- Email: [email protected]
A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases
Biochimica et Biophysica Acta, 1171 (1993) 299-303
299
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 90434
Short Sequence-Paper
A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases Ulla Magdolen a, Giinter Miiller b, Viktor Magdolen c and Wolfhard Bandlow Institut fiir Genetik und Mikrobiologie, Miinchen (Germany), b c / o Hoechst Aktiengesellsehaft Frankfurt am Main, Frankfurt (Germany) and " Max-Planck-lnstitut fiir Biochemie, Martinsried (Germany)
(Received 28 September 1992)
Key words: Bleomycinhydrolasegene; Antitumor drug; Cysteineproteinase; (S. cerevisiae) We have purified bleomycin hydrolase from yeast (molecular mass 55000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blhl mutant cells. Bleomycin is a mixture of closely related glycopeptide antibiotics secreted from Streptomyces verticillus [1]. It causes radiomimetic D N A damage in bacteria and eukaryotes. It also inhibits growth of a number of squamous and small cell type tumors in situ and in vitro, although its exact mechanism of action is unknown. Due to its lack of hepatic, renal and bone marrow toxicities, the drug is extensively used in tumor therapy [1,2]. A more general use of the drug is, however, primarily hampered by two drawbacks, (i) the pulmonary toxicity [3] and (ii) the resistance of certain cell types and tissues to the drug due to high intracellular activities of the thiol proteinase bleomycin hydrolase [4-6]. The ubiquitous and abundant occurrence of this enzyme in all animal tissues and in microorganisms points to an important, although as yet unknown function. In order to study bleomycin resistance, to identify the physiological role of the proteinase and to understand why certain tumors are refractory to therapy by the drug, it may be helpful that we have isolated a yeast gene displaying high homology
Correspondence to: W. Bandlow, Institut fiir Genetik und Mikrobiologie, Maria-Ward-Strasse la, D-8000 Miinchen 19, Germany. The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number X69124.
of the encoded protein to bleomycin hydrolase from rabbit. We have isolated the bleomycin hydrolase protein from yeast during a study of a cAMP receptor glycolipid-anchored to plasma membranes [7]. We have enriched the latter protein by isolating crude plasma membranes from yeast spheroplasts by Percoll gradient centrifugation and sequential purification of the detergent-solubilized protein by cAMP-agarose affinity chromatograpy, molecular sieving and phenyl Sepharose lipophilic chromatography which uses the hydrophobicity of the glycolipid anchor-containing protein as a parameter for separation. When the eluted proteins were separated on a sodium dodecylsulfate polyacrylamide gel, one of the major bands comigrated with the authentic, 8-N3-[32p]cAMP photoaffinity-labeled product (apparent molecular mass 54 kDa, as deduced from electrophoretic mobility) [7]. This material was excised from the gel, eluted and cleaved either with trypsin or CNBr. The peptides were separated by high performance liquid chromatography. Two major fragments (one from each cleavage) were sequenced (indicated in Fig. 2). It turned out from the sequence data (see below) that they obviously do not belong to the glycosyl-phosphatidylinositol-anchored cAMP-binding protein from yeast plasma membranes. Whether the enriched protein with homology to bleomycin hydrolase binds to cAMP agarose and phenyl Sepharose directly or interacts with the native cAMP receptor protein
300 fortuitously or in a functional complex remains to be established in the future. The corresponding gene was isolated by using the polymerase chain reaction method. Since the intramolecular arrangement of the two eluted and sequenced peptides was unknown, three primers were designed from each of them, two overlapping primers in the one and a third one in the reverse direction. Pairwise amplification by polymerase chain reaction and crosswise hybridization led to the identification of a DNA fragment, which encoded sequences from both protein fragments. The genomic DNA clone, pPMY 11.0, subsequently isolated from a gene bank constructed from BamHI-digested yeast DNA, contained about 11 kbp of genomic DNA and was characterized by restriction and hybridization analyses. The region cross-hybridizing to the amplified DNA fragment could be narrowed down to two HindIII fragments, which were sequenced subsequently from the central HindIII site in both directions to yield the complete DNA sequence of the yeast BLH1 gene (Fig. I A, B). The nucleotide sequence analysis and the deduced primary structure of the protein are displayed in Fig. 2. The open reading frame comprises 483 triplets with a deduced molecular mass of the encoded protein of 55.5 Da, the calculated isoelectric point is at pH 9.58. Structural analyses by using the algorithms of Kyte and Doolittle, and Hopp and Woods do not reveal any striking structural features and suggest a globular, hydrophilic protein. Southern hybridization with a labeled DNA fragment, derived from the coding region of the BLH1 gene shows that BLH1 occurs in single copy in the yeast genome (one example is given in Fig. 1D, lane 1). The codon bias index [8] of 0.35 points to a relatively high expression. The DNA sequence flanking the gene at its 5' side displays no obvious T A T A box. However, many yeast genes are known which lack obvious T A T A sequences. In the putative promoter region of BLH1 a perfect consensus sequence for an abundant DNA binding protein, Grf2p [9], is found, immediately followed by a pyrimidine-rich sequence (Fig. 2). Grf2p may have a role in nucleosome positioning by excluding nucleosomes from the vicinity of its binding site. The same combination of sequence motifs as in the BLHl-upstream region also occurs in the promoter region of the yeast profilin gene, PFY1 [10]. Deletion of these elements in the PFY1 promoter dramatically reduce transcription of PFY1 (H. Herrmann and W. Bandlow, unpublished results). Data base searches using the Genetics Computer Group (GCG) Sequence Analysis Software Package [11] reveal significant sequence similarities to bleomycin hydrolase, a cysteine proteinase isolated from rabbit lung and liver. A cDNA fragment encoding the central portion of this enzyme has been obtained from rabbit liver and sequenced [6]. The alignment of the deduced
A E
HH
EHE
I
II
III
H I
I
I lOOO bp I
B
I I
N
I
C
Jl
EH
[
Jl
E
11oo bp
4
I 500 bp
CN b
C I N
""~-~--Jj C
EH
I
E
# I~ it
N
C
2500 bp
EH
I E
blh 1
II CN
3500 bp
D 1
Jme
2
3
4
5
~
~
~
3.5
kbp
~
~
~
2.5
kbp
i
m
I.I
kbp
Fig. h Inactivation of the BLH1 gene. (A) Genomic restriction map of the BLH1 locus. The arrow indicates direction of transcription. (B) Restriction map of the sequenced region. Restriction sites relevant for gene disruption are shown. (C) Genomic organization after integration of the linearized plasmid pPMP9-A at the BLH1 locus by homologous recombination. Vector sequences between the duplicated BLHl-coding sequences (indicated by thick bars) are not drawn to scale. (D) Genomic Southern blot from wild-type and disruption mutants. Genomic DNA was isolated from the haploid wild-type strain W303-1A (lane 1), from haploid disruption strains (lanes 2 and 3), from heterozygous diploid strains (lanes 4 and 5) and restricted with Ndel. The blots were probed with a digoxigeninlabeled fragment corresponding to the BLH1 fragment cloned into pPMP9-A (indicated by thick bars in (C), C, Clal; E, EcoRI; H, HindIlI; N, Ndel.
rabbit and yeast protein sequences is shown in Fig. 3A. Within the region of the known rabbit sequence, the homology is highly significant (40 S.D. units, using the program A L I G N [12] with the mutation data matrix, 100 random runs and a penalty for a gap of 20). The yeast sequence extends beyond the known rabbit sequence by 21 amino acids to the N-terminus and 158 residues to the C-terminus. The overlap of the two proteins contains 43% identical amino acids and 22% conservative exchanges at comparable positions (Fig.
301 3A). The search for protein motifs and domains reveals that a stretch of 17 amino acids (pos. 92-108 of Blhlp) is highly homologous to the region containing the cysteine residue conserved in the catalytic centre of eukaryotic thiol proteinases (Fig. 3B). These include calpain, plant and protozoan cystein proteinases, vertebrate lysosomal and secretory cathepsins, and two developmentally co-regulated cysteine proteinases from the slime mold Dictyostelium discoideum. In addition,
it can be seen from Fig. 3B that Blhlp from yeast also has significant homology to two sequence blocks of other cysteine proteinases (which were previously unknown from the rabbit enzyme) implied to contribute to the active centre, but conserved only in a subset of thiol proteinases. In order to gain some information about the physiological role of the protein, we replaced the structural BLH1 gene with two different incomplete blhl alleles
260 GRF2 ............. C G T A T A T T C A G T G C T T T T T G A A A T T T A G A A A A T A G T C G 6 ~ T T A T T G A T T A T G C T A C A T A T G T T A T T T T G C T T T A G T A T G T G T C A A A T A G C A T G C A A T C G C C C G G T A C T G A G T G A C A C A T C CTTT'~FFCCC
C T C C C A C C A C A A A G T C A C C T G C T C T C T T A A T C A A T G G G G T T G C A T G T G G T A A T C A A T T G A T A A C G C C G G C T G A C A A G T C G C C G A C G G G C A C C C A T A A A T A A A C G A T A A A T AGCCC~'FGCT C T T T T G T T A C
ATG C T T C C T A C T T C T G T T T C C CGG T C C TTG T A T CTC AAG ACG TTT AGA AGC CAC CTT T T A CGA G C A C C A C A A A T T G T T T T A A A A A G A ATG T C C T C T T C C ATC Met Leu Pro T h r Set Val Set Arg Set Leu Tyr Leu Lys Thr Phe Arg Set His Leu Leu Arg Ala Pro Gin lle Val Leu Lys Arg Met Set Ser Ser Ile
+103 GAT A T C A G T A A G A T C A A C T C T TGG A A C A A A G A G T T T CAA TCC G A C T T A ACC CAT C A A TTG GCA A C C A C T G T C C T T A A G A A T T A T A A T G C C G A T G A T G C A CTG A s p Ile Ser Lys Ile Asn Ser T r p Ash Lys Glu Phe Gin Set A s p Leu Thr His Gln Leu Ala Thr T h r Val Leu Lys Ash Tyr A s n A l a A s p A s p Ala Leu
~205 TTC A A C AAG A C T A G A CTG C A A A A G CAA G A T A A C A G G G T T TTC AAC A C T G T T G T C TCT ACT GAT TCC A C T C C A G T C A C C A A C C A A A A A A G C T C T G G T A G A TGT Leu Ash Lys Thr A r g Leu Gln LyS Gln A s p Ash Arg Val Phe Ash Thr Val Val Set Thr Asp Set Thr Pro Val Thr A s n G i n Lys Ser Set G I y A r g Cys
+307 TGG T T G T T T G C C G C T A C C A A T C A A C T G CGC TTG A A T G T T CTT TCT GAA TTG A A C T T A A A A GAA T T C C A A CTG T C C C A A G C T T A C T T G T T C ~ T A C G A T AAG Trp Leu Phe Ala Ala Thr Ash Gln Leu A r g Leu Ash Val Leu Ser Glu Leu Ash Leu Lys Glu Phe C l u Leu Set Gln A l a T y r Leu Phe Phe T y r A S p Lys
+409 TTG G A A AAG G C C A A C T A T T T C TTG G A C CAA A T C G T C T C C TCG G C T G A T C A A G A C A T C G A C TCA C C T C T C GTG CAA T A C T T G T T A G C A G C T C C A A C A G A A GAC Leu G l u Lys Ala Ash Tyr Phe Leu A s p Gln Ile Val Ser Set Ala A s p Gln A s p Ile Asp Ser A r g Leu Val Cln Tyr Leu Leu Ala Ala Pro Thr Glu ASp
+511 GGT G G C C A A T A C A G C A T G q ~ T TTG A A C C T A G T C A A G AAG TAT G G C C T T ATG CCC AAA G A T TTA T A C G G G G A T C T A C C T T A T T C T A C T A C T G C G T C C A G A AAA Gly Gly Gln Tyr Ser Met Phe Leu Ash Leu Val Lys Lys Tyr Gly L e u Met Pro Lys A s p Leu Tyr Cly A s p Leu Pro Tyr Ser Thr Thr A l a Set Arg Lys
+613 TGC A A T T C T C T G TTG A C T A C T A A A C T G A G A G A A T T T G C C GAG A C T C T A AGA A C A G C T TTG AAA G A G C G T T C T G C C C A T G A T T C C A T A A T T G T C A C T C T G A G A Trp Asn Set Leu Leu Thr Thr Lys Leu A r g Glu Phe Ala Glu Thr Leu Arg Thr Ala Leu Lys Glu Arg S~r Ala A S p Ash S~r Ile Ile Val Thr Leu Arn primer I-A +715 GAG C A A A T G C A A A G A G A A A T C T T C A G G T T G ATG TCG TTG TTC A T G GAC A T A C C T C C A GTG CAA C C A A A C GAG C A A T T C A C T T G G G A A T A C G T T G A C A A A GAC Glu Gln M~t Gln Arm Clu Ii~ Phe Aro Leu Met get Leu Phe Met A s p Ile Pro Pro Val Gln Pro A s n Glu Gln Phe Thr T r p Glu T y r Val A s p Lys ASp
+817 AAG AAA A T C C A C A C T A T C A A A TCG A C T C C G T T A G A A T T T GCC T C C A A A T A C GCA A A A T T G GAC C C T T C C A C G CCA G T C T C A T T G A T C A A T G A T C C A A G A CAC Lys Lys Ile His Thr Ile Uys Set Thr Pro Leu Glu Phe Ala Ser Lys Tyr Ala Lys Leu Asp Pro Ser Thr Pro Val Set Leu Ile A s n A s p Pro Arg His
+919 CCA T A T G G T A A A T T A A T T AAG A T C G A T C G T T T A G G A A A C GTC C T T GGC GGA G A T G C C CTG A T T T A C T T A A A T C T T G A C A A T G A A A C A C T A T C T A A A TTG G T T Pro Tyr G l y Lys Leu Ile Lys Ile A s p Arg Leu Gly Ash Val Leu Cly Gly Asp Ala Val Ile Tyr Leu A s n Val A s p Asn C l u T h r Leu Set Lya Leu Val
+1021 Wv GTT A A G A G A T T A C A A A A T A A C A A A G C T G T C T T T T T T GGA T C ~ C A C A C T CCA A A G T T C A T G GAC A A G A A A A C T G G T G T C A T G G A T A~'F G A A T T G T G G A A C TAT Val Lys A r g Leu Gln ASh Asn Lys Ala Val Phe Phe Cly Set His Thr Pro Lys Phe Met A s p Lys Lys Thr Gly Val M~t A s h II~ ~ ] u Leu T r D ASh "~vr p r i m e r 2-A
/
primer 2-C
C C T G C C A T C G G C T A T A A T ~ A C C T C A G C A A AAG G C A TCG CGT A T T AGA T A C C A T GAA A G T TTG A T G A C T C A T G C T ATG ~ G A T C A C T G G C T G C C A C G T C G A T Pro A l a If@ G l v Tvr Ash Leu Pro C]n Gln Lvs A l a Sat Arg Ile Arg Tyr His GIu Set Leu Met Thr His A l a Met Leu Ile Thr G l y CyS His Val Asp
+1225 GAA A C G T C T A A A q~FA C C A C T T C G C T A C CGC G T T G A A A A T TCC TGG G G T A A A G A C T C C G G T A A A G A C G G A T T A T A C G T G A T G A C T C A A A A G T A C T T C G A G GAG Clu T h r Ser Lys Leu Pro Leu A r g Tyr A r g Val Glu A S h Set Trp GIy Lys A s p Set Gly Lys A s p G l y Leu Tyr Val Met Thr G l n Lys ~ r Phe Glu Glu
+1327 TAC T G C T T T C A A A T T G T G G T C G A T A T C A A T G A A T T G C C A AAA GAG CTG G C ~ TCA AAA T T C ACC T C A G G T A A G G A A GAG C C G A T T G T C T T G C C C A T C TGG G A C Tyr Cys Phe Gln Ile Val Val A s p Ile A S h Glu Leu Pro Lys Glu Leu Ala Set Lys Phe Thr Set Gly Ly$ G l u G l u Pro Ile Val Leu Pro Ile T r p Asp
+1429 CCA A T G G G T G C T TTG G C C A A A TAA Pro Met G I y Ala Leu A l a Lys ***
ATAGTT TCAGCAGCTC TGATGTAGAT ACACGTATCT CGACATGTTT TATTTTTACT ATACATACAT AAAAAGAAAT AAAAAATGAT AACGTGTATA
Fig. 2. Nucleotide and deduced amino acid sequences of BLH1. The putative Orf2p-binding site is overlined, the adjacent pyrimidine-rich region is indicated by dots. The two peptides sequenced from the purified protein are underlined. The DNA regions corresponding to the primers effective in PCR amplification are overlined by arrows. The sequences of these degenerate oligonucleotides were: 5' CAA/GATGCAA/GC/ A G I / C G A A / G A T (I-A); 5' CCIATI/CGCI/CGGA/GTAA/GTT (2-A); 5' TGT/CTGI/CGGI/CAA/GA/GTTA/GTA (2-C). I denotes inosine.
302 as detailed in Fig. 1C. For this purpose the coding sequence contained between the ClaI site at pos. + 100 and the EcoRI site at pos. +615 was cloned into the yeast integration vector pRS306 harbouring the URA3 gene as a selective marker [13] to yield clone pPMP9-A. After cleavage of pPMP9-A with HindlII (pos. +383 of the coding region of BLH1) the resulting linear plasmid DNA was transformed into either haploid or diploid yeast wild-type ceils. The correct integration of the plasmid by homologous recombination at the BLH1 locus leads to the duplication of those BLH1 DNA sequences cloned into pRS306, the resulting two incomplete copies being separated by sequences from the vector (Fig. 1C). The integration event was proven by Southern blotting of genomic DNA restricted with NdeI. The blot was probed with the BLHl-derived sequences cloned into pPMP9-A. DNA from wild-type cells yields a single signal corresponding to 1.1 kbp (Fig. ID, lanes 1), whereas the haploid disruption mutants display two signals as expected, corresponding to 2.5 and 3.5 kbp (Fig. 1D, lanes 2 and 3) instead of the 1.1 kbp band (cf. Fig. 1B and C). Heterozygous diploids contain both, the wild-type and the disrupted alleles. Consequently, all three signals can be seen with DNA from heterozygous diploid disruption strains (Fig. 1D, lanes 4 and 5).
blhl disruption mutants were found to be viable in the haploid state. When the growth behaviour of the haploid mutant and the isogenic wild-type strains was compared, no differences were observed on glucose, galactose and glycerol media. Also long-term experiments, where cells were shifted from one carbon source to another (eight changes of media), revealed no disadvantage for the blhl mutant strains compared to wildtype cells. In addition, heterozygous diploids sporulate well, and the spores germinate. It must be concluded from these results that the gene is dispensable for vegetative and meiotic growth, cell viability and differentiation. Finally, the haploid wild-type strain (with a functional BLH1 allele) was compared to the disruption strain with respect to growth in the presence of bleomycin. For this purpose co-cultivation experiments of the haploid strain W303-1A and of the isogenic blhl mutant strain were performed: cells of both strains pre-grown overnight in the presence of bleomycin were mixed and incubated for 2 days in the absence of 3.5 mg bleomycin/ml medium (which leads to about 50% growth inhibition). The ratio of the wild-type and mutant cells was monitored by replica-plating at the beginning of the experiment and after two days of growth by testing uracil auxotrophy (URA3 has been used as the selective marker to inactivate BLH1). The initial
A - - -A~QI~I~:KRMSS S DISKINSWNKEFQSDLTHQEATTVLKNYNADD~LLNKTR HMD~LD I CERRAT D ~. . . . F ~ ; ~ ; A Q N V G . - . . . . . . . . . . . . . . . .
yeast B l h l p
rabbit BLM hydrolase
rabbit BLM hydrolase
L O K Q D N R~ N T ~V S T D S T ~ - - - - - Q HV~Q H V~ P Q E G
yeast Blhlp rabbit BLM hydrolase
L ; S Q A Y,L E ~ Y ;D;K L E K A N Y F E D F~S ~V ~ ~W~V E R C Y FF~N
yeast B l h l p
rabbit BLM hydrolase
M F L MLVN
yeast Blhlp rabbit BLM hydrolase
SADDS VT LR EQMQRE G~ T K A Ei S A T O D T M M EE
yeast Blhlp
S - ~P~EFA P I ~..... E F Y
yeasl BIhlp
rabbit BLM hydrolase
......
L~ A A T Q L N V L S E ~N L K S~ E I F ; S C L;N V M R L ; P F M K K ~ N I E
Q I VS S ~D Q D - - DSR A FVD T~Q K K E P EbG~
L V K ~ YG L MPK D L Y G D L P Y S T T ASR ~ ~E~(~VV KCFP~SH T ~ E ~ R
S K YAKR Q H V~P
QG~
~ ~O¥ L L A A P T E OiGG~Y VO~ L LM N P ~NDGG~WD
S
K WN S L L T T K L R E F A E T LR T A L K E R L N H K MREF C I R ~R N - M VHS
R MN D.
~R L M S L FMD I PP VQ P NEQF~E V V C I C L G NP P .... E T F~W
¥ VDKDK K I H TIK E Y ROK D K N Y O K | G
- L D P S T P V S L INOPR - - H P~G K~I K I DR EGN V L~GD L F N M E D K I C F VN~P R P Q H KYN REY T VD YESNM V~R
A - - K
B Blhlp
~
BLM hydrolase cathepsin H cathepsin L cathepsin S CP1 CP2 cruzain papain aLeurain oryzain y calpain
.l!~SC
L 133aa - - V N H ~ V ~ A V~ 130u- - L DH'GV;~!VV~-
~
-
-
-P'V K D'OG - T D I C~G
H C~
S!~P
A L~D~W~
S T ,T ~.is~ m. ~s~v S T ~ ~ S TT L ~ I -
131=a- - V N H G V ~ V V , f ~ Z34a=L DI;fG I ~ ! i V Y 130aa -
L D H G V ~ V V~G -
126aa131aa132aa149aa-
L DHG V~ L V~VDB~VAAV~VNH~V~AV~V N~V~A V~KG~Y SV~A -
Z2eaa
11aa - - : ~ K P ~ . S .
N W~N N~Y
F -
:1.5. . . .
~ K ~ E W ~ M ~ Y I ~ l a a - -'VK,N~_~p~I~L H F ~ D Q ~ Y I ZGa=- - ~ K I t ~ W ; ~ G A B W ~ E Q E Y I 4eaa - S Zl . . . . I KN,~T
7aaZlaa11aa 11aa-
W~ I K~Y
T QW~E
E~Y N~Y - I K~S~W~A~D'WQDN~Y - I KN ~Wi~AOWGD NGY - I R~ pW!~QV EWTGAWS
- I KN~'8~,GTGW~E
I -
I I F F
-
Fig. 3. Comparison of the deduced amino acid sequence of BLH1 to other cysteine proteinases: (A) residues identical to the known sequence of rabbit bleomycin hydrolase [6] at corresponding positions are shaded. (B) Alignment of Blhlp with other cysteine proteinases in the regions containing the putative active-site cysteine, histidine and asparagine (indicated by stars) [14]. Amino acids identical to Blhlp are shaded. Sequences were taken from [6,14-19].
303
ratio of 55% blhl mutant and 45% wild-type cells changed after two days (i.e., 26 generations) to 27% mutant and 73% wild-type cells, indicating a slightly higher sensitivity of the mutant cells to the drug. In contrast, the ratios of both types of ceils did not change significantly in the absence of bleomycin (55%/45% versus 56%/44% blhl/BLH1 cells). These results make it likely that BLH1, in fact, encodes a cysteine proteinase capable of hydrolyzing bleomycin. The lack of a detectable phenotype in blhl mutant cells in the absence of bleomycin as well as the only slightly increased sensitivity to bleomycin suggest that yeast may contain other proteinases with overlapping substrate specificities in addition to bleomycin hydrolase. We thank E.-M. Wetekam for excellent technical assistence. The work was supported by a grant of the Deutsche Forschungsgemeinschaft to W.B. (Ba415/194).
References 1 Umezawa, H., Maeda, K., Takeuchi, T. and Akami, Y. (1966) J. Antibiot. Ser. A 19, 200-209. 2 Povirk, L.F. (1983) In Molecular Aspects of Anti-Cancer Drug Action (Neidle, S. and Waring H., eds.), pp. 157-181, Macmillan Publisher, London.
3 Lazo, J.S. and Humphreys, C.J. (1983) Proc. Natl. Acad. Sci. USA 80, 3064-3068. 4 Akiyama, S.I., Ikezaki, K., Kuramochi, H., Takahashi, K. and Kuwano, M. (1981) Biochem. Biophys. Res. Commun. 101, 55-60. 5 Lazo, J.S., Sebti, S.M. and Filderman, A.E. (1987) In Metabolism and Mechanism of Action of Anti-Cancer Drugs (Powis, G. and Prough, R.A., eds.), pp. 194-210, Taylor and Francis, London. 6 Sebti, S.M., Mignano, J.E., Jani, J.P., Srimatkandada, S. and Lazo, J.S. (1989) Biochemistry 28, 6544-6548. 7 Miiller, G. and Bandlow, W. (1991) Biochemistry 30, 10181-10190. 8 Bennetzen, J.L. and Hall, B.D. (1982) J. Biol. Chem. 257, 30263031. 9 Buchman, A.R. and Kornberg, R.D. (1990) Mol. Cell. Biol. 10, 887-897. 10 Magdolen, V., Oechsner, U., Miiller, G. and Bandlow, W. (1988) Mol. Cell. Biol. 8, 5108-5115. 11 Devereux, J., Haeberli, P. and Marquess, P. (1984) Nucleic Acids Res. 12, 387-395. 12 George. D.G., Barker, W.C. and Hunt, L.T. (1986) Nucleic Acids Res. 14, 11-17. 13 Sikorski, R.S. and Hieter, P. (1989) Genetics 122, 19-27. 14 Eakin, A.E., Mills, A.A., Harth, G., McKerrow, J.H. and Craik, C.S. (1992)J. Biol. Chem. 267, 7411-7420. 15 Higgins, D.G., McConnell, D.J. and Sharp, P.M. (1989) Nature 340, 604. 16 Watanabe, H., Abe, K., Emori, Y., Hosoyama, H. and Arai, S. (1991) J. Biol. Chem. 266, 16897-16902. 17 Ritonja, A., Colic, A., Dolenc, I., Ogrinc, T., Podobnik, M. and Turk, V. (1991) FEBS Lett. 283, 329-331. 18 Pears, C.J., Mahbubani, H.M. and Williams, J.G. (1985) Nucleic Acids Res. 13, 8853-8866. 19 Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984) Nature 312, 566-570.
299
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 90434
Short Sequence-Paper
A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases Ulla Magdolen a, Giinter Miiller b, Viktor Magdolen c and Wolfhard Bandlow Institut fiir Genetik und Mikrobiologie, Miinchen (Germany), b c / o Hoechst Aktiengesellsehaft Frankfurt am Main, Frankfurt (Germany) and " Max-Planck-lnstitut fiir Biochemie, Martinsried (Germany)
(Received 28 September 1992)
Key words: Bleomycinhydrolasegene; Antitumor drug; Cysteineproteinase; (S. cerevisiae) We have purified bleomycin hydrolase from yeast (molecular mass 55000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blhl mutant cells. Bleomycin is a mixture of closely related glycopeptide antibiotics secreted from Streptomyces verticillus [1]. It causes radiomimetic D N A damage in bacteria and eukaryotes. It also inhibits growth of a number of squamous and small cell type tumors in situ and in vitro, although its exact mechanism of action is unknown. Due to its lack of hepatic, renal and bone marrow toxicities, the drug is extensively used in tumor therapy [1,2]. A more general use of the drug is, however, primarily hampered by two drawbacks, (i) the pulmonary toxicity [3] and (ii) the resistance of certain cell types and tissues to the drug due to high intracellular activities of the thiol proteinase bleomycin hydrolase [4-6]. The ubiquitous and abundant occurrence of this enzyme in all animal tissues and in microorganisms points to an important, although as yet unknown function. In order to study bleomycin resistance, to identify the physiological role of the proteinase and to understand why certain tumors are refractory to therapy by the drug, it may be helpful that we have isolated a yeast gene displaying high homology
Correspondence to: W. Bandlow, Institut fiir Genetik und Mikrobiologie, Maria-Ward-Strasse la, D-8000 Miinchen 19, Germany. The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number X69124.
of the encoded protein to bleomycin hydrolase from rabbit. We have isolated the bleomycin hydrolase protein from yeast during a study of a cAMP receptor glycolipid-anchored to plasma membranes [7]. We have enriched the latter protein by isolating crude plasma membranes from yeast spheroplasts by Percoll gradient centrifugation and sequential purification of the detergent-solubilized protein by cAMP-agarose affinity chromatograpy, molecular sieving and phenyl Sepharose lipophilic chromatography which uses the hydrophobicity of the glycolipid anchor-containing protein as a parameter for separation. When the eluted proteins were separated on a sodium dodecylsulfate polyacrylamide gel, one of the major bands comigrated with the authentic, 8-N3-[32p]cAMP photoaffinity-labeled product (apparent molecular mass 54 kDa, as deduced from electrophoretic mobility) [7]. This material was excised from the gel, eluted and cleaved either with trypsin or CNBr. The peptides were separated by high performance liquid chromatography. Two major fragments (one from each cleavage) were sequenced (indicated in Fig. 2). It turned out from the sequence data (see below) that they obviously do not belong to the glycosyl-phosphatidylinositol-anchored cAMP-binding protein from yeast plasma membranes. Whether the enriched protein with homology to bleomycin hydrolase binds to cAMP agarose and phenyl Sepharose directly or interacts with the native cAMP receptor protein
300 fortuitously or in a functional complex remains to be established in the future. The corresponding gene was isolated by using the polymerase chain reaction method. Since the intramolecular arrangement of the two eluted and sequenced peptides was unknown, three primers were designed from each of them, two overlapping primers in the one and a third one in the reverse direction. Pairwise amplification by polymerase chain reaction and crosswise hybridization led to the identification of a DNA fragment, which encoded sequences from both protein fragments. The genomic DNA clone, pPMY 11.0, subsequently isolated from a gene bank constructed from BamHI-digested yeast DNA, contained about 11 kbp of genomic DNA and was characterized by restriction and hybridization analyses. The region cross-hybridizing to the amplified DNA fragment could be narrowed down to two HindIII fragments, which were sequenced subsequently from the central HindIII site in both directions to yield the complete DNA sequence of the yeast BLH1 gene (Fig. I A, B). The nucleotide sequence analysis and the deduced primary structure of the protein are displayed in Fig. 2. The open reading frame comprises 483 triplets with a deduced molecular mass of the encoded protein of 55.5 Da, the calculated isoelectric point is at pH 9.58. Structural analyses by using the algorithms of Kyte and Doolittle, and Hopp and Woods do not reveal any striking structural features and suggest a globular, hydrophilic protein. Southern hybridization with a labeled DNA fragment, derived from the coding region of the BLH1 gene shows that BLH1 occurs in single copy in the yeast genome (one example is given in Fig. 1D, lane 1). The codon bias index [8] of 0.35 points to a relatively high expression. The DNA sequence flanking the gene at its 5' side displays no obvious T A T A box. However, many yeast genes are known which lack obvious T A T A sequences. In the putative promoter region of BLH1 a perfect consensus sequence for an abundant DNA binding protein, Grf2p [9], is found, immediately followed by a pyrimidine-rich sequence (Fig. 2). Grf2p may have a role in nucleosome positioning by excluding nucleosomes from the vicinity of its binding site. The same combination of sequence motifs as in the BLHl-upstream region also occurs in the promoter region of the yeast profilin gene, PFY1 [10]. Deletion of these elements in the PFY1 promoter dramatically reduce transcription of PFY1 (H. Herrmann and W. Bandlow, unpublished results). Data base searches using the Genetics Computer Group (GCG) Sequence Analysis Software Package [11] reveal significant sequence similarities to bleomycin hydrolase, a cysteine proteinase isolated from rabbit lung and liver. A cDNA fragment encoding the central portion of this enzyme has been obtained from rabbit liver and sequenced [6]. The alignment of the deduced
A E
HH
EHE
I
II
III
H I
I
I lOOO bp I
B
I I
N
I
C
Jl
EH
[
Jl
E
11oo bp
4
I 500 bp
CN b
C I N
""~-~--Jj C
EH
I
E
# I~ it
N
C
2500 bp
EH
I E
blh 1
II CN
3500 bp
D 1
Jme
2
3
4
5
~
~
~
3.5
kbp
~
~
~
2.5
kbp
i
m
I.I
kbp
Fig. h Inactivation of the BLH1 gene. (A) Genomic restriction map of the BLH1 locus. The arrow indicates direction of transcription. (B) Restriction map of the sequenced region. Restriction sites relevant for gene disruption are shown. (C) Genomic organization after integration of the linearized plasmid pPMP9-A at the BLH1 locus by homologous recombination. Vector sequences between the duplicated BLHl-coding sequences (indicated by thick bars) are not drawn to scale. (D) Genomic Southern blot from wild-type and disruption mutants. Genomic DNA was isolated from the haploid wild-type strain W303-1A (lane 1), from haploid disruption strains (lanes 2 and 3), from heterozygous diploid strains (lanes 4 and 5) and restricted with Ndel. The blots were probed with a digoxigeninlabeled fragment corresponding to the BLH1 fragment cloned into pPMP9-A (indicated by thick bars in (C), C, Clal; E, EcoRI; H, HindIlI; N, Ndel.
rabbit and yeast protein sequences is shown in Fig. 3A. Within the region of the known rabbit sequence, the homology is highly significant (40 S.D. units, using the program A L I G N [12] with the mutation data matrix, 100 random runs and a penalty for a gap of 20). The yeast sequence extends beyond the known rabbit sequence by 21 amino acids to the N-terminus and 158 residues to the C-terminus. The overlap of the two proteins contains 43% identical amino acids and 22% conservative exchanges at comparable positions (Fig.
301 3A). The search for protein motifs and domains reveals that a stretch of 17 amino acids (pos. 92-108 of Blhlp) is highly homologous to the region containing the cysteine residue conserved in the catalytic centre of eukaryotic thiol proteinases (Fig. 3B). These include calpain, plant and protozoan cystein proteinases, vertebrate lysosomal and secretory cathepsins, and two developmentally co-regulated cysteine proteinases from the slime mold Dictyostelium discoideum. In addition,
it can be seen from Fig. 3B that Blhlp from yeast also has significant homology to two sequence blocks of other cysteine proteinases (which were previously unknown from the rabbit enzyme) implied to contribute to the active centre, but conserved only in a subset of thiol proteinases. In order to gain some information about the physiological role of the protein, we replaced the structural BLH1 gene with two different incomplete blhl alleles
260 GRF2 ............. C G T A T A T T C A G T G C T T T T T G A A A T T T A G A A A A T A G T C G 6 ~ T T A T T G A T T A T G C T A C A T A T G T T A T T T T G C T T T A G T A T G T G T C A A A T A G C A T G C A A T C G C C C G G T A C T G A G T G A C A C A T C CTTT'~FFCCC
C T C C C A C C A C A A A G T C A C C T G C T C T C T T A A T C A A T G G G G T T G C A T G T G G T A A T C A A T T G A T A A C G C C G G C T G A C A A G T C G C C G A C G G G C A C C C A T A A A T A A A C G A T A A A T AGCCC~'FGCT C T T T T G T T A C
ATG C T T C C T A C T T C T G T T T C C CGG T C C TTG T A T CTC AAG ACG TTT AGA AGC CAC CTT T T A CGA G C A C C A C A A A T T G T T T T A A A A A G A ATG T C C T C T T C C ATC Met Leu Pro T h r Set Val Set Arg Set Leu Tyr Leu Lys Thr Phe Arg Set His Leu Leu Arg Ala Pro Gin lle Val Leu Lys Arg Met Set Ser Ser Ile
+103 GAT A T C A G T A A G A T C A A C T C T TGG A A C A A A G A G T T T CAA TCC G A C T T A ACC CAT C A A TTG GCA A C C A C T G T C C T T A A G A A T T A T A A T G C C G A T G A T G C A CTG A s p Ile Ser Lys Ile Asn Ser T r p Ash Lys Glu Phe Gin Set A s p Leu Thr His Gln Leu Ala Thr T h r Val Leu Lys Ash Tyr A s n A l a A s p A s p Ala Leu
~205 TTC A A C AAG A C T A G A CTG C A A A A G CAA G A T A A C A G G G T T TTC AAC A C T G T T G T C TCT ACT GAT TCC A C T C C A G T C A C C A A C C A A A A A A G C T C T G G T A G A TGT Leu Ash Lys Thr A r g Leu Gln LyS Gln A s p Ash Arg Val Phe Ash Thr Val Val Set Thr Asp Set Thr Pro Val Thr A s n G i n Lys Ser Set G I y A r g Cys
+307 TGG T T G T T T G C C G C T A C C A A T C A A C T G CGC TTG A A T G T T CTT TCT GAA TTG A A C T T A A A A GAA T T C C A A CTG T C C C A A G C T T A C T T G T T C ~ T A C G A T AAG Trp Leu Phe Ala Ala Thr Ash Gln Leu A r g Leu Ash Val Leu Ser Glu Leu Ash Leu Lys Glu Phe C l u Leu Set Gln A l a T y r Leu Phe Phe T y r A S p Lys
+409 TTG G A A AAG G C C A A C T A T T T C TTG G A C CAA A T C G T C T C C TCG G C T G A T C A A G A C A T C G A C TCA C C T C T C GTG CAA T A C T T G T T A G C A G C T C C A A C A G A A GAC Leu G l u Lys Ala Ash Tyr Phe Leu A s p Gln Ile Val Ser Set Ala A s p Gln A s p Ile Asp Ser A r g Leu Val Cln Tyr Leu Leu Ala Ala Pro Thr Glu ASp
+511 GGT G G C C A A T A C A G C A T G q ~ T TTG A A C C T A G T C A A G AAG TAT G G C C T T ATG CCC AAA G A T TTA T A C G G G G A T C T A C C T T A T T C T A C T A C T G C G T C C A G A AAA Gly Gly Gln Tyr Ser Met Phe Leu Ash Leu Val Lys Lys Tyr Gly L e u Met Pro Lys A s p Leu Tyr Cly A s p Leu Pro Tyr Ser Thr Thr A l a Set Arg Lys
+613 TGC A A T T C T C T G TTG A C T A C T A A A C T G A G A G A A T T T G C C GAG A C T C T A AGA A C A G C T TTG AAA G A G C G T T C T G C C C A T G A T T C C A T A A T T G T C A C T C T G A G A Trp Asn Set Leu Leu Thr Thr Lys Leu A r g Glu Phe Ala Glu Thr Leu Arg Thr Ala Leu Lys Glu Arg S~r Ala A S p Ash S~r Ile Ile Val Thr Leu Arn primer I-A +715 GAG C A A A T G C A A A G A G A A A T C T T C A G G T T G ATG TCG TTG TTC A T G GAC A T A C C T C C A GTG CAA C C A A A C GAG C A A T T C A C T T G G G A A T A C G T T G A C A A A GAC Glu Gln M~t Gln Arm Clu Ii~ Phe Aro Leu Met get Leu Phe Met A s p Ile Pro Pro Val Gln Pro A s n Glu Gln Phe Thr T r p Glu T y r Val A s p Lys ASp
+817 AAG AAA A T C C A C A C T A T C A A A TCG A C T C C G T T A G A A T T T GCC T C C A A A T A C GCA A A A T T G GAC C C T T C C A C G CCA G T C T C A T T G A T C A A T G A T C C A A G A CAC Lys Lys Ile His Thr Ile Uys Set Thr Pro Leu Glu Phe Ala Ser Lys Tyr Ala Lys Leu Asp Pro Ser Thr Pro Val Set Leu Ile A s n A s p Pro Arg His
+919 CCA T A T G G T A A A T T A A T T AAG A T C G A T C G T T T A G G A A A C GTC C T T GGC GGA G A T G C C CTG A T T T A C T T A A A T C T T G A C A A T G A A A C A C T A T C T A A A TTG G T T Pro Tyr G l y Lys Leu Ile Lys Ile A s p Arg Leu Gly Ash Val Leu Cly Gly Asp Ala Val Ile Tyr Leu A s n Val A s p Asn C l u T h r Leu Set Lya Leu Val
+1021 Wv GTT A A G A G A T T A C A A A A T A A C A A A G C T G T C T T T T T T GGA T C ~ C A C A C T CCA A A G T T C A T G GAC A A G A A A A C T G G T G T C A T G G A T A~'F G A A T T G T G G A A C TAT Val Lys A r g Leu Gln ASh Asn Lys Ala Val Phe Phe Cly Set His Thr Pro Lys Phe Met A s p Lys Lys Thr Gly Val M~t A s h II~ ~ ] u Leu T r D ASh "~vr p r i m e r 2-A
/
primer 2-C
C C T G C C A T C G G C T A T A A T ~ A C C T C A G C A A AAG G C A TCG CGT A T T AGA T A C C A T GAA A G T TTG A T G A C T C A T G C T ATG ~ G A T C A C T G G C T G C C A C G T C G A T Pro A l a If@ G l v Tvr Ash Leu Pro C]n Gln Lvs A l a Sat Arg Ile Arg Tyr His GIu Set Leu Met Thr His A l a Met Leu Ile Thr G l y CyS His Val Asp
+1225 GAA A C G T C T A A A q~FA C C A C T T C G C T A C CGC G T T G A A A A T TCC TGG G G T A A A G A C T C C G G T A A A G A C G G A T T A T A C G T G A T G A C T C A A A A G T A C T T C G A G GAG Clu T h r Ser Lys Leu Pro Leu A r g Tyr A r g Val Glu A S h Set Trp GIy Lys A s p Set Gly Lys A s p G l y Leu Tyr Val Met Thr G l n Lys ~ r Phe Glu Glu
+1327 TAC T G C T T T C A A A T T G T G G T C G A T A T C A A T G A A T T G C C A AAA GAG CTG G C ~ TCA AAA T T C ACC T C A G G T A A G G A A GAG C C G A T T G T C T T G C C C A T C TGG G A C Tyr Cys Phe Gln Ile Val Val A s p Ile A S h Glu Leu Pro Lys Glu Leu Ala Set Lys Phe Thr Set Gly Ly$ G l u G l u Pro Ile Val Leu Pro Ile T r p Asp
+1429 CCA A T G G G T G C T TTG G C C A A A TAA Pro Met G I y Ala Leu A l a Lys ***
ATAGTT TCAGCAGCTC TGATGTAGAT ACACGTATCT CGACATGTTT TATTTTTACT ATACATACAT AAAAAGAAAT AAAAAATGAT AACGTGTATA
Fig. 2. Nucleotide and deduced amino acid sequences of BLH1. The putative Orf2p-binding site is overlined, the adjacent pyrimidine-rich region is indicated by dots. The two peptides sequenced from the purified protein are underlined. The DNA regions corresponding to the primers effective in PCR amplification are overlined by arrows. The sequences of these degenerate oligonucleotides were: 5' CAA/GATGCAA/GC/ A G I / C G A A / G A T (I-A); 5' CCIATI/CGCI/CGGA/GTAA/GTT (2-A); 5' TGT/CTGI/CGGI/CAA/GA/GTTA/GTA (2-C). I denotes inosine.
302 as detailed in Fig. 1C. For this purpose the coding sequence contained between the ClaI site at pos. + 100 and the EcoRI site at pos. +615 was cloned into the yeast integration vector pRS306 harbouring the URA3 gene as a selective marker [13] to yield clone pPMP9-A. After cleavage of pPMP9-A with HindlII (pos. +383 of the coding region of BLH1) the resulting linear plasmid DNA was transformed into either haploid or diploid yeast wild-type ceils. The correct integration of the plasmid by homologous recombination at the BLH1 locus leads to the duplication of those BLH1 DNA sequences cloned into pRS306, the resulting two incomplete copies being separated by sequences from the vector (Fig. 1C). The integration event was proven by Southern blotting of genomic DNA restricted with NdeI. The blot was probed with the BLHl-derived sequences cloned into pPMP9-A. DNA from wild-type cells yields a single signal corresponding to 1.1 kbp (Fig. ID, lanes 1), whereas the haploid disruption mutants display two signals as expected, corresponding to 2.5 and 3.5 kbp (Fig. 1D, lanes 2 and 3) instead of the 1.1 kbp band (cf. Fig. 1B and C). Heterozygous diploids contain both, the wild-type and the disrupted alleles. Consequently, all three signals can be seen with DNA from heterozygous diploid disruption strains (Fig. 1D, lanes 4 and 5).
blhl disruption mutants were found to be viable in the haploid state. When the growth behaviour of the haploid mutant and the isogenic wild-type strains was compared, no differences were observed on glucose, galactose and glycerol media. Also long-term experiments, where cells were shifted from one carbon source to another (eight changes of media), revealed no disadvantage for the blhl mutant strains compared to wildtype cells. In addition, heterozygous diploids sporulate well, and the spores germinate. It must be concluded from these results that the gene is dispensable for vegetative and meiotic growth, cell viability and differentiation. Finally, the haploid wild-type strain (with a functional BLH1 allele) was compared to the disruption strain with respect to growth in the presence of bleomycin. For this purpose co-cultivation experiments of the haploid strain W303-1A and of the isogenic blhl mutant strain were performed: cells of both strains pre-grown overnight in the presence of bleomycin were mixed and incubated for 2 days in the absence of 3.5 mg bleomycin/ml medium (which leads to about 50% growth inhibition). The ratio of the wild-type and mutant cells was monitored by replica-plating at the beginning of the experiment and after two days of growth by testing uracil auxotrophy (URA3 has been used as the selective marker to inactivate BLH1). The initial
A - - -A~QI~I~:KRMSS S DISKINSWNKEFQSDLTHQEATTVLKNYNADD~LLNKTR HMD~LD I CERRAT D ~. . . . F ~ ; ~ ; A Q N V G . - . . . . . . . . . . . . . . . .
yeast B l h l p
rabbit BLM hydrolase
rabbit BLM hydrolase
L O K Q D N R~ N T ~V S T D S T ~ - - - - - Q HV~Q H V~ P Q E G
yeast Blhlp rabbit BLM hydrolase
L ; S Q A Y,L E ~ Y ;D;K L E K A N Y F E D F~S ~V ~ ~W~V E R C Y FF~N
yeast B l h l p
rabbit BLM hydrolase
M F L MLVN
yeast Blhlp rabbit BLM hydrolase
SADDS VT LR EQMQRE G~ T K A Ei S A T O D T M M EE
yeast Blhlp
S - ~P~EFA P I ~..... E F Y
yeasl BIhlp
rabbit BLM hydrolase
......
L~ A A T Q L N V L S E ~N L K S~ E I F ; S C L;N V M R L ; P F M K K ~ N I E
Q I VS S ~D Q D - - DSR A FVD T~Q K K E P EbG~
L V K ~ YG L MPK D L Y G D L P Y S T T ASR ~ ~E~(~VV KCFP~SH T ~ E ~ R
S K YAKR Q H V~P
QG~
~ ~O¥ L L A A P T E OiGG~Y VO~ L LM N P ~NDGG~WD
S
K WN S L L T T K L R E F A E T LR T A L K E R L N H K MREF C I R ~R N - M VHS
R MN D.
~R L M S L FMD I PP VQ P NEQF~E V V C I C L G NP P .... E T F~W
¥ VDKDK K I H TIK E Y ROK D K N Y O K | G
- L D P S T P V S L INOPR - - H P~G K~I K I DR EGN V L~GD L F N M E D K I C F VN~P R P Q H KYN REY T VD YESNM V~R
A - - K
B Blhlp
~
BLM hydrolase cathepsin H cathepsin L cathepsin S CP1 CP2 cruzain papain aLeurain oryzain y calpain
.l!~SC
L 133aa - - V N H ~ V ~ A V~ 130u- - L DH'GV;~!VV~-
~
-
-
-P'V K D'OG - T D I C~G
H C~
S!~P
A L~D~W~
S T ,T ~.is~ m. ~s~v S T ~ ~ S TT L ~ I -
131=a- - V N H G V ~ V V , f ~ Z34a=L DI;fG I ~ ! i V Y 130aa -
L D H G V ~ V V~G -
126aa131aa132aa149aa-
L DHG V~ L V~VDB~VAAV~VNH~V~AV~V N~V~A V~KG~Y SV~A -
Z2eaa
11aa - - : ~ K P ~ . S .
N W~N N~Y
F -
:1.5. . . .
~ K ~ E W ~ M ~ Y I ~ l a a - -'VK,N~_~p~I~L H F ~ D Q ~ Y I ZGa=- - ~ K I t ~ W ; ~ G A B W ~ E Q E Y I 4eaa - S Zl . . . . I KN,~T
7aaZlaa11aa 11aa-
W~ I K~Y
T QW~E
E~Y N~Y - I K~S~W~A~D'WQDN~Y - I KN ~Wi~AOWGD NGY - I R~ pW!~QV EWTGAWS
- I KN~'8~,GTGW~E
I -
I I F F
-
Fig. 3. Comparison of the deduced amino acid sequence of BLH1 to other cysteine proteinases: (A) residues identical to the known sequence of rabbit bleomycin hydrolase [6] at corresponding positions are shaded. (B) Alignment of Blhlp with other cysteine proteinases in the regions containing the putative active-site cysteine, histidine and asparagine (indicated by stars) [14]. Amino acids identical to Blhlp are shaded. Sequences were taken from [6,14-19].
303
ratio of 55% blhl mutant and 45% wild-type cells changed after two days (i.e., 26 generations) to 27% mutant and 73% wild-type cells, indicating a slightly higher sensitivity of the mutant cells to the drug. In contrast, the ratios of both types of ceils did not change significantly in the absence of bleomycin (55%/45% versus 56%/44% blhl/BLH1 cells). These results make it likely that BLH1, in fact, encodes a cysteine proteinase capable of hydrolyzing bleomycin. The lack of a detectable phenotype in blhl mutant cells in the absence of bleomycin as well as the only slightly increased sensitivity to bleomycin suggest that yeast may contain other proteinases with overlapping substrate specificities in addition to bleomycin hydrolase. We thank E.-M. Wetekam for excellent technical assistence. The work was supported by a grant of the Deutsche Forschungsgemeinschaft to W.B. (Ba415/194).
References 1 Umezawa, H., Maeda, K., Takeuchi, T. and Akami, Y. (1966) J. Antibiot. Ser. A 19, 200-209. 2 Povirk, L.F. (1983) In Molecular Aspects of Anti-Cancer Drug Action (Neidle, S. and Waring H., eds.), pp. 157-181, Macmillan Publisher, London.
3 Lazo, J.S. and Humphreys, C.J. (1983) Proc. Natl. Acad. Sci. USA 80, 3064-3068. 4 Akiyama, S.I., Ikezaki, K., Kuramochi, H., Takahashi, K. and Kuwano, M. (1981) Biochem. Biophys. Res. Commun. 101, 55-60. 5 Lazo, J.S., Sebti, S.M. and Filderman, A.E. (1987) In Metabolism and Mechanism of Action of Anti-Cancer Drugs (Powis, G. and Prough, R.A., eds.), pp. 194-210, Taylor and Francis, London. 6 Sebti, S.M., Mignano, J.E., Jani, J.P., Srimatkandada, S. and Lazo, J.S. (1989) Biochemistry 28, 6544-6548. 7 Miiller, G. and Bandlow, W. (1991) Biochemistry 30, 10181-10190. 8 Bennetzen, J.L. and Hall, B.D. (1982) J. Biol. Chem. 257, 30263031. 9 Buchman, A.R. and Kornberg, R.D. (1990) Mol. Cell. Biol. 10, 887-897. 10 Magdolen, V., Oechsner, U., Miiller, G. and Bandlow, W. (1988) Mol. Cell. Biol. 8, 5108-5115. 11 Devereux, J., Haeberli, P. and Marquess, P. (1984) Nucleic Acids Res. 12, 387-395. 12 George. D.G., Barker, W.C. and Hunt, L.T. (1986) Nucleic Acids Res. 14, 11-17. 13 Sikorski, R.S. and Hieter, P. (1989) Genetics 122, 19-27. 14 Eakin, A.E., Mills, A.A., Harth, G., McKerrow, J.H. and Craik, C.S. (1992)J. Biol. Chem. 267, 7411-7420. 15 Higgins, D.G., McConnell, D.J. and Sharp, P.M. (1989) Nature 340, 604. 16 Watanabe, H., Abe, K., Emori, Y., Hosoyama, H. and Arai, S. (1991) J. Biol. Chem. 266, 16897-16902. 17 Ritonja, A., Colic, A., Dolenc, I., Ogrinc, T., Podobnik, M. and Turk, V. (1991) FEBS Lett. 283, 329-331. 18 Pears, C.J., Mahbubani, H.M. and Williams, J.G. (1985) Nucleic Acids Res. 13, 8853-8866. 19 Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984) Nature 312, 566-570.