eur j biochem 1991 nakayama

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Eur. J . Biochem.202, 569-574 (1991)(i) FEBS 1991

The medium chainsof the mammalian clathrin-associated proteinshave a homolog in yeastYdsuhiro NAKAY AMA, Mark GOEBL, Betsy OBRINE GRECO, andy LEMMON4,Elizabeth PINGCHANG CHOW nd Tomas KIRCHHAUSEN

Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, USADepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, USABiogen lnc., Cambridge, USADepartmentof Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, USA(Received June lX/August 13, 1991)- EJ B 91 0791

We have cloned and sequenced mouse brain AP47, the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1. The predicted protein sequence of AP47 is closely related to rat and calf brainAP50, the corresponding medium chain of the plasma-membrane clathrin-associated protein complex AP-2. Wehave also identified in the yeast genome an open reading frame encoding a protein of previously unknown function.Referred to here as YAP54, its predicted protein sequence displays a striking homology to AP47. We thereforepropose that Yap54 is the medium chain subunit of a putative AP-1 complex in yeast. From the analyses of theoptimized sequence alignments of AP47, AP50 and Yap54p, we suggest a model for the domain organization ofthe medium chains.

AP-1 and AP-2 are the two main classes of clathrin-associ-ated protein complexes [AP; also referred to as assembly pro-teins or adaptors (Ahle et al., 1988; Robinson and Pearse,1986; Zaremba and Keen, 1983; Pearse, 1988)]. They arefound in clathrin-coated pits and vesicles of the trans-Golginetwork (AP-1) and at the plasma membrane (AP-2) of mam-malian cells (Ahle et al., 1988; Robinson and Pearse, 1986).These complexes are heterotetrameric structures composed oftwo large chains of z 100 kDa (y and /3 in AP-1; C and /3 inAP-2), one medium chain of z 50 kDa (AP47 in AP-1 orAP50 in AP-2) and one small chain of z 17 kDa (AP19 inAP-1 or AP17 in AP-2) (see schematic representationin Fig. 4;Ahle et al., 1988; Virshup and Bennett, 1988; Matsui andKirchhausen, 1990). Although the functionof these complexesis still not well understood, they are believed to promotethe assembly of clathrin coats and to direct the sorting ofmembrane proteins transported by coated vesicles (Zarembaand Keen, 1983; Pearse, 1988; Glickman, 1989; Kirchhausenet al., 1989).Recent invitroexperiments suggest that the medium chainsmay be involved in a regulatory switch of A P activity. Anumber of studies have shown that the mammalian AP50 selfphosphorylates and that it is a good substrate for phos-phorylation by a serinelthreonine kinase found in coated ves-Correspondence toT . Kirchhausen, Harvard Medical School, De-partment of Anatomy and Cellular Biology, 220 Longwood Ave,Boston, MA 02115, USAAbbreviations.AP, clathrin-associated protein complex; AP-I andAP-2, AP on clathrin-coated structures from the trans-golgi networkand plasma membrane, respectively; AP47 and AP50, medium chainsfrom AP-1 and AP-2, respectively.Note. The novel nucleotide sequence data published here havebeen deposited with the EMBL/GeneBank sequence data bank and

are available under accession number M 62419 for AP47 and X602XXfor YAP54.

icles (Pauloin et al., 1982; Campbell et aI., 1984; Manfrediand Barzari, 1987; Keen et al., 1987). Furthermore, AP47 maybe a serine kinase capable of phosphorylating the cytoplasmictail of the mannose 6-phosphate receptor (Meresse et al.,1990).Although yeast cells also contain clathrin-coated vesicles(Mueller and Branton, 1984; Payne and Schekman, 1985;Lemmon et al., 1988), the existenceof AP in yeast has notbeen documented directly. However, their existence has beenpostulated based on the recent identification of putativehomologs of the mammalian PIP large chains (K irchhausen,1990) and AP17/AP19 small chains (Kirchhausen et al., 1991)by computer searches of the published yeast genome DNAdata base.Previously we cloned a cDNA for the rat brain AP50subunit of AP-2 and found that the predicted sequence wasessentially identical to the partially sequenced bovine brainAP50 (Thurieau et al., 1988). This remarkable sequence con-servation among mammalian species has also been observedwith the large chains (K irchhausen et al., 1989; Ponnambalamet al., 1990; Robinson, 1989; Tucker et al., 1990) and smallchains (Kirchhausen et al., 1991) of the AP complexes. Wehave assumed that this theme of sequence conservation alsoholds for the medium AP47 chain and have therefore usedcalf brain AP50 to obtain protein sequence information suit-able for oligonucleotide synthesis and cDNA library screens.We now report the cloning and sequencing of a mouse brainAP47 cDNA which encodes a protein closely related to themammalian AP50. Neither protein has significant sequencesimilarity with known kinases. We also report a computersearch that has uncovered from the yeast genome a previouslypublished sequence (Daignan-Fornier and Bolotin-Fukuhara,1989), here referred to as YAP.54, that encodes Yap54p, aprotein displaying very extensive protein sequence identitywith the mammalian AP47 and AP50 chains. Analysis of the


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< - s t a r t Regi on IAGCTTGCTGAACGCCCGCAGTTCCTGCCGTTGCCGCCGCCCTCGGCTACTGCCGAGACCT TCCAAAGCCGCCATGTCCGCCAGCGCCGTCTACGTACTG

M S A S A V Y V LGATCTGAAGGGCAAGGTGCTCATCTGCAGGAACTACCGTGGGGATGTGGACATGTCAGAGGTGGAGCACT TCATGCCCAT TC TGATGGAGAAGGAGGAGD L K G K V L I C R N Y R G D V D M S E V E H F M P I L M E K E EG A G G G C A T G C T G T C A C C T A T C T T G G C C C A T G G T G G C G T T C G T T T C A T G T G G A T T A A G C A C A A C A A C C T G T A C C T G G T C G C C A C T T C A A A A A A G A A T G C TE G M L S P I L A H G G V R F M W I K H N N L Y L V A T S K K N AT G T G T G T CA C T G G T G T T CT C C T TC C T C T A C A A G G T G G T A C A G G T CT T C T CC G A G T A C T T T A A G G A G T T G G A G G A G G A G A G C A T C C G A G A C A A C T T T G T CC V S L V F S F L Y K V V Q V F S E Y F K E L E E E S I R O N F VA T C A T C T A C G A G C T G CT A G A T G A G C T C A T G G A C T T TG G C T A C C C G C A G A C C A C TG A C A G C A A G A T C T T G C A G G A G T A C A T C A C T C A G G M G G C C A C M Gl I Y E L L D E L M D F G Y P Q T T D S K l L P E Y 1 T Q E G H K- - - - - - - - - - - - - - - - - - - - - - - - x - - - - - - - - -x - - - - - - - - - - - - - - - - - - - - - - - - - - - - - x - - -CTGGAAACGGGGGCCCCTAGGCCCCCAGCCACAGTCACCMTGCTGTGTCCTGGCGTTCAGAAGGCATCMGTATCGGAAGAATGAAGTAT TCCT GGATL E T G A P R P P A T V T N A V S W R S E G I K Y R K N E V F L D- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - x - - - - - x - - - - - - - - - 47.3.2GTCATTGAGGCTGTTMCCTCTTGGTCAGTGCCMTGGCAACGTGCTGCGCAGTGAGATTGTGGGTTCCATCAAGATGCGGGTCTTCCTCTCAGGCATGV I E A V N L L V S A N G N V L R S E I V G S I K M R V F L S G MCCTGAGTTACGCCTGGGTCTCAATGACAAGGTCCTCTTCGACAACACACCCCGACGGMGACCMGTCAGTGGAGCTGGAGGATGTGAAATTCCACCAGP E L R L G L N D K V L F D N T G R G K S K S V E L E D V K F H PT G T G T G C G G C T G T C A C G T T T T G A G MC G A C C G C A C T A T C T C C T T C A T C C C A C C C G A C G G A G A G T T T G A A C T C A T G T C C T A C C G C C T C MC A C C C A T G T GC V R L S R F E N D R T I S F I P P D G E F E L M S Y R L N T H V- - - - - - - - - x - - -A A G C C T T T G A T C T G G A T T G A G T C C G T G A T T G A G M G C A T T C C C A C A G C C G C A T T G A G T A C A T G G T C M G G C C M G A G C C A G T T C M G A G G C G G T C M C AK P L I U I E S V I E K H S H S R I E Y M V K A K S Q F K R R S T- - - - - - - - - - - - x - - - - - x - - x - - - - - - - - -7. .5 - - _ -GCCAACMTGTAGAGATCCATAT ACCAGTCCCCAACGATGCTGATTCACCCAAGTTCAAGACTACAGTGGGGAGTGTCMGTGGGTCCCTGMAACAGTA N N V E I H I P V P N D A D S P K F K T T V G S V K W V P E N S- - - - - - x - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - x - - - - - x - - - - - -b 7 2 3 - _ _ _ _ _ _ - - - _ _ _ - _ _GAGATCGTGTGGTCCGTCMGTCCTTTCCGGGTGGCAAGGAGTACCTGATGCGGGCCCACTTTGGCCTTCCCAGTGTGGAAGCTGMGACAAGGAGGGAE I V W S V K S F P G G K E Y L M R A H F G L P S V E A E D K E G_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - Tb84.85 - - - x - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCCCCCCATCAGCGTCMGTT TGAGATCCCCTACTTCACT ACCTCTGGCATCCAGGTGCGCTACCTGAAAATCAT TGAGMGAGTGGGTACCAGGCCK P P I S V K F E I P Y F T T S G I P V R Y L K I I E K S G Y Q A

- - - - - - - - - - - - - - - - - - - - - x - - - - - - - - - - - - - - - - - - 47 3 2 - _ _ _ _ - - - - _ _ _ -

47.3.1- - _ _ - - _ _ _ _ _ - _ _ _ _ _ _ _ _ _

Ta84.85. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Ta72.73. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R e g i o n I end-, CTGCCCTGGGTACGATATATCACACAGMCGGAGATTATCAGCTCCGGACCCAGTGAGAGGCCTCTGCCACCAGCCCAGCCCACCTAGCCTCAGGGACAL P U V R Y I T Q N G D Y Q L R T QT121.122. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T77_ _ - - - - - _ _ - - -C C T G C A C A C T C A C C A A A A C T G A A G C T A G A G G G T G G C C C T G G A C A T G C A G C C A C C C T C T C C T C A A G C C T G A G T G G A C T C A C A C A G MC C C C T T C C C C G G TC C C A T T C T G A T C C G A G G G T G G G A A G G A A G G G C C T G C C A G C C T C C C C C A G G G A C A A G C C A G C T G G A G C T G T G C T C C T G G T G C C T C A T G A C T G G C C A C C C AC C A C C C T G T A G A C G C C A T C C C T G C C C A C C T C C G A A G C C T C C T T C T G T T G C C A T T T T G C T G A G C A T A G T T G G C T C A T T G T C A T T G T C A T C T G T G G C T G T CCTGTCACCTTTCTTCAAGTGTCCTGTGCAGCTGCCATGCTGCACTTAATGAGGGCAGCTGTCCCCTATCCATGCTGGTCTGTATGCCGGATAGTTGCTCTGCCTGGGCCGGCTGTGCCTCCCTTGCCAGCCCTTGACCACATCAGTGTTTTCTCAGAAGGGCACATGGCCTCAGCCTTTGCCCTAAAATTCCTGGGCAGGCACAGGCTACCCTCACTGTACAAGGCCTCGTTGTCCTGGACCCATGTGTGTGTGTGACAGTTAT~ATCCATTT GTTACAAAAAAAAAAAAAAA A A A A 1913

2791264222575324108423141522174

621207720240819273918306101733911163721215405

131442314131512161I171018091908

Fig. 1. Complete nucleotide and deduced protein sequence qf the mouse brain cDNA fo r AP47. The nucleotide sequence corresponds to thelongest cDN A clone (pYN lO l ) and is otherwise identical to three other shorter and overlapping cDNA clones. The underl ined sequencesindicate the identity between the N-terminal sequences determined from selected tryptic fragments of calf brain AP47 and the corrcspondingregions of the mouse cDNA ; X denotes amino acids whose identity by Edman degradation was ambiguous. The putative polyadenylationsignal ATTAAA is underlined. Regions I and I1 define the two most conserved portions among the medium chains (see sequence alignmentin Fig. 3)

alignment of AP47, AP50 and YAP54 sequences lead us toidentify portions of the proteins that may be specific for thefunction of AP47 and AP50 and to proposeahypothesis fora possible domain organization of the medium chains in theAP complexes.

MATERIALS AND METHODSPurification of calf'brain AP47

Samples of AP complexes were obtained from depolymer-ized coats of calf-brain coated vesicles by sizing chromatog-raphy as described previously (Matsui and Kirchhausen,1990). AP-1 complexes were separated from AP-2 complexes

using Sepharose-Q (Ahle et al., 1988; Matsui and Kirch-hausen, 1990) followed by hydroxylapatite ionic-exchangechromatography (Pearse and Robinson, 1984). The samplescontaining AP-1 complexes were pooled and its polypeptidechains fractionated by preparative 6% or 12% SDSjPAGE(Laemmli, 1970). The band corresponding to calf brain AP47was obtained from each type of gel by either overnight dif-fusion at 37C (into10vols 20 mM Tris, 1 mM CaCl,, 0.01%SDS, pH 7.9) or electroelution at 25C (into 50 mM Tris,384mM glycine, 0.01% SDS, pH 8.3) and concentrated bycentrifugation in a Centricon-30 filter (Amicon). Internalamino acid sequence information for the purified calf brainAP47was obtained from the automated Edman degradationof fragments generated by tryptic digestion and HPLC frac-


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571tionation (Huang et al., 1986). N-terminal sequencing of theintact AP47 was performed after preparative 6%SDSjPAGE(Laemmli, 1970) and electrotransfer onto a poly(viny1di-fluoride) membrane (Immobilon, Milipore) (Matsudaira,1987; Kirchhausen et al., 1989; Kirchhausen et al., 1991).cDNA isolation, DNA sequencing and DNA blots

Based on the sequences of the tryptic fragments TI 21.122,47.3.1 and Tb84.85, three oligonucleotide probes of degener-ate sequence 3-TTI AGI CCI ATG/A GTC/T CGI GAI GGIACC CAI G/TCI ATG/A TAI TGI GTC/T TTG/A CCI CTG/CCI AT1 T/AC/GI GT l AA and 5-TGG GTI CCI GAG/AAAC/T T/AC/GI GAG/A AT1 GTI TGG T/AC/GI AT1 AAwere prepared and used at low stringency conditions to screena mouse brain cDNA library inserted in the uniZapXR vector(Stratagene). The probes were labeled with [p3P]ATP andT4 kinase and used for overnight hybridization at 37 C n6xNaCl/Cit (3 M NaCl, 0.3M NaCit), 0.5% SDS, 25 pg/mlboiled tRNA, 2 x Denhardts solution (0.24xNaCl/Cit,0.02% SDS, 0.4 g/1 Ficoll, 0.4 g/1 polyvinylpyrrolidone, 0.4 g/lserumalbumin). Nitrocellulose filters were washed with 2xNaC1/Cit, 0.5% SDS at 42C. Limited sequencing of twohybridizing uniZapXR phage clones (pYN611 and pYN614),converted to plasmid cDNA by rescue excision according tothe instructions of the manufacturer, indicated that they werepartial length cDNA. The full length clone pYNlOl wasobtained from an additional screen of the same mouse cDNAlibrary using as a probe the anti-sense oligonucleotide 5-AGTGAT GTA CTC CTG CAA GAT CTT GCT GTC AGT GGstarting at nucleotide position 408. The complete sequenceof pYNlOl was determined in both strands by the chaintermination method using synthetic 17-mer primers (Sangeret a]., 1977). Genomic mouse DNA blots were carried out aspreviously described (Reed and Mann, 1985) using a cDNAAP47 probe spanning nucleotide positions 535- 913.

A ATG/A GTC/T CAI , 5-AAG/A TGG GGI AAG/A CCI

RESULTS AND DISCUSSIONMolecular cloningand the sequence of the mouse AP47 medium chain

Since the N terminus of AP47 purified from AP-1complexes of calf-brain clathrin-coated vesicles appears to beblocked, we obtained internal amino acid sequence infor-mation from chemical microsequencing of several tryptic frag-ments and used T/21.122,47.3.1 and Tb84.85 (Fig. 1) to designthe oligonucleotide probes described in Materials andMethods. Using these probes, we screened a mouse braincDNA library, and identified three hybridizing independentand overlapping clones corresponding to AP47. The sequenceof the longest cDNA clone pYN101, shown in Fig. 1, contains1985 nucleotides and predicts a protein of 423 amino acids( M ,48543; ~17.32). he first ATG translational start codonat position 1 of the nucleotide sequence partially conforms tothe consensus sequence for eukaryotic translational start sitesGCCATGAiG (Kozak, 1989). Use of a more 5-proximal ATGstart codon is unlikely given that upstream of the suggestedstart codon there is no significant protein sequence relation-ship with the related mammalian AP50 chain nor with theputative yeast Yap54p homolog (see below). The 3-untranslated region (625 nucleotides) contains a possiblepolyadenylation signal (ATTAAA) at nucleotide position 1876followed by a poly(A) tail.

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572gene (Thurieau et al., 1988) since a different pattern wasobtained in the mouse genomic DNA blot probed with thecoding region of rat brain AP50 (data not shown).Recently it was reported that calf AP47 binds GTP andphosphorylates the cytoplasmic tail of the mannose 6-phos-phate receptor (Meresse et al., 1990).To determine if AP47 isa member of the family GTP-binding proteins with kinaseactivity, we performed several computer searches usingFASTA and TFASTA (Lipman and Pearson, 1985; Pearsonand Lipman,1988).We could not detect significant alignmentswith known GTP-binding proteins, kinases or indeed with anyother proteins, (except mammalian AP50 and yeast Yap54p;see below) whose sequence has been deposited in the proteindata bank (National Biomedical Research Foundation, ver-sion 25). We also could not find alignments with any openreading frame contained in the DNA data bank (GenBank,version 66). Moreover, inspection of the predicted proteinsequenceof mouse AP47 did not reveal the characteristic setof three consensus sequence elements GXXXXGK, DXXGand N K X D normally found in the GTP-binding domain ofproteins having a high affinity for GTP (reviewed in Dever eta\., 1987), nor did it show the consensus sequence GXGXXGfollowed by a downstream lysine 15 -20 residues away thatcan be found in the nucleotide binding domain of other pro-teins (reviewed by Hunter, 1987). These results are consistentwith our analysis of the mammalian AP50 (Thurieau et al.,1988), which also lacks detectable sequence similarity withknown kinases and nucleotide binding proteins, although it isconsidered to have autokinase activity (Campbell et al., 1984;Keen et al., 1987).Identification in the yeast genome of YapS4p, a putativehotnolog of the mammalian AP47 and APSO medium chainso f the A P- and AP -2 complexes

Recently, Daignan-Fornier and Bolotin-Fukuhara havereported the sequence of a yeast genomic DNA clone whichhas not yet appeared in the DNA data bank with three openreading frames (A, B, C)but only frameC encodedaproteinof known function (Daignan-Fornier and Bolotin-Fukuhara,1989). Using the program T FASTA (Deveraux et al., 1984;Pearson and L ipman, 1988) to compare the predicted proteinsequences of the open reading frames A andB against a limitedprotein data base compiled by one of us(M.G.), it was noticedthat the open reading frame A is a yeast homolog of themammalian AP47 and AP50 chains. The deduced protein,referred to here as Yap54p contains 475 amino acids and hasa predicted M , 53865 (P I 9.43). I ts expression in yeast cellsis likely, since a mRNA of the expected size is transcribed(Daignan-Fornier and Bolotin-Fukuhara, 1989). Using theprograms GAP or BESTFIT (Deveraux et al., 1984), we foundthat the optimal alignment of Yap54p and mouse AP47 intro-duces eight relatively small gaps and results in 56% sequenceidentity and 75% sequence similarity allowing for conserva-tive changes (Fig. 3). The alignment of Yap54p with rat AP50( M ,49612; pl 10.35) (Thurieau et al., 1988) introduces eightgaps and shows 39% sequence identity and 59% sequencesimilarity. This level of relationship is similar to the relatednessbetween the mammalian AP47 and AP50 chains, whose opti-mal alignment requires five gaps for 40% sequence identityand 64% sequence similarity.Based on this striking pattern of sequence similarities, wesuggest that Yap54p is the yeast homolog of mammalianAP47. Direct biochemical confirmation that Yap54p is a con-stituent of yeast-coated vesicles s not yet available. However,

additional dentification in the yeast genome of the open read-ing frames encoding for Yap8Op and Yapl7p as homologs ofthe mammalian PIP large chains (Kirchhausen, 1990) andof the AP17/AP19 small chains (Kirchhausen et al., 1991),respectively, provides further support for the presence of APcomplexes in yeast. The alignment of the Yap8Op and Yap1 7pwith their mammalian counterparts did not allow the estab-lishment of whether these yeast homologs are clearly morerekated to the AP-1 or AP-2 components. In contrast thehomology of Yap54p is much closer to the AP47 subunit,which suggests yeast cells may contain a clathrin-associatedAP-1 complex involved in vesicular traffic from the trans-Golgi network. It is interesting to note that although thecomplete role for clathrin in yeast has not been elucidated,recent studies indicate that clathrin plays a key role in theselective retention of membrane proteins in an intracellulartrans-Golgi compartment (Payne and Schekman, 1989). Whatare the chances that yeast cells contain more than one type ofAP complex? Assuming that the proposed yeast AP complexalso contains four polypeptide chains, that yeast cells contain5000 distinct proteins and that only about 10% of the yeastgenomeiscurrently available for sequence searches in differentdata bases, then the probability of randomly identifying anythree out four chains from an AP complex is about (l/lO)(l /10)(1/10)(9/10)(4)=M 1/300. Against these odds, it is remark-able that three homologies were found. This calculation istherefore consistent with the possibility that yeast cells, likemammalian cells, also contain several types of AP complexesso that one might expect to find in the future other geneversions of YAP l 7, YAP.54, YAP80 as well as the missinghomologs of the mammalian CI and y large chains.Sequence analysis o the medium chains;possible domain organization

The high level of sequence relationship among Yap54p,AP47 and AP50 allows us to detect those regions that maycontain sequence information unique to each class of chains.Based on the optimal alignment of their sequences (Fig. 3),we can divide the primary structures into two regions of simi-lar length but with distinct characteristics. Region I , locatedat the N terminus, spans about 230 amino acids that aremostly conserved among the three chains. Region I of eachchain is separated from the C-terminal region I 1 by differentlinkers of 10- 2 amino acids in length. Region I1spans about190amino acids and in contrast to region I, contains severalsequence stretches specific to AP50 interspersed with otherstrongly conserved sections common to all three chains. Oneprediction from this analysis is that the linker between regionsI and I1may declineate the boundary between two domainsin the medium chains. Indeed, limited tryptic proteolysis ofAP-2 complexes bound to clathrin coats results in the cleavageand degradation of the C terminus of AP50 while the Nterminus, of about M , 24000, remains intact (Matsui andKirchhausen, 1990). A second prediction is that the interac-tions determining the specificity of association of the mediumchains with the other subunits of AP-1 or AP-2 complexesmay principally be located in the more constant region I . Themore variable region I1could participate in interactions withother proteins to be found in specific association with coatedstructures at either the plasma membrane or the trans-Golginetwork.Useof genomic data bases

The yeast DNA sequences deposited in the available databases correspond mostly to specific genes and their flanking


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