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VOL. 8: 673-680 (1992)
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Saccharomyces cerevisiae Contains a Homolog of Human FKBP- 13, a Membrane-associated FK506/Rapamycin Binding Protein JUDITH A. PARTALEDIS, MARK A. FLEMING, MATTHEW W. HARDING AND VIVIAN BERLIN* Vertex Pharmaceuticals Inc., 40 Allston Street, Cambridge MA 02139-421 I , USA. FKB2 encodes a homolog of human FKBP-13, a membrane-associated binding protein for the immunosuppressantsFK506 and rapamycin. FKB2 is located on the right arm of chromosome IV and contains an open reading frame of 135 amino acids, of which the first 17 residues comprise a putative hydrophobic leader peptide. Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity) and to FKBP-12, the major cytosolic receptor for FK506.In the alignment of
FKBP-13 and FKBP-12 sequences, there are 28 invariant residues. Among these conserved residues are those that comprise the drug binding and peptidyl-prolyl cis-trans isomerase active site of FKBP-12. The phylogenetic conservation of the FKBP family suggests that the proteins are involved in a basic cellular function. KEY WORDS-Immunosuppressant drugs; membrane proteins; S. cerevisiae; chromosome IV. INTRODUCTION FK506 and rapamycin are potent immunosuppressive agents that block distinct signal transduction pathways required for T-cell activation. FK506 blocks an early, Ca2+-dependent step required for interleukin 2 transcription (Tocci et al., 1989; Bierer et al., 1990a; Mattila et al., 1990; Flannagan et al., 1991), whereas rapamycin blocks lymphokine-induced T cell proliferation (Dumont et al., 1990a, b). Both FK506 and rapamycin bind with high affinity to a family of receptor proteins (Fretz et al., 1991) of which FKBP12 (Mr=12 000) is the best characterized (Harding et al., 1989; Siekierka et al., 1989; Bierer et al., 1990b). Like FK506, cyclosporin A (CsA; Handschumacher et al., 1984) is a potent immunosuppressant that inhibits Ca2+-dependent signalling pathways (Emmel et al., 1989; Johansson and Moller, 1990; Lin et al., 1991) and binds to an abundant cytosolic protein, cyclophilin (Handschumacher et al., 1984). The FKBPs and cyclophilin proteins constitute a growing class of immunosuppressant-binding proteins (immunophilins), all of which have been shown to catalyse the cis-trans isomerization of peptidyl-prolyl amide bonds (Fischer et al., 1989; Takahashi et al., 1989; Siekierka et *Addressee for correspondence. 0749-503X/92/080673-08$09.00 01992 by John Wiley & Sons Ltd
al., 1989; Harding et al., 1989; Galat et al., 1992). FK506, rapamycin and CsA all inhibit the peptidyl prolyl cis-trans isomerase (PPIase) activity of their cognate receptors. However, inhibition of PPIase activity is insufficient for immunosuppression; rather the complex of an immunophilin with drug appears to be the active entity (Bierer et al., 1990a, b; Hultsch et al., 1991). Consistent with this notion is the recent finding that FKBP- 12 and cyclophilin, bound to FK506 and CsA, respectively, inhibit calcineurin, a Ca2+-dependent phosphatase whose role in T cell activation is being investigated (Liu et al., 1991). The immunophilins FKBP-12 and cyclophilin are ubiquitous, cytosolic proteins whose primary structure is conserved between species (Franc0 et al., 1991; Hasel et al., 1991; Galat et al., 1992). FKBP-12 and cyclophilin define two structurally unrelated families of proteins widely distributed in tissues and subcellular compartments. The phylogenetic conservation of the FKBPs and cyclophilins suggests they are involved in fundamental cellular processes; their PPIase activity implicates the immunophilins in protein folding (reviewed by Gething and Sambrook, 1992). However, with the exception of the ninaA gene of Drosophila melanogaster, which encodes a cyclophilin required for assembly of rhodopsin in photo-
674 receptor cells (Stammnes et al., 1991; Colley et al., 1991), the in vivo function and physiological substrates of proteins with PPIase activity remain to be established. No function has been ascribed to the cyclophilin or FKBP- 12 proteins of Saccharomyces cerevisiae, since single or multiple deletions of the genes encoding these proteins produce no marked cellular phenotype (Koltin et al., 1991; Heitman et al., 1991; Wiederrecht et al., 1991; Koser et al., 1991; J.A. Partaledis and V. Berlin, unpublished results). Recently, additional members of the FKBP family have been isolated from mammalian tissue on the basis of their affinity to FK506 or rapamycin (Fretz et al., 1991). The human gene encoding one of these specifies a 13-kDa protein, designated FKBP-13, which is 43% identical to FKBP-12 (Jin et al., 1991). Using an FK506 affinity matrix, we have isolated a 13-kDa protein from yeast that is the homolog of human FKBP-13. MATERIALS AND METHODS Strains and growth media
LZAPI library screening and in vivo excision were performed with Escherichia coli strain BB4: (Stratagene) LE392.23 [FlacPZAM1.5, proAB, TnlO (tet‘)]. Plasmid DNA was propagated in E. coli strain DH5a: F- $80dlacZAM15 A(lacZYA-argF)U169 endA 1 recA 1 hsdR 17(rK- mK+)deoR thi- 1 supE44 AgyrA96 relAl. The S. cerevisiae strain used for protein isolation was F37 (formerly BJ2168, gift of E.W. Jones): Mata leu2 trpl ura3-52 prbl-1122 pep4-3 prcl-407. Yeast genomic DNA was from strain VBY25: Mata ura3-52 leu2-3,112 trplAl lys2A201 his3A200 ade2 cyh2 canl. The yeast strains used for mapping the FKB2 gene were VB49-1B: Mata ura3 trplAl leu2-3,112 cyh2, VB88-4B: Mata ura3-52 lys2A201 his3A200 leu2-3,112 fkb2-l::LEU2, F133 (formerly 10208-6A, gift of G. R. Fink): Mata trp4 ade8 r a 3 leu2-3. YPD contained 1% yeast extract, 2% Bacto-peptone (Difco) and 2% glucose. Minimal medium contained 0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose, and was supplemented with the required amino acids (Sherman et al., 1986). Protein preparation and amino acid sequencing of FKBP-13
JUDITH A. PARTALEDIS ETAL..
fluoride (Sigma), 0.5 mM-diisopropyl fluorophosphate (Sigma). Cells were disrupted using a Bead Beater (Biospec Products), and the cell debris was pelleted twice at 4000 x g for 5 min. The supernatant was clarified by successive centrifugations at 47,000 x g for 30 min, 48,000 x g for 45 rnin and 100,OOO x g for 60 min. Cytosol (350 mg total protein) was passed over a 10 ml column of end-capped Affigel 10 resin (Biorad, Richmond, CA) and then over a 2 ml Affigel 10-FK506 column (0.5 mg FK506/ml resin) at 4°C. A 32-[4’-(4-aminophenyl)butyryl]-FK506derivative was coupled to Affigel 10 resin as described by Fretz and coworkers (1991). After extensive washing with phosphate buffered saline-0.2% Tween 20, proteins were batch eluted by overnight incubation with FK506 (1.0 mg in 5 ml). Eluted proteins were dialysed against 10 mM-Tris-HC1, pH 7.4, lyophilysed and resolved by electrophoresis in a 12% sodium dodecyl sulfate-polyacrylamide gel SDS-PAGE (Laemmli et al., 1970). After electrotransfer of proteins, a 15-kDa band was excised from Immobilon-P membrane (0.45 p pore size, Millipore, Bedford, MA) (Matsudaira et al., 1987), and was subjected to amino-terminal sequence analysis using an Applied Biosystems protein sequencer (Model 477A). Library screening and DNA sequencing The N-terminal amino acid sequence of yeast FKBP13 was used to generate probes to isolate FKB2 cDNAs. Degenerate oligonucleotides were used to synthesize two products, 105 bp and 63 bp in length, by the polymerase chain reaction (PCR). The 105 bp PCR product was synthesized using oligos 543:
5’-GG’ITCITTXTCIGAYTTXGAXATIGGTAT-3’ (I = inosine, X = purine, Y = pyrimidine) and 545: 5’-ACCIGTXTAXTGXACYTTXACYTTXATYYXTCAC-3’. The 63 bp PCR product was synthesized using oligos 546: 5’-CCIGTYGAXGAYTGYTTXATIAAXGCIATGCC-3’ and 545. Approximately 10 ng of yeast genomic DNA prepared from strain VBY25 was used as the template for the PCR reactions. PCR reactions were performed using an Eppendorf Microcycler E for 35 cyclTs consisting of 94°C for 1.5 min to denature, 42 C for 2.0 min to anneal and 72°C for 1.5 rnin to polymerize. A single cycle at 72°C for 10 rnin was used to com-
Strain F37 was grown to mid-log phase in YPD. Cells were harvested (35 g wet weight) and suspended in 50 mM-potassium phosphate, pH 7.2, 0.9% potassium chloride, 10 mM EDTA, 1 mM-phenylmethylsulfonyl plete extension of the chains.
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The PCR products, labeled with 32P, were used to screen a ?ZAP1 cDNA library (provided by Jeff Kuret, Cold Spring Harbor Laboratory). Approximately 100,OOO plaques were screened using methods described by Stratagene (La Jolla, CA). Three putative FKB2 cDNAs were excised from the hZAPI vector as pBluescript plasmids and were designated 213-2,Z13-5 and 213-7. All cDNA clones were characterized further by sequencing. Sequences of the cDNAs were used to generate a robe to isolate the FKB2 gene. The probe was a 520 bp y2P-labeled PCR product corresponding to a region of FKB2 extending from positions +41 to +561 (Figure 1). The 32P-PCR product, generated from genomic DNA, was used to screen a yeast genomic library in the vector YEp24 (Carlson and Botstein, 1982). The methods used for library screening were those of Berent and coworkers (1985). Eighteen plasmids were isolated, purified and their inserts mapped by restriction analysis. The plasmid Y 13-8A was further characterized by sequencing. FKB2 cDNA and genomic clones were sequenced using the dideoxy chain termination method with a modification by Hattori and Sakaki (1986) for double stranded DNA plasmids. All sequencing primers were either purchased from Stratagene (La Jolla, CA) or custom made on an Applied Biosystems oligonucleotide synthesizer (model 380A). All sequencing reactions were performed using reagents from the Sequenase DNA sequencing kit (United States Biochemical, Cleveland, OH). Computer analysis Nucleotide and amino acid sequences were analysed with the sequence analysis software package of the Genetics Computer Group, University of Wisconsin, Madison (version 6.1). The default parameters of the BESTFIT program were used for amino acid sequence comparisons. The GenBank (release no. 69) and the European Molecular Biology Laboratory (EMBL, release no. 28) nucleic acid data bases, and the National Biomedical Research Foundation (NBRF) protein (release no. 30) data base were searched with the DNASTAR Macintosh Molecular Biology Software program with GENEMAN (version 1.26). Genetic mapping To determine the chromosomal position of FKB2, a 32P-labeled, 520-bp PCR fragment (also used to isolate the FKB2 gene; see preceding section) was
hybridized to a chromoblot purchased from Clontech (lot no.17037) and to a set of bacteriophage h clones (gift of L. Riles and M. V. Olson) representing the entire yeast genome (Link and Olson, 1991). Linkage to known markers was determined by tetrad analysis. RESULTS AND DISCUSSION Isolation of the yeast gene encoding FKBP-I3 Using an FK506 affinity matrix, a 15-kDa protein was purified from yeast and the N-terminal amino acid sequence: GSLSDLEIGIIKRIPVEDCLKAMPWKVKV~ GxLB
was determined. Of these 39 residues, 17 (those underlined) were identical to those in the amino terminus of human FKBP-13 (Jin et al., 1991). Complementary DNA and genomic clones were isolated using probes specific for the amino terminal sequence of the 15-kDa yeast protein and for the cDNA, respectively. Both the cDNA and genomic clones contained an open reading frame in which amino acids 18 through 56 were identical to the N-terminal amino acid sequence of the purified 15-kDa FK506-binding protein of yeast. This observation confirmed that we had cloned the gene (FKB2) for a second m 5 0 6 binding protein in yeast. The protein encoded by FKB2 shares significant homology with human FKBP- 13 (see succeeding section). The complete nucleotide sequence of FKB2 and the deduced amino acid sequence of its product, FKBP13, are shown in Figure 1. FKB2 has a single open reading frame of 135 amino acids containing two consecutive in-frame AUGs which could function as initiation codons. The sequence context of the second AUG codon in FKB2 is more favorable than the first for translation initiation (Kozak, 1983; Hamilton et al., 1987). A comparison of the deduced amino acid sequence and the actual N-terminal amino acid sequence indicates FKBP- 13 contains a putative leader peptide of 17 amino acids with a predicted cleavage occurring between Ala-17 and Gly-18. The hydrophobic nature of the leader peptide suggests it may function as a signal sequence to direct FKBP-13 to the endoplasmic reticulum (ER). In fact, human FKPB-13 is enriched in the heavy membrane fraction of Jurkat T cells and has a hydrophobic leader peptide that can be cleaved in the presence of microsomal membranes (Jin et al., 1991). The mature form of yeast FKBP-13 beginning with Gly-18 consists of 118 amino acids and has a calculated M, of 12,447. This is 2.5 kDa smaller
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-250
CGTATATATT
-240
ACGTGTAAAAATTGATCTGATTACTGCATCGTTTCGTTTCTGGAAG~TAGCTAATATTCGTT
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ATGATGTTTAATATTTACCTTTTCGTCACTTTTTTTTCCACCATTCTTGCAGGTTCCCTG M
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TCAGGCACATATCTGCAACAAGATGCATGGAGTACGTATGTGGCAAACT~TTTTTATAG
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541
TGTGTATATTATGTTTGCAGCAAAATAAAATAAAAAGGGCTCTAACTACAATATATTAATA
601
TAAAGAAATGTTATTCAATCTAGTTTGGGTATGAATTTTTTCCCTCTTAAT~TAGT
661
TTAAGTAGTTTGTTCAGTCTTCTTATCGCTGGTAAACATTTTTTGGG~TTTGGTTTT~
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ACTATCAMLAWLATCCTGAAAAN~GTCTTCTTCAAAGGAATTGMTTGGTCGACG
S. CEREVISIAE CONTAINS A HOMOLOG OF HUMAN FKBP-13
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than the mass of FKBP-13 estimated from its mobility Table 1. Chromosomal position of FKBZ in SDS-PAGE. The basis of this discrepancy, which TETRAD TYPES also exists for human FKBP-13 (Jin et al., 1991), is LOCI PD NPD Ta LINKAGE(CM)b unknown. 33 28 ma3 ade8 25 0 The DNA sequence of FKB2 contains putative 13 0 44 39 recognition sites frequently found in the 5' and 3' ends ma3 fkb2-l::LEU2 45 >50 7 5 of yeast genes (Figure 1). Potential TATA sequences for adeSfkbZ-l::LEU2 transcription initiation (Struhl, 1987) are located at a PD is parental ditype tetrad, NPD is nonparental ditype tetrad, positions -72, -89 and -248. The sequences required for and T is tetratype tetrad. The values are the total number of tetrads mRNA 3' end formation in yeast, TAG ...TA(T) in each catagory. b Genetic distance is given in centimorgans (cM) and calculated GT...TIT(Zaret and Sherman, 1982) and " T A T A (Henikoff and Cohen, 1984) are found between according to the formula 100(T + 6NPD)/2(PD + NPD + T) positions +406 to +587 in FKB2. The eukaryotic (Perkins 1949). consensus sequence for polyadenylation, AATAAA (Proudfoot and Brownlee, 1976; Bimstiel et al., 1985), Homology of yeast FKBP-13 to human FKBP-I3 and usually absent in yeast genes, is found at position FKBP-12 +569 of FKB2. Comparison of the genomic and cDNA sequences of FKB2 indicates that poly(A) Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity and 66% similarity), yeast addition begins after the thymidine at position +621. FKBP-12 (52% amino acid identity and 67% similarity), human FKBP-12 (48% amino acid identity and 62% similarity) and Neurospora crassa FKBP-12 Chromosomal location of FKB2 (46% amino acid identity and 64% similarity). In the Hybridization of an FKB2 probe to a chromoblot and alignment of the FKBP proteins, there are 28 invarian ordered set of bacteriophage h clones representing ant residues and 3 1 positions in which at least four of the yeast genome indicated FKBZ is located on the five aligned residues are identical or are conservaChromosome IV or VII. The ambiguity of this prelim- tive changes (Figure 2). Yeast FKBP-13 also contains inary analysis was resolved by performing a set of the two cysteine residues present in human FKBP- 13 crosses. Since one of the two bacteriophage h clones (cys20 and cys75), which may form a disulfide bond to which FKB2 hybridized contained CYH2, on chro- (Jin et al., 1991); these cysteine residues are absent in mosome VII (L. Riles, personal communication), a FKBP-12. Among the residues conserved in all the cross was performed between strains VB49-1B and FKBPs are those that form a hydrophobic pocket in VB88-4B to examine linkage between FKBZ and FKBP-12 that bind FK506 and rapamycin or interact CYH2. This cross established that the two genes were with the drugs via hydrogen bonds (Van Duyne et unlinked, eliminating the possibility that FKB2 al., 1991a; Figure 2). The conservation of amino acids resided on chromosome VII. It is not clear whether that interact with FK506 and rapamycin suggests hybridization of the FKB2 probe to the chromosome these residues may be involved in binding to an VII clone was non-specific or was due to the presence endogenous ligand. of sequences sharing homology to FKB2. The location of FKB2 on chromosome IV was further refined FKBP-13 is a member of a family of related proteins by a cross between strains F133 and VB88-4B. Tetrad analysis indicated FKBZ was linked to rna3 and Like FKBP-12, FKBP-13 has PPIase activity that is unlinked to ade8 on chromosome IV, placing FKBZ to inhibited by rapamycin and FK506. The Ki values for the right of rna3 on the right arm of chromosome IV the two drugs determined with bovine FKBP-13 are (Table 1). 3.6 nM and 38 nM, respectively (M.W. Harding, J.A. Figure I . DNA sequence of the FKB2 chromosomal region and the deduced amino acid sequence of the FKBP-13 protein. Numbers in the left margin refer to the f i s t nucleotide in each line. Negative numbers indicate nucleotide positions upstream from the putative translation start site at +l. The single open reading frame is translated into predicted amino acids and the hydrophobic signal sequence (amino acids 1-17) is underlined. Amino acids 18-56 were confirmed by N-terminal amino acid sequencing. The putative TATA sequences at the 5' end of FKBZ are indicated in bold letters. Sequences located between positions +406 and +587 that are possibly involved in mRNA 3'-end formation include: (1) TAG ...TA(T)GT...TIT (Zaret and Sherman, 1982) found in the coding (underlined) and non-coding (underscored with broken lines) DNA strands (Imiger et al., 1991); ( 2 ) TTITTATA (Henikoff and Cohen, 1984), indicated by a line above the sequence; (3) AATAAA (Proudfoot and Brownlee, 1976; Bimstiel et al., 1985). which is underlined. Poly(A) addition occurs after the thymidine at position +621 shown in bold.
JUDITH A. PARTALEDIS ETAL.
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x x x x x NcFK12 HuFK12 ScFK12 ScFK13 HuFK13
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Figure 2. Alignment of FKBP-13 and FKBP-12 sequences. The alignment of amino acid sequences was performed by hand, based on multiple binary comparisons generated by BESTFIT. The sequences compared were: Neurospora crassa FKBP-12 (NcFK12; Tropscbug et al., 1990), human FKBP-12 (HuFK12; Standaert et a/., 1990; Maki er al., 1990). Saccharumyces cerevisiae FKBP-12 (ScFK12; Wiederecht et al., 1991; Koltin et al., 1991; Heitman et a/., 1991), S. cerevisiae FKBP-13 (ScFK13, this study), human FKBP-13 (HuFK13; Jin et al., 1991). Invariant residues are shadowed. X’s above the alignment denote positions in which at least four of the five residues are similar or identical. Under the alignment are shown the residues known to contact FK506 or rapamycin by hydrophobic interactions or hydrogen bonding (Michnick etal., 1991; Van Duyne et al., 1991a, b; Moore e t a / . , 1991).
Lippke, M. Fleming, 0. Futer, M.T. Decenzo, D.J. Livingston, D.A. Peattie, unpublished results). Recently two other FKBPs, FKBP-25 (Galat et al., 1992) and FKBP-52 (M. Benasutti, M.W. Harding, M.A. Fleming, M.T. Decenzo, J.A. Lippke, D.J. Livingston, D.A. Peattie, unpublished results), have been characterized, which also have PPIase activity inhibited by rapamycin and FK506. Many of the conserved residues highlighted in Figure 2 are present in FKBP-25 and FKBP-52. The potential ER targeting sequence in FKBP-13 (Jin et al., 1991 and this study) and potential nuclear targeting sequence in FKBP-25 (Galat et al., 1992) suggest the FKBPs are localized to different subcellular compartments. The role of the various FKBPs in mediating the action of FK506 and rapamycin has yet to be determined. With the cloning of the yeast genes for FKBP-12 (Wiederecht et al., 1991; Koltin et al., 1991; Heitman et al., 1991; J.A. Partaledis and V. Berlin, unpublished results) and now FKBP13, genetic analysis can be undertaken to elucidate the physiological role and to identify the natural ligands of the FKBPs. ACKNOWLEDGEMENTS The authors would like to thank Jeff Kuret for the yeast cDNA library in AZAPI. We would also like to thank David Armistead for synthesis of 32-[4’-(4aminophenyl)butyryl]-FK506 and Arlene Semerjian and David Livingston for critical reading of the manuscript.
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Dumont, F.J., Staruch, M.J., Koprak, S.L., Melino, M.R. and Sigal, N.H. (1990b). Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK506 and rapamycin. J . Immunol. 144, 25 1-258. Emmel, E.A., Venveij, C.L., Durand, D.B., Higgins, K.I.M., Lacy, E., and Crabree, G.R. (1989). Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science 246, 1617-1620. Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. and Schmid, F.X. (1989). Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337,476-478. Flannagan, W.M., Corthesy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352, 803-807. Franco, L., Jimenez, A., Demolder, J., Molemans, F., Fiers, W. and Contreras, R. (1991). The nucleotide sequence of a third cyclophilin-homologous gene from Saccharomyces cerevisiae. Yeast 7,971-979. Fretz, H., Albers, M.W., Galat, A., Standaert, R.F., Lane, W.S., Burakoff, S.J., Bierer, B.E. and Schreiber, S.L. ( 1991). Rapamycin and FK506 binding proteins (immunophilins). J . Am. Chem. SOC.113,1409-141 1. Galat, A., Lane, W.S., Standaert, R.F. and Schreiber, S.L. ( 1992). A rapamycin-selective 25-kDa immunophilin. Biochemistry 31,2427-2434. Gething, M.J. and Sambrook, J. (1992). Protein folding in the cell. Nature 355,3345. Hamilton, R., Watanabe, C.K. and de Boer, A. (1987). Compilation and comparison of the sequence context around the AUG start codons in Saccharomyces cerevisiae mRNAs. Nucl. Acids Res. 15, 3581-3593. Handschumacher,R.E., Harding, M.W., Rice, J., Drugge, R.J. and Speicher, D.W. (1984). Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226,544-546. Harding, M.W., Galat, A,, Uehling, D.E. and Schreiber, S.L. (1989). A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature (London) 341,758-760. Hasel, K.W., Glass, J.R., Godbout, M. and Sutcliffe, G. (1991 ). An endoplasmic reticulum-specific cyclophilin. Mol. Cell. Biol. 11,3484-3491. Hattori, Y. and Sakaki, Y. (1986). Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152,232-238. Heitman, J., Movva, N.R., Hiestand, P.C. and Hall, M.N. (1991). FK506-binding protein proline rotamase is a target for the immunosuppressive agent FK506 in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 1948-1 952.
679 Henikoff, S. and Cohen, E. H. (1984). Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1515-1 520. Hultsch, T., Albers, M.W., Schreiber, S.L. and Hohman, R. J. (1991). Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proc, Natl. Acad. Sci. USA 88, 6229-6233. Imiger, S., Egli, C.M. and Braus, G.H. (1991). Different classes of polyadenylation sites in the yeast Saccharomycescerevisiae.Mol.Cell. Biol. 11,3060-3069. Jin, Y.J., Albers, M.W., Lane, W.S., Bierer, B.E., Schreiber, S.L. and Burakoff, S.J. (1991). Molecular cloning of a membrane-associated human FK506- and rapamycinbinding protein, FKBP-13. Proc. Natl. Acad. Sci. CJSA. 88,6677-668 1. Johansson, A. and Moller, E. (1990). Evidence that the immunosuppressive effects of FK506 and cyclosporine are identical. Transplantation 50, 1001-1007. Koltin, Y., Faucette, L., Bergsma, D.J., Levy, M.A., Cafferkey, R., Koser, P.L., Johnson, R.K. and Livi, G.P. (1991). Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein. Mol. Cell. Biol. 11, 1718-1723. Koser, P.L., Bergsma, D.J., Cafferkey, R., Eng, W.-K., Mclaughlin, M.M., Ferrara, A., Silverman, C., Kasyan, K., Bossard, M.J., Johnson, R.K., Porter, T.G., Levy, M.A. and Livi, G.P. (1991). The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin Asensitive peptidyl-prolyl cis-trans isomerase with an Nterminal signal sequence. Gene 108,73-80. Kozak, M. (1983). Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol. Rev. 47, 1-45. Laemmli, U. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Lin, C.S., Boltz, R.C., Siekierka, J.J. and Sigal, N.H. (1991). FK506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes. Cell. Immunol. 133,269-284. Link, A.J. and Olsen, M.V. (1991). Physical map of the Saccharornyces cerevisiae genome at 1 10-kilobase resolution. Genetics 127,681-698. Liu, J., Farmer, J.D., Lane, W.S., Friedman, J., Weissman, I. and Schreiber, S.L. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66,807-815. Maki, N., Sekiguchi, J., Nishimaki, J., Miwa, K., Hayano, T., Takahashi, N. and Suzuki, M. (1990). Complementary DNA encoding the human T-cell
680 FK506-binding protein, a peptidylprolyl cis -trans isomerase distinct from cyclophilin. Proc. Natl. Acad. Sci. USA 87,5440-5443. Matsudaira, P. (1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol.Chem. 262, 10035. Mattila, P.S., Ullman, K.S., Fiering, S., Emmel, E.A., McCutcheon, M., Crabtree, G.R., and Herzenberg, L.A. (1990). The actions of cyclosporin-A and FK506 suggest a novel step in the activation of T lymphocytes. EMBO J. 9 , 4 4 2 5 4 3 1. Michnick, S.W., Rosen M.K., Wandless, T.J., Karplus, M. and Schreiber, S.L. (1991). Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin. Science 252,836-839. Moore, J.M., Peattie, D.A., Fitzgibbon, M.J. and Thomson, J.A. (1991). Solution structure of the major binding protein for the immunosuppressant FK506. Nature 351, 248-250. Perkins, D.D. (1949). Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34, 607-629. Proudfoot, N.J. and Brownlee. (1976). 3’ non-coding region sequences in eukaryotic messenger RNA. Nature 263, 21 1-214. Sherman, F., Fink, G.R. and Hicks, J.B. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Siekierka, J.J., Hung, S.H.Y., Poe, M., Lin, C.S. and Sigal, N.H. (1989). A cytosolic protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature (London) 341, 758-760. Stamnes, M.A., Shieh, B.-H., Chuman, L., Harris, G.L. and Zuker, C.S. (1991). The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of drosophila rhodopsins. Cell 65,219-227. Standaert, R.F., Galat, A., Verdine, G.L. and Schreiber, S.L. (1990). Molecular cloning and expression of the human FK506-binding protein, FKBP. Nature 346,671-674. Struhl, K. (1987). Promoters, activator proteins, and the mechanism of transcriptional initiation in yeast. Cell 49,295-297. Takahashi, N., Hayano, T., and Suzuki, M. (1989). Peptidyl-prolyl cis-trans isomerase is the cyclosporin
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Nature 337, A-binding protein cyclophilin. 473475. Tocci, M.J., Matkovich, D.A., Collier, K.A., Kwok, P., Dumont, F. Lin, S. Degudicibus, S., Siekierka, J.J., Chin, J. and Hutchinson, N.I. (1989). The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. J. Immunol. 143,7 18-726. Tropschug, M., Nicholson, D.W., Hartl, F.U., Kohler, H., Pfanner, N., Wachter, E. and Neupert, W. (1988). Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondria1 forms. J. Biol. Chem. 263, 14433-14440. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. and Clardy, J. (1991a). Atomic structure of FKBPFK506, an immunophilin-immunosuppressant complex. Science 252,839-842. Van Duyne, G.D., Standaert, R.F., Schreiber, S.L. and Clardy, J. (1991b). Atomic structure of the rapamycin human immunophilin FKBP-12 complex. J. Am. Chem. SOC.113,7433-7434. Wiederecht, G., Brizuela, L., Elliston, K., Sigal, N.H., and Siekierka, J.J. (1991). FKBl encodes a nonessential FK506-binding in Saccharomyces cerevisiae and contains regions suggesting homology to the cyclophilins. Proc. Natl. Acad. Sci. USA 88, 1029-1033. Zaret, K.S. and Sherman, F. (1982). DNA sequences required for efficient transcription termination in yeast. Cell 28,563-573.
NOTE ADDED IN PRESS We would like to note that the sequence in Fig. 1 contains an error. Nucleotides -227 to -250 correspond to sequence from the noncoding rather than the coding strand. A correction of the sequence will appear in a later issue of YEAST. Moreover we have determined that the EUGl gene is directly upstream of FKBZ. EUGl encodes an ER protein, related to PDII which specifies protein disulfide isomerase, an enzyme involved in protein folding (Gething and Sambrook, 1992). Therefore we have identified a region of the genome encoding two genes, FKB2 and EUGl, both of which may play a role in protein folding in the same subcellular compartment.
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Saccharomyces cerevisiae Contains a Homolog of Human FKBP- 13, a Membrane-associated FK506/Rapamycin Binding Protein JUDITH A. PARTALEDIS, MARK A. FLEMING, MATTHEW W. HARDING AND VIVIAN BERLIN* Vertex Pharmaceuticals Inc., 40 Allston Street, Cambridge MA 02139-421 I , USA. FKB2 encodes a homolog of human FKBP-13, a membrane-associated binding protein for the immunosuppressantsFK506 and rapamycin. FKB2 is located on the right arm of chromosome IV and contains an open reading frame of 135 amino acids, of which the first 17 residues comprise a putative hydrophobic leader peptide. Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity) and to FKBP-12, the major cytosolic receptor for FK506.In the alignment of
FKBP-13 and FKBP-12 sequences, there are 28 invariant residues. Among these conserved residues are those that comprise the drug binding and peptidyl-prolyl cis-trans isomerase active site of FKBP-12. The phylogenetic conservation of the FKBP family suggests that the proteins are involved in a basic cellular function. KEY WORDS-Immunosuppressant drugs; membrane proteins; S. cerevisiae; chromosome IV. INTRODUCTION FK506 and rapamycin are potent immunosuppressive agents that block distinct signal transduction pathways required for T-cell activation. FK506 blocks an early, Ca2+-dependent step required for interleukin 2 transcription (Tocci et al., 1989; Bierer et al., 1990a; Mattila et al., 1990; Flannagan et al., 1991), whereas rapamycin blocks lymphokine-induced T cell proliferation (Dumont et al., 1990a, b). Both FK506 and rapamycin bind with high affinity to a family of receptor proteins (Fretz et al., 1991) of which FKBP12 (Mr=12 000) is the best characterized (Harding et al., 1989; Siekierka et al., 1989; Bierer et al., 1990b). Like FK506, cyclosporin A (CsA; Handschumacher et al., 1984) is a potent immunosuppressant that inhibits Ca2+-dependent signalling pathways (Emmel et al., 1989; Johansson and Moller, 1990; Lin et al., 1991) and binds to an abundant cytosolic protein, cyclophilin (Handschumacher et al., 1984). The FKBPs and cyclophilin proteins constitute a growing class of immunosuppressant-binding proteins (immunophilins), all of which have been shown to catalyse the cis-trans isomerization of peptidyl-prolyl amide bonds (Fischer et al., 1989; Takahashi et al., 1989; Siekierka et *Addressee for correspondence. 0749-503X/92/080673-08$09.00 01992 by John Wiley & Sons Ltd
al., 1989; Harding et al., 1989; Galat et al., 1992). FK506, rapamycin and CsA all inhibit the peptidyl prolyl cis-trans isomerase (PPIase) activity of their cognate receptors. However, inhibition of PPIase activity is insufficient for immunosuppression; rather the complex of an immunophilin with drug appears to be the active entity (Bierer et al., 1990a, b; Hultsch et al., 1991). Consistent with this notion is the recent finding that FKBP- 12 and cyclophilin, bound to FK506 and CsA, respectively, inhibit calcineurin, a Ca2+-dependent phosphatase whose role in T cell activation is being investigated (Liu et al., 1991). The immunophilins FKBP-12 and cyclophilin are ubiquitous, cytosolic proteins whose primary structure is conserved between species (Franc0 et al., 1991; Hasel et al., 1991; Galat et al., 1992). FKBP-12 and cyclophilin define two structurally unrelated families of proteins widely distributed in tissues and subcellular compartments. The phylogenetic conservation of the FKBPs and cyclophilins suggests they are involved in fundamental cellular processes; their PPIase activity implicates the immunophilins in protein folding (reviewed by Gething and Sambrook, 1992). However, with the exception of the ninaA gene of Drosophila melanogaster, which encodes a cyclophilin required for assembly of rhodopsin in photo-
674 receptor cells (Stammnes et al., 1991; Colley et al., 1991), the in vivo function and physiological substrates of proteins with PPIase activity remain to be established. No function has been ascribed to the cyclophilin or FKBP- 12 proteins of Saccharomyces cerevisiae, since single or multiple deletions of the genes encoding these proteins produce no marked cellular phenotype (Koltin et al., 1991; Heitman et al., 1991; Wiederrecht et al., 1991; Koser et al., 1991; J.A. Partaledis and V. Berlin, unpublished results). Recently, additional members of the FKBP family have been isolated from mammalian tissue on the basis of their affinity to FK506 or rapamycin (Fretz et al., 1991). The human gene encoding one of these specifies a 13-kDa protein, designated FKBP-13, which is 43% identical to FKBP-12 (Jin et al., 1991). Using an FK506 affinity matrix, we have isolated a 13-kDa protein from yeast that is the homolog of human FKBP-13. MATERIALS AND METHODS Strains and growth media
LZAPI library screening and in vivo excision were performed with Escherichia coli strain BB4: (Stratagene) LE392.23 [FlacPZAM1.5, proAB, TnlO (tet‘)]. Plasmid DNA was propagated in E. coli strain DH5a: F- $80dlacZAM15 A(lacZYA-argF)U169 endA 1 recA 1 hsdR 17(rK- mK+)deoR thi- 1 supE44 AgyrA96 relAl. The S. cerevisiae strain used for protein isolation was F37 (formerly BJ2168, gift of E.W. Jones): Mata leu2 trpl ura3-52 prbl-1122 pep4-3 prcl-407. Yeast genomic DNA was from strain VBY25: Mata ura3-52 leu2-3,112 trplAl lys2A201 his3A200 ade2 cyh2 canl. The yeast strains used for mapping the FKB2 gene were VB49-1B: Mata ura3 trplAl leu2-3,112 cyh2, VB88-4B: Mata ura3-52 lys2A201 his3A200 leu2-3,112 fkb2-l::LEU2, F133 (formerly 10208-6A, gift of G. R. Fink): Mata trp4 ade8 r a 3 leu2-3. YPD contained 1% yeast extract, 2% Bacto-peptone (Difco) and 2% glucose. Minimal medium contained 0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose, and was supplemented with the required amino acids (Sherman et al., 1986). Protein preparation and amino acid sequencing of FKBP-13
JUDITH A. PARTALEDIS ETAL..
fluoride (Sigma), 0.5 mM-diisopropyl fluorophosphate (Sigma). Cells were disrupted using a Bead Beater (Biospec Products), and the cell debris was pelleted twice at 4000 x g for 5 min. The supernatant was clarified by successive centrifugations at 47,000 x g for 30 min, 48,000 x g for 45 rnin and 100,OOO x g for 60 min. Cytosol (350 mg total protein) was passed over a 10 ml column of end-capped Affigel 10 resin (Biorad, Richmond, CA) and then over a 2 ml Affigel 10-FK506 column (0.5 mg FK506/ml resin) at 4°C. A 32-[4’-(4-aminophenyl)butyryl]-FK506derivative was coupled to Affigel 10 resin as described by Fretz and coworkers (1991). After extensive washing with phosphate buffered saline-0.2% Tween 20, proteins were batch eluted by overnight incubation with FK506 (1.0 mg in 5 ml). Eluted proteins were dialysed against 10 mM-Tris-HC1, pH 7.4, lyophilysed and resolved by electrophoresis in a 12% sodium dodecyl sulfate-polyacrylamide gel SDS-PAGE (Laemmli et al., 1970). After electrotransfer of proteins, a 15-kDa band was excised from Immobilon-P membrane (0.45 p pore size, Millipore, Bedford, MA) (Matsudaira et al., 1987), and was subjected to amino-terminal sequence analysis using an Applied Biosystems protein sequencer (Model 477A). Library screening and DNA sequencing The N-terminal amino acid sequence of yeast FKBP13 was used to generate probes to isolate FKB2 cDNAs. Degenerate oligonucleotides were used to synthesize two products, 105 bp and 63 bp in length, by the polymerase chain reaction (PCR). The 105 bp PCR product was synthesized using oligos 543:
5’-GG’ITCITTXTCIGAYTTXGAXATIGGTAT-3’ (I = inosine, X = purine, Y = pyrimidine) and 545: 5’-ACCIGTXTAXTGXACYTTXACYTTXATYYXTCAC-3’. The 63 bp PCR product was synthesized using oligos 546: 5’-CCIGTYGAXGAYTGYTTXATIAAXGCIATGCC-3’ and 545. Approximately 10 ng of yeast genomic DNA prepared from strain VBY25 was used as the template for the PCR reactions. PCR reactions were performed using an Eppendorf Microcycler E for 35 cyclTs consisting of 94°C for 1.5 min to denature, 42 C for 2.0 min to anneal and 72°C for 1.5 rnin to polymerize. A single cycle at 72°C for 10 rnin was used to com-
Strain F37 was grown to mid-log phase in YPD. Cells were harvested (35 g wet weight) and suspended in 50 mM-potassium phosphate, pH 7.2, 0.9% potassium chloride, 10 mM EDTA, 1 mM-phenylmethylsulfonyl plete extension of the chains.
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S. CEREVISIAE CONTAINS A HOMOLOG OF HUMAN FKBP-13
The PCR products, labeled with 32P, were used to screen a ?ZAP1 cDNA library (provided by Jeff Kuret, Cold Spring Harbor Laboratory). Approximately 100,OOO plaques were screened using methods described by Stratagene (La Jolla, CA). Three putative FKB2 cDNAs were excised from the hZAPI vector as pBluescript plasmids and were designated 213-2,Z13-5 and 213-7. All cDNA clones were characterized further by sequencing. Sequences of the cDNAs were used to generate a robe to isolate the FKB2 gene. The probe was a 520 bp y2P-labeled PCR product corresponding to a region of FKB2 extending from positions +41 to +561 (Figure 1). The 32P-PCR product, generated from genomic DNA, was used to screen a yeast genomic library in the vector YEp24 (Carlson and Botstein, 1982). The methods used for library screening were those of Berent and coworkers (1985). Eighteen plasmids were isolated, purified and their inserts mapped by restriction analysis. The plasmid Y 13-8A was further characterized by sequencing. FKB2 cDNA and genomic clones were sequenced using the dideoxy chain termination method with a modification by Hattori and Sakaki (1986) for double stranded DNA plasmids. All sequencing primers were either purchased from Stratagene (La Jolla, CA) or custom made on an Applied Biosystems oligonucleotide synthesizer (model 380A). All sequencing reactions were performed using reagents from the Sequenase DNA sequencing kit (United States Biochemical, Cleveland, OH). Computer analysis Nucleotide and amino acid sequences were analysed with the sequence analysis software package of the Genetics Computer Group, University of Wisconsin, Madison (version 6.1). The default parameters of the BESTFIT program were used for amino acid sequence comparisons. The GenBank (release no. 69) and the European Molecular Biology Laboratory (EMBL, release no. 28) nucleic acid data bases, and the National Biomedical Research Foundation (NBRF) protein (release no. 30) data base were searched with the DNASTAR Macintosh Molecular Biology Software program with GENEMAN (version 1.26). Genetic mapping To determine the chromosomal position of FKB2, a 32P-labeled, 520-bp PCR fragment (also used to isolate the FKB2 gene; see preceding section) was
hybridized to a chromoblot purchased from Clontech (lot no.17037) and to a set of bacteriophage h clones (gift of L. Riles and M. V. Olson) representing the entire yeast genome (Link and Olson, 1991). Linkage to known markers was determined by tetrad analysis. RESULTS AND DISCUSSION Isolation of the yeast gene encoding FKBP-I3 Using an FK506 affinity matrix, a 15-kDa protein was purified from yeast and the N-terminal amino acid sequence: GSLSDLEIGIIKRIPVEDCLKAMPWKVKV~ GxLB
was determined. Of these 39 residues, 17 (those underlined) were identical to those in the amino terminus of human FKBP-13 (Jin et al., 1991). Complementary DNA and genomic clones were isolated using probes specific for the amino terminal sequence of the 15-kDa yeast protein and for the cDNA, respectively. Both the cDNA and genomic clones contained an open reading frame in which amino acids 18 through 56 were identical to the N-terminal amino acid sequence of the purified 15-kDa FK506-binding protein of yeast. This observation confirmed that we had cloned the gene (FKB2) for a second m 5 0 6 binding protein in yeast. The protein encoded by FKB2 shares significant homology with human FKBP- 13 (see succeeding section). The complete nucleotide sequence of FKB2 and the deduced amino acid sequence of its product, FKBP13, are shown in Figure 1. FKB2 has a single open reading frame of 135 amino acids containing two consecutive in-frame AUGs which could function as initiation codons. The sequence context of the second AUG codon in FKB2 is more favorable than the first for translation initiation (Kozak, 1983; Hamilton et al., 1987). A comparison of the deduced amino acid sequence and the actual N-terminal amino acid sequence indicates FKBP- 13 contains a putative leader peptide of 17 amino acids with a predicted cleavage occurring between Ala-17 and Gly-18. The hydrophobic nature of the leader peptide suggests it may function as a signal sequence to direct FKBP-13 to the endoplasmic reticulum (ER). In fact, human FKPB-13 is enriched in the heavy membrane fraction of Jurkat T cells and has a hydrophobic leader peptide that can be cleaved in the presence of microsomal membranes (Jin et al., 1991). The mature form of yeast FKBP-13 beginning with Gly-18 consists of 118 amino acids and has a calculated M, of 12,447. This is 2.5 kDa smaller
676
JUDITH A. PARTALEDIS ET AL
-250
CGTATATATT
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S. CEREVISIAE CONTAINS A HOMOLOG OF HUMAN FKBP-13
677
than the mass of FKBP-13 estimated from its mobility Table 1. Chromosomal position of FKBZ in SDS-PAGE. The basis of this discrepancy, which TETRAD TYPES also exists for human FKBP-13 (Jin et al., 1991), is LOCI PD NPD Ta LINKAGE(CM)b unknown. 33 28 ma3 ade8 25 0 The DNA sequence of FKB2 contains putative 13 0 44 39 recognition sites frequently found in the 5' and 3' ends ma3 fkb2-l::LEU2 45 >50 7 5 of yeast genes (Figure 1). Potential TATA sequences for adeSfkbZ-l::LEU2 transcription initiation (Struhl, 1987) are located at a PD is parental ditype tetrad, NPD is nonparental ditype tetrad, positions -72, -89 and -248. The sequences required for and T is tetratype tetrad. The values are the total number of tetrads mRNA 3' end formation in yeast, TAG ...TA(T) in each catagory. b Genetic distance is given in centimorgans (cM) and calculated GT...TIT(Zaret and Sherman, 1982) and " T A T A (Henikoff and Cohen, 1984) are found between according to the formula 100(T + 6NPD)/2(PD + NPD + T) positions +406 to +587 in FKB2. The eukaryotic (Perkins 1949). consensus sequence for polyadenylation, AATAAA (Proudfoot and Brownlee, 1976; Bimstiel et al., 1985), Homology of yeast FKBP-13 to human FKBP-I3 and usually absent in yeast genes, is found at position FKBP-12 +569 of FKB2. Comparison of the genomic and cDNA sequences of FKB2 indicates that poly(A) Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity and 66% similarity), yeast addition begins after the thymidine at position +621. FKBP-12 (52% amino acid identity and 67% similarity), human FKBP-12 (48% amino acid identity and 62% similarity) and Neurospora crassa FKBP-12 Chromosomal location of FKB2 (46% amino acid identity and 64% similarity). In the Hybridization of an FKB2 probe to a chromoblot and alignment of the FKBP proteins, there are 28 invarian ordered set of bacteriophage h clones representing ant residues and 3 1 positions in which at least four of the yeast genome indicated FKBZ is located on the five aligned residues are identical or are conservaChromosome IV or VII. The ambiguity of this prelim- tive changes (Figure 2). Yeast FKBP-13 also contains inary analysis was resolved by performing a set of the two cysteine residues present in human FKBP- 13 crosses. Since one of the two bacteriophage h clones (cys20 and cys75), which may form a disulfide bond to which FKB2 hybridized contained CYH2, on chro- (Jin et al., 1991); these cysteine residues are absent in mosome VII (L. Riles, personal communication), a FKBP-12. Among the residues conserved in all the cross was performed between strains VB49-1B and FKBPs are those that form a hydrophobic pocket in VB88-4B to examine linkage between FKBZ and FKBP-12 that bind FK506 and rapamycin or interact CYH2. This cross established that the two genes were with the drugs via hydrogen bonds (Van Duyne et unlinked, eliminating the possibility that FKB2 al., 1991a; Figure 2). The conservation of amino acids resided on chromosome VII. It is not clear whether that interact with FK506 and rapamycin suggests hybridization of the FKB2 probe to the chromosome these residues may be involved in binding to an VII clone was non-specific or was due to the presence endogenous ligand. of sequences sharing homology to FKB2. The location of FKB2 on chromosome IV was further refined FKBP-13 is a member of a family of related proteins by a cross between strains F133 and VB88-4B. Tetrad analysis indicated FKBZ was linked to rna3 and Like FKBP-12, FKBP-13 has PPIase activity that is unlinked to ade8 on chromosome IV, placing FKBZ to inhibited by rapamycin and FK506. The Ki values for the right of rna3 on the right arm of chromosome IV the two drugs determined with bovine FKBP-13 are (Table 1). 3.6 nM and 38 nM, respectively (M.W. Harding, J.A. Figure I . DNA sequence of the FKB2 chromosomal region and the deduced amino acid sequence of the FKBP-13 protein. Numbers in the left margin refer to the f i s t nucleotide in each line. Negative numbers indicate nucleotide positions upstream from the putative translation start site at +l. The single open reading frame is translated into predicted amino acids and the hydrophobic signal sequence (amino acids 1-17) is underlined. Amino acids 18-56 were confirmed by N-terminal amino acid sequencing. The putative TATA sequences at the 5' end of FKBZ are indicated in bold letters. Sequences located between positions +406 and +587 that are possibly involved in mRNA 3'-end formation include: (1) TAG ...TA(T)GT...TIT (Zaret and Sherman, 1982) found in the coding (underlined) and non-coding (underscored with broken lines) DNA strands (Imiger et al., 1991); ( 2 ) TTITTATA (Henikoff and Cohen, 1984), indicated by a line above the sequence; (3) AATAAA (Proudfoot and Brownlee, 1976; Bimstiel et al., 1985). which is underlined. Poly(A) addition occurs after the thymidine at position +621 shown in bold.
JUDITH A. PARTALEDIS ETAL.
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x x x x x NcFK12 HuFK12 ScFK12 ScFK13 HuFK13
x
Y
x x x x NcFK12 HuFK12 ScFK12 ScFK13 HuFK13
x
x
MTIPQLDGLQ------1EVQQEGQGTRE GVQ------VETISPGDGRTF MSEVIEGNVK------1DRISPGDGATF MMFNIYLFVTFFSTIL--------AGSLSDLEIGIIKRIPVEDCLIK MRLSWFRVLTVLSICLSAVATATGAEGKRKLQIGVKKR--VDHCPIK
/1/ /1/ /1/ /1/ /1/
x x xxxxx
/61/ /45/ /61/
x
FD
xxxx x x
xx xxx
LGMK
G IKGVQKGE
IS
AQMS
PKLS AGMC LGMC
/00/
/85/
F
QEVI
W
1s
K IERRTE L Y
I
F
Figure 2. Alignment of FKBP-13 and FKBP-12 sequences. The alignment of amino acid sequences was performed by hand, based on multiple binary comparisons generated by BESTFIT. The sequences compared were: Neurospora crassa FKBP-12 (NcFK12; Tropscbug et al., 1990), human FKBP-12 (HuFK12; Standaert et a/., 1990; Maki er al., 1990). Saccharumyces cerevisiae FKBP-12 (ScFK12; Wiederecht et al., 1991; Koltin et al., 1991; Heitman et a/., 1991), S. cerevisiae FKBP-13 (ScFK13, this study), human FKBP-13 (HuFK13; Jin et al., 1991). Invariant residues are shadowed. X’s above the alignment denote positions in which at least four of the five residues are similar or identical. Under the alignment are shown the residues known to contact FK506 or rapamycin by hydrophobic interactions or hydrogen bonding (Michnick etal., 1991; Van Duyne et al., 1991a, b; Moore e t a / . , 1991).
Lippke, M. Fleming, 0. Futer, M.T. Decenzo, D.J. Livingston, D.A. Peattie, unpublished results). Recently two other FKBPs, FKBP-25 (Galat et al., 1992) and FKBP-52 (M. Benasutti, M.W. Harding, M.A. Fleming, M.T. Decenzo, J.A. Lippke, D.J. Livingston, D.A. Peattie, unpublished results), have been characterized, which also have PPIase activity inhibited by rapamycin and FK506. Many of the conserved residues highlighted in Figure 2 are present in FKBP-25 and FKBP-52. The potential ER targeting sequence in FKBP-13 (Jin et al., 1991 and this study) and potential nuclear targeting sequence in FKBP-25 (Galat et al., 1992) suggest the FKBPs are localized to different subcellular compartments. The role of the various FKBPs in mediating the action of FK506 and rapamycin has yet to be determined. With the cloning of the yeast genes for FKBP-12 (Wiederecht et al., 1991; Koltin et al., 1991; Heitman et al., 1991; J.A. Partaledis and V. Berlin, unpublished results) and now FKBP13, genetic analysis can be undertaken to elucidate the physiological role and to identify the natural ligands of the FKBPs. ACKNOWLEDGEMENTS The authors would like to thank Jeff Kuret for the yeast cDNA library in AZAPI. We would also like to thank David Armistead for synthesis of 32-[4’-(4aminophenyl)butyryl]-FK506 and Arlene Semerjian and David Livingston for critical reading of the manuscript.
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NOTE ADDED IN PRESS We would like to note that the sequence in Fig. 1 contains an error. Nucleotides -227 to -250 correspond to sequence from the noncoding rather than the coding strand. A correction of the sequence will appear in a later issue of YEAST. Moreover we have determined that the EUGl gene is directly upstream of FKBZ. EUGl encodes an ER protein, related to PDII which specifies protein disulfide isomerase, an enzyme involved in protein folding (Gething and Sambrook, 1992). Therefore we have identified a region of the genome encoding two genes, FKB2 and EUGl, both of which may play a role in protein folding in the same subcellular compartment.
