMARDLFNA
`1301
`AFVSCWSELN
`RSCWALAQAY
`EDQQDELIRS TELALTSQDI
`1400
`below) was conditioned with [100 nM), glutathione-S-transferase-
`PLPLRDONGI
`13521
`AEFMEHSDKG
`VLLGERAAKC RAYAKALHYK
`AEVTQTLLENL
`1450
`1401
`FKBP12 fusion protein (GFK), GFK-FK506 or GFA-Rap. Complexes with
`AILESLISIN
`GVLEYAMKHE GELETOATWY
`NELQQPEAAA
`ELEFOKGPTP
`i500
`1452
`VAYOKKMOTN
`KODPELMLGR:
`EKLHEWEDAL
`fusion proteins were recovered by glutathione-affinity chromatography
`MRCLEALGEW GOLNQQCCERK
`1501
`1550
`EYTCMIPROT HDGAFYRAVL
`KMARMAAAAA
`WGLGQWDSME
`WTLVNDETQA
`and detected as described in Fig 1 legend. We also found FRAP in
`1600
`1551
`LOAELTAMAG
`ESYSRAYGAM VSCHMLSELE
`ALHQDLFSLA
`QOCTOMARDL
`16590
`1601
`bovineliver and thymus.b, Predicted translational product of the human
`EVIQYKLWPE
`RRETIRQIWW
`ERLQGCORLYEDWOKILMVR SLYVSPHEDM
`GKSGRLALAH
`i700
`
`KILVLLLGYDPSROLDHPLP21651TWHPQVTYAY
`
`FRAP cDNA clone. Bovine FRAP peptide sequences aligned to human
`RIWLEYASLE
`i750
`1701
`MKNMIKSARK
`TDAFQHMQHF
`VQTNQQQAQH
`ATATEDQQHK
`QELHKLMARC
`FRAPare indicated by underlined segments. in the reading frame shown
`HAWAVMNEE
`1e00
`1751
`EHORSWYKAW
`FLKLGEWQLN
`LOGINESTIP KVLQYYSAAT
`18590
`TASTEGSNSE
`translational stop codons were not encountered upstream ofthe initiat-
`1901
`RDEKKKLRHA SGANITNATT
`AATTAATATT
`VLHYKHQNOA
`19900
`1851
`STSLSRGNNL
`SEAESTENSP
`TVPAVQGFFR
`TPSPLQKKYI_EDLSKTLLMY
`ing methionine, c, Northern biot analysis of human tissue, Jurkat T cell
`19590
`1901
`FOYGHAPOVN EALVEGVKAT
`QIDTWLQVIP
`OQLIARIDTPR
`QOTLRVLTLW
`and MG-63 cell poly (A)' RNA. The Jurkat/MG-63 and multiple tissue
`2000
`1951
`ANKILKNMCE
`KSTTTARHNA
`PLVGRLTHOL
`LTDIGRYHPQ ALIYPLTVAS
`2001
`2050
`MVSEELIRVA ILWHEMWHEG
`LEEASRLYFG ERNVKGMFEV
`Northern blots (Clontech) were hybridized with **P-labelled probes
`HSNTLVQQAM
`2100
`2051
`LEPLHAMMER
`AQEWCRKYMK SGNVKOLTQA
`GPQTLKETSE NOAYGROLME
`derived from the 182 bp PCR fragment and the 5.5 kb clone (text),
`21590
`2101
`WOLYYRVFRR
`LMCRDLELAYPGTYDPNOPT
`ISKQLPQLTS LELQYVSPKL
`respectively, Hybridization to human fj-actin probe is shown as an inter-
`2200
`2151
`EFVFLLEGHE DLRQOERVMQ
`ORPR KLTLMGSNGH
`2250
`LFGLVNTLLA
`2202
`nal control for loading.
`STNSGLIGWY PHCOTLHALT
`NDPTSLRKNLSIORYAVIPL
`2300
`2251
`LNIEHRIMLR MAPDYOHLTL
`RDYREKKKIL
`MOKWEWEEHAVANTAGDDLA
`METHODS. Bovine FRAP waspurified by grinding 900 g of bovine brain
`2350
`2301
`GYILGLGORH PSNLMLORLS
`EVWFDRRTNY TRSLAVMSHV
`BLLWLKSPSS
`in blender with 1 litre of PIP (0.3% Triton X-100, 50 mM sodium phos-
`2400
`2351
`RMLTNAMEVT GLOGNYRITC
`GKILHIDFGD
`CFEVAMTREK FPEKIPFRLT
`2450
`2401
`phate, pH 7.2, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 100 uM Nas VO,
`HTVMEVLREH
`KDSVMAVLEA FVYDPLLWWR
`LMDTNTKGNK BSRTBTDSYS
`2500
`2451
`LGEPAHKK
`p
`KAIQL
`25 mM 2-glycerophosphate, 1mM PMSF, 1g mi! leupeptin, 1 ug
`2501
`2549
`INRVRDOKLTG
`ROFSHDOTLO VPTQVELLIA
`QATSHENLCQ CYLGWCPFW
`mi! pepstatin A, 1 mM benzamidine and 2 mM DTT). The homogenate
`was centrifuged at 25,000g and the supernatant(20gtotal
`protein) was loaded onto a 1 litre S-Sepharose (Pharmacia)
`column, The column was then washed with PIP and eluted
`with PINP (PIP with 1M NaCl). GFA-rapamycin was added
`to the pooled eluate to a final concentration of 100 nM
`and recovered by glutathione-affinity chromatography. FRAP
`was resolved by SDS-PAGE and transferred to PVDF, Fol-
`lowing digestion with trypsin or endoproteinase Lys-C
`(Boehringer Mannheim)
`bFRAP peptides were micro-
`sequenced". The Jurkat T cell cDNAlibrary (Stratagene)
`was constructed through random and oligo dT priming a
`of cytoplasmic oligo dT purified RNA (ref. 25). cDNA
`ala
`screening, Jurkat and MG-63 RNA isolation and northern
`blotting and were performed by procedures similar to those
`previously described**. A 182 bp fragment was amplified
`from a human brain stem library (Stratagene) and labelled
`by incorporation of *°P-dCTPin the courseof reamplification
`by PCR. The sequences were analysed using BLAST(ref.
`26) and the University of Wisconsin GCG (ref. 27) software.
`The human FRAP cDNA sequence has been submitted to
`Genbank.
`
`| o5kb— bee
`75 kb—
`gS
`
`|
`
`|
`
`¢
`
`& aeea
`24kh— %
`
`TSE tec 1.95thee
`
`75kb—
`=
`sak |
`
`2akbo—
`1.35 ko
`
`‘a
`
`aa kb
`
`24k
`
`We used two structural variants of rapamycin, 16-keto-
`rapamycin (S. D, Meyer and §.L.S., manuscript in preparation)
`and 25,26-iso-rapamycin*', to identify any biologically relevant
`targets of the FKBP-rapamycin complex. Both variants bind
`tightly to human FKBP12, as shown by their ability to inhibit
`rolamase activity of the recombinant protein (A; values were
`0.2nM for rapamycin’, 2nM for keto-rapamycin, and 0.1 nM
`for iso-rapamycin). But the variants are about two orders of
`magnitudeless potent than rapamycin in preventing the progres-
`sion through Gl of MG-63 human osteosarcoma cells. The
`values of IC.) (half-maximal inhibitory concentration) estimated
`from dose-response curves are 0.1 nM. 7.5 nM and 50 nM for
`rapamycin, keto- and iso-rapamycin, respectively. Thus the com-
`plexes of iso- and keto-rapamycin with FKBP1I2 should bind to
`NATURE + VOL 369 - 30 JUNE 1994
`
`less effectively than FKBP12-
`
`the FKBP12-rapamycin target
`rapamycinitself.
`A fusion protein of glutathione-S-transferase with FKBP12
`(GFK) was used to identify candidates for the biologically
`relevant targets of FKBP12 rapamycin. MG-63 cells were lysed
`by detergent and complexes of GFK- rapamycin, GFK-FK506
`or GFK alone were added individually to clarified lysate at a
`final concentration of 500 nM or 2.5 4M (Fig. la), A protein of
`approximate relative molecular mass 220,000 (A4,~220K) was
`detected in the GFK-rapamycin sample by SDS-PAGE and
`silver staining (Fig.
`la, lane 3). This FKBP-rapamycin-assoc-
`iated protein (FRAP) was not retained with GFK-FK506 or
`GFK alone(Fig. la, lanes | and 2). No other rapamycin-specific
`proteins were detected by silver staining (Fig. la) or by a similar
`757
`
`© 1994 Nature Publishing Group
`
`West-Ward Pharm.
`Exhibit 1006
`Page 002
`
`West-Ward Pharm.
`Exhibit 1006
`Page 002
`
`

`

`LETTERS TO NATURE
`
`
`
`
`affinity purification procedure using lysates from [*°S] methio-
`nine-labelled cells (data not shown). The GFK-ketorapamycin
`and GFK-isorapamycin complexes bound FRAP lesseffectively
`than GFK-rapamyein; at concentrations of 500 nM, the keto-
`and iso- complexes were unable to retain the 220K protein (Fig.
`la, lanes 4, 5), whereas at higher concentrations of the complexes
`(2.5 uM) detectable quantities of FRAP were retained (Fig. la,
`lanes 4,5). This is consistent with the finding that these com-
`pounds are still strong cell-cycle inhibitors, albeit less potent
`than rapamycin itself. Thus, the binding of GFK-ligand com-
`plexes to FRAP correlates with the ability of the ligands to
`impede G1 progression in MG-63cells. FRAP wasalso detected
`in Jurkat T-lymphocyte cells and rat basophilic leukaemiacells
`(Fig. 15), two mammalian cell
`lines that are also sensitive to
`rapamycin®**, No other rapamycin-specific bands were observed
`in cach case.
`FRAP purified from bovine brain (bFRAP) had a similar
`specificity for GFK-ligand (Fig. 2a), Microsequencing of
`bFRAP proteolytic fragments (298 amino acids in total, Fig.
`2h) led to the design of a pair of degenerate oligonucleotides for
`use in the polymerase chain reaction (PCR). A 182 bp PCR
`product allowed for the isolation of overlapping clones from a
`human Jurkat T cell AZAP I] cDNA library, yielding 7.6 kb of
`contiguous sequence. Using these cDNA sequences as probes, a
`band migrating at approximately 8.5 kilobases was detected by
`Northern blot analysis of oligo dT purified RNA isolated from
`a variety of humantissues and cell lines (Fig. 2c). The human
`cDNA sequence encodes an amino-acid open reading frame
`(ORF) and aligns with 99% identity to the bFRAP peptides
`(Fig. 25), As N-terminal peptide sequence from purified bovine
`FRAP wasnot obtained, the initiating methionine shown in Fig.
`26 is unconfirmed. The predicted molecular mass of this ORF
`(~300K) is greater than that inferred by the mobility of FRAP
`during SDS-PAGE (above).
`Human FRAP is highly related to the DRR//TORI and
`DRR2/TOR2 gene products. Overall
`it
`is 44% identical
`to
`DRR1/TORI and 46%identical to DRR2/TOR2. The region
`of greatest homology to DRR1I/TORI and DRR2/TOR2 lies
`in the C-terminal 660 amino acids of human FRAP (57% and
`59%identical, respectively). In addition, this region has homo-
`logy to several known phosphatidylinositol kinases (21% identity
`on average),
`including mammalian phosphatidylinositol
`3-
`kinase'”'* (PI3K), a yeast PISK VPS34 (refs. 17 and 18) and
`PIKI (ref. 20), These similarities indicate that FRAP mayalso
`have phosphatidylinositol kinase activity.
`Through the introduction of minute structural changes in
`rapamycin, this study implicates FRAP as a mediator of G1 cell
`cycle progression in mammalian cells, Identification of FRAP
`as the target of FKBP12-rapamycin together with the earlier
`demonstration of calcineurin as the target of FKBP12-FK506
`(ref. 2) addresses a fascinating aspect of immunophilin research,
`namely that the immunophilin FKBP12 can bind two distinct
`natural products and thereby gain the ability to bind twodistinct
`signalling molecules involved in cell cycle entry and progression.
`Further biochemical characterization ofthis unique mammalian
`protein should elucidate its role in propagating the mitogen-
`initiated signals that lead to the activation of p70*** and cyclin-
`Cdk complexes.
`
`Received 3 May; accepted 20 May 1994.
`1. Schreiber, S. L. Science 264, 283-287 (1991).
`2. Schreiber, S. L. Cel! 70, 365-368 (1992).
`3. Albers, M. W. et al. Ann. N.Y. Acad. Sei. 698, 54-62 (1993).
`4, Francavilla, A. et al. Hepatology 25, 871-877 (1992).
`5. Price, 0. J., Grove, J. R.. Calvo. V., Avruch, J. & Blerer, B. E. Science 287, 973-977 (1992).
`6. Bierer, 8. E et al. Proc, natn. Acad. Sci. U.S.A. 87, 9231-9235 (1990),
`7, Dumont, F. J, Staruch, M. J, Koprak, SL, Molino, M. R. & Sigal, N.H. J. Immun, 144,
`251-258 (1990)
`8 Heitman, J., Movva, N. R. & Hall, M. N. Science 283, 905-909 (1991),
`9. Lane, H. A, Fernandez, 4., Lamb, N, J. C. & Thomas, G. Nature 363, 170-172 (1993).
`10. Norbury ©. & Nurse, P. A. Rev. Biochem. 61, 441-470 (1992).
`41. Chung. J.. Kuo, C. 1, Crabtree, GR. & Blenis, J. Cell @9, 1227-1236 (1992).
`12. Kuo, C J. et al. Nature 368, 70-73 (1992).
`
`13. Calvo, V., Crews, C, M., Vik, T. A. & Bierer, B. Proc. natn. Acad. Sci, U.S.A, 89, 7571-7575
`(1992),
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`ACKNOWLEDGEMENTS. M.W.A. and E.1.6. have made an equal contribution to this research.
`We thank 5. J. Danishefsky and C. M. Hayward for isorapamycin: S. D. Meyer for preparing
`kelorap: A, F. Standgert
`for advice and assistance; P. A. Beal
`for critically reading the
`manuscript: Anjana Rao and Lewis Kunkel for helpful discussions; and R. Robinson, R. Chicz
`and V. Bailey for their assistance. This work was supported by a grant from the NIH (5.L.S.),
`MLW.A.
`iS a Howard Hughes Medica! Institute predoctoral fellow, EJ.8. ts an NIH predoctoral
`fellow, KI, is @ visiling scientist sponsored by Sankyo Co. Ltd. and 5.L.5. is an Investigator with
`the Howard Hughes Medical Institute.
`
`
`
`Functional dissection
`of the yeast Cyc8-Tup1
`transcriptional
`co-repressor complex
`
`Dimitris Tzamarias & Kevin Struhi*
`
`Department of Biological Chemistry and Molecular Pharmacology,
`240 Longwood Avenue, Harvard Medical School, Boston,
`Massachusetts 02115, USA
`
`DNA-BINDING repressor proteins mediate regulation of yeast genes
`bycell type (Mem1/a2 and al/a2), glucose (Migl) and oxygen
`(Rox!) (refs 1-4 respectively). An unusual feature of all these
`regulatory pathwaysis that transcriptional repression requires two
`physically associated proteins” that do not bind DNA Cyc&(Ssn6)
`and Tupl. The Cyc8-Tup1 complex has been proposed to be a co-
`repressorthat is recruited to target promoters by pathway-specific
`DNA-binding proteins®, but the specific functions of the individual
`proteins are unknown. Here we show that when it
`is bound
`upstream ofa functional promoter through the LexA DNA-binding
`domain, Tup] represses transcription in the absence of Cyc8. De-
`letion analysis indicates that Tupl contains at least two non-over-
`lapping transcriptional repression regions with minimal primary
`sequence similarity, and a separable Cyc8-interaction domain.
`These Tupl domains, which do not
`include the f-transducin
`motifs’, are necessary and partially sufficient for Top! function.
`We suggest that Tupl performs the repression function of the
`Cyc8-Tup! co-repressor complex, and that Cyc8 serves as a link
`with the pathway-specific DNA-binding proteins.
`It has been previously shown that Cyc8 can repress transcrip-
`tion in a Tup!-dependent manner when bound upstream of the
`intact CYC/ promoter through the heterologous LexA DNA-
`binding domain®. Similarly, a LexA-Tup! hybrid protein confers
`a 16-fold reduction of expression from a promoter containing
`four LexA operators upstream of the CYC/ promoter (Table
`1). LexA-Tup] and LexA-Cyc8 also repress expression of a
`his3 gene containing a single LexA operator upstream of the Tz
`TATAelement (Fig. 1a), suggesting that they can inhibit basal
`transcription, Surprisingly, LexA-Tup] retains almost its entire
`*To whom correspondence should be addressed.
`
`758
`
`© 1994 Nature Publishing Group
`
`NATURE + VOL 369 - 30 JUNE 1994
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`West-Ward Pharm.
`Exhibit 1006
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