γ-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p

Volume 314, number 2, 195-198 FEBS 11860 © 1992 Federation oi" European Biochemical Societies 00145793/92/$5.00

December 1992

7-COP, a coat subunit of non-clathrin-coated vesicles with homology to See2 lp Gudrun

S t e n b e c k ~, R u p e r t

S c h r e i n e r " , D o r i s H e r r m a n n ~, S y l v i a A u e r b a c h ~', F r i e d r i c h L o t t s p e i c h b , J a m e s E. R o t h m a n ~ a n d F e l i x T . W i e l a n d ~

alnstitut far IUoehemte I der Umversitat Heidelberg, Im Neuenheuner Feld 328, D-6900 Heulelberg, Germato,, bMar-Planck-lnstitut flit' Blochemie. Genzentrum, Am KIopferspttz. D-8033 Martinsrted, Germany and ~Sloan-Ketteri~g Cancer Research Center, Rockefeller Research Laboratory, 1275 York A veuue, New York, N Y 10021, USA

Received 26 October 1992 Constitutive secretory transport in eukaryotes is hkdy to be mediated by non-clathrin-eoated v¢~icle~,which have been b.olatcd and characterized [(1989) Cell 58, 329- 336; (1991) Nature 349, 215-220]. They contain a set of coat ploteins (COPs) which are aho likely to exist in a preformed cyto~olic complex named eoatomer [(1991) Nature 349, 248-250]. From peptide sequence and eDNA ~trueture eomparmons evidence is presented that one of the subumts of coatomer, 7-COP, is a true constituent of non-clathrin-coated vehicles, and that ?'-COP is related to sec 21. a secretory mutant of tile yeast Saccharono, ce~ cervhlae. Endoplasmic reticulum, Golgi, Vesicular transport, Coatomer; See21, Coat protein ?'-COP

1. I N T R O D U C T I O N In e u k a r y o t i c cells, constitutive secretory p r o t e i n t r a n s p o r t occurs f r o m the e n d o p l a s m i c reticulum ( E R ) via the various stations o f the Golgi a p p a r a t u s to the p l a s m a m e m b r a n e . Individual steps o f this t r a n s p o r t have b e e n reconstituted in vitro (see for example [4-6]), and a variety o f its biochemical p a r a m e t e r s have been elucidated (for a review see [7]). N e w l y synthesized proteins a p p e a r to be t r a n s p o r t e d t h r o u g h the G o l g i stack in n o n . c l a t h r i n - e o a t e d vesicles, which have been isolated a n d characterized [2]. T h e y c o n t a i n a set o f c o a t proteins ( C O P s ) (~-, ,6'-, 2,-, ~-, ~- a n d ~'-COP with molecttlar weights of' 160, 107, 98, 61, 36, and 20 k D a , respectively). T h e s e p r o t e i n s show similarity in molecular weight but are i m m u n o l o g i c a l l y unrelated to the subunits o f the clathrin c o a t involved in e n d o c y t o t i c m e m b r a n e traffic, f l - C O P has b e e n characterized at a m o l e c u l a r level a n d shows s o m e h o m o l o g y to fladaptin, a protein involved in the clathrin system. By peptide sequence c o m p a r i s o n [2,8], f l - C O P was p r o v e n to r e p r e s e n t a c o m p o n e n t o f b o t h the non-clathrinc o a t e d t r a n s p o r t vesicles a n d a cytosolic complex, the c o a t o m e r . C o a t o m e r consist~ o f subunits o f m o l e c u l a r weights identical to ct-, fl-, 7'-, ~-, a- a n d ~'-COPs, indicating that it is a p r e o r g a n i z e d assembly o f the c o a t o f n o n - c l a t h r i n - c o a t e d vesicles. W e have isolated the indi-

Corresponcience addreaa. F.T. Wi¢land, Iantltut fill Btoeltufme I tier Universitat Heidelberg, ln~ Neuenhmmer Feld 328, D-6900 l-Icicle|berg, Germany. Fa~: (49) (6221) 564 366.

Pubhshed by Elsevier Science Publishers 17 V

vidual C O P s f r o m Golgi-derived n o n - c l a t h r i n - e o a t e d vesicles, as well as their c o u n t e r parts f r o m c o a t o m e r . H e r e we r e p o r t t h a t 7 - C O P is a constituent o f b o t h the c o a t o m e r and the vesicles, a n d that this G o l g i vesiclederived c o a t s u b u n i t is related to Sec21p, a protein e n c o d e d by a gene required for vesicle b u d d i n g in E R to Golgi t r a n s p o r t [9,10]. T h i s finding p r o v i d e s strong evidence that the c o a t o m e r is required for vesicular t r a n s p o r t in vivo, and in a d d i t i o n that C O P - c o a t e d vesicles mediate t r a n s p o r t b o t h f r o m the E R t o the Golgi, as well as within the G o l g i stack. 2. M A T E R I A L S A N D M E T H O D S Preparation of non-clathrm-coated Ool~-derived transport vesicles, of coatomer, lsolauon ol"?'-COP, preparation of tryptic peptides and sequenein8 was as described [2,3]. For cloning of ?,-COP eDNAs a degenerated ohgonucleotide probe was designed TG(T,C)TC(C,T)TG(A,G)AA(T,G,A)AT(T,C)TC(T,C)TGOCQGGTGGCAGC, corresponding to the 7-COP-peptide, AATRQEIFQEQ. This probe was used to screen a 2gtl0 library from bovine brain (random primed, Clontech). Three independent clones were sequenced in the M I3 mpl 8 system with Sequenase (USB). For the production of anti-?'-COP peptide antibodie~, the dodecapeptide, VAATRQEIFQEQ, according to amino acid positions 254--265 of the partial sequence was synthesized (kindly performed by Dr. R. Frank, Heidelberg), and coupled to persucemylated bovine serum albumin as a earri.-r [I II, which was activated with i~obutyl chloroformale. About 20 tool ofpeptide was c~valently linkg"dto I tool of carrier. This product 'w~ injected into rabbits, and the antiserum obtained was depleted of antibodies directed against the carrier protern by at'finity adsorpuon to persuccmylated bovine serum albumin bound to nitrocellulose. The resulting supernatant was affinity puri-%e o p I t fled b)' adsorpt:on :o m..o..!.ulose bound antigen and ~ub~qoent ¢lution with 50 mM citrate bulTer, pH 2 3 [12].The eluant was quickly neutralized by the addition of 1 M Tris, pH 8.3 ~.

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3. R E S U L T S A N D D I S C U S S I O N In our attempt to characterize the individual nonclathrin-coated vesicle coat subunits, we isolated ),-COP from non-elathrin-coated Golgi-derived vesicles [2], as well as From coatomer [3], by SDS-gel eleetrophoresis. The protein was submitted to tryptie digestion and the resulting peptides were purified by reverse-phase HPLC on RP 18 columns, and sequenced. A de~ene='at¢d oligonucleotide probe was synthesized correspondin~ to one of the peptides (see Materials and Methods) and used to screen a random-primed bovine brain e D N A library (Clonteeh). The largest insert found was cloned into M 13 mpl 8 and sequenced. This e D N A comprises an open reading frame o f 1,569 bp, with a stop codon at position 1,570 (Fi~. 1). Four peptides known from

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D~ember 1992

mierosequencing were found in the derived amino acid sequence (underlined in Fig. 1 .). The dodecapeptide corresponding to the segment from amino acid 254--265 in the derived protein sequence was synthesized, linked to a carrier, and used to raise antibodies in a rabbit. Immunological analysis o f isolated coatomer with this antiserum is shown in Fig. 2. In the gel system used the migration offl- and y-COP is reversed as compared to the system used in [2]. Ia lane 2, eoatomer was stained with Coomassie brilliant blue alter separation. The positions of the COP~ are indicated. As a control, Western blotting was performed with the monoclonal antibody, M3AS, directed against,6'-COP (and kindly provided by Thomas Kr¢is) (lane 3 in Fig. 2). The antiserum against the dodeeapeptide, Y A A T R Q E I F Q E Q , reacted clearly and specifically with ?'-COP, as shown in lane 4 in Fig.

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Fig. 1. Partial eDN A sequence of y-COP. A random-primedA,gtl 0 eDNA hbraryfrom bovine brain (Clontech) was ~creened with an oh~onucleotld¢ probe as descrtb~d in Materials and Methods, and three independentclones were sequenced. Tryptic peptides from ;,- COP, i~olated as described m [2,3], were m~crosequencedand compared with the 7-DNA-derived protein .~¢quenc¢.The four peptide~ found in the derived sequence am u,d~rlined. Amino acids are ~iven in the three letter code. 196

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Fig. 2. Ant=bod~¢s against a eDNA-derived syntheue pepude react with ;,-COP. Antibod=es against the peptide, VAATRQEIFQEQ, were prepared as described in Materials and Methods. Coatomcr isolated according to [3] wab separated by SDS-8¢Ieleetrophorcbison a 6% aerylam=du 8el with a ratlo of" monomer to N,N'-methyleneb=~acrylamid¢ of 100.1, wlth 6 M urea in the separating gel. Lane 1, molecular weight standard proteins; lane 2, coatomer; lane 3, immuno~tamzng after electrotran~fer to lmmobilon ~upport ofeoatomer with the monoclonal aatibody, M3AS, directed a~ainst If-COP (kindly provided by Thoma~ Kr¢i~), lane 4, sample a~ m Lane3 ~mmunostamed with affinity-purified (see Matermls and Method~)antibod=esdirected against the eDNA-derived dod¢capcptide. 2. We take this as confirmation that the e D N A shown in Fig. 1 encodes a 524 amino acid region comprising part of this coat protein. The missing portion o f the full-length e D N A is not present in the e D N A library used, as re-screening o f a ,~.gtl 1 e D N A library made from denatured RNA, r a n d o m plus oligo-dT-primed (Clontech), did not yield inserts that would have completed the full e D N A of ?'-COP. N o r did application of the R A C E procedure [13] with bovine mamarian gland epithelial cell R N A lead to additional e D N A information. Computer-assisted structure comparison revealed a striking homology to S¢c21p, as shown in Fig. 3. The Bestfit program was used that introduces gaps for the optimal alignment o f the sequences. In a stretch o f 569 positions 28.3% of the amino acids are identical, and a similarity of 52.3% is obtained if conserved amino acids are considered. Individual peptide stretches o f between 40 and 60 amino acids are found with identitie~ o f more than 50% at either side o f the two major gaps. In the Blast P program for protein comparison [14] a high score for the relation o f ?'-COP to See21 p o f 204 is obtained, with a smallest Poisson probability o f 2.5 x 10 -~-~. F o r comparison, the next best score was 56 for a hypothetical 119.5 k D a protein [15] with a smallest Poisson -p-' ^t u" o- '~- u: 'l:~+L". r O f .~. 7 × .tn-o . . . . . . " t h o s e results, together with the identities and similarities shown in Fig. 3, provide a statistically firm evidence that Sec21p and ?.-COP are related proteins.

December 1992

Sec21p [16] is a protein o f 935 amino acids with a molecular weight of a b o u t 103 kDa. This is in clos~ agreement to the apparent molecular weight o f a b o u t 100 k D a for ?'-COP, as estimated by SDS-gel electrophoresis. The alignment o f our partial protein sequence with See21 p is optimal in the C-terminal part o f See21 p. This is in good agreement with a stop c o d o n found at position 1,570 of the e D N A sequence, and indicates that o u r e D N A codes for a C-terminal part o f T-COP, c o m prising almost two thirds o f the entire protein. W h a t implications do our findings have on o u r knowledge on the mechanism of vesicular biosynthetic protein transport? First, 7/-COP is the second sabunit o f the cytosolie coatomer complex that is n o w shown to be present in transport vesicles as well. Thus, the remaining subuntts of coatomer are also highly likely to represent the coat o f non-clathrin-coated transport vesicles. Second, and more importantly, the similarity o f a c o a t o m e r subunit from animal transport vesicles to a yeast secretory mutant protein offers strong confirmation that coatomer plays a functional role m vesicular y-cop

t TGSEGSIDRLMKQISSFMSEISDEFKWVVQAISALCQKYPRKflAVL~tF5B II I I: 1 I: : : i l : l l : : : . : l : I: :1 ::11 sec21 369 TGrSKNISSLISTITNF%HBVSDDFKIIIIDAVRTLSLNFPQEWKSILNF418 y-cop

51 LFSMLRE.EGGFEYKRAIVDCIISIIEENAESKETGLSHLCEFIEDCEFT99 I: ; t : ; III1:1 I1::1 l: ::111:1 :ll:lllllll see21 419 LIDVLKNSEGGFKFKNSIVEALID/VSFVPQSKELALENLCOFIEDCEFN468

~-cop 1¢0 VLATRILHLLGQEGPGPATLPSTSASSTTAWCWRPPRSRR.PVSALAKFG14~ : Illltll II1:: I :1 I1 I 1 : secZ1 469 EILVRILHLLGKEGPSAPNPgL~VPdtlYNRVVLENSIZRgAAWALgKFA518 ~-ccp 149 AQNEE,,HLPSILVLLKRCViVDDDNEVRDRATFYLNVLE ..QKQKALNA 193 .: : I1: IlII I I:1111111: I: :: 1: : see21 5Z9 LTKNDPTLYESIISLLKRIANDKDDEVRDRATZALEFIDSARNKODVIAQ568 y-cop 194 GYILNGLAVSIPGLERALQQYT. . . . . . . . . . . . . . . . . LCPSEKPFD 224 I I1:11 I L I I::: sec2$ 569 NLIESKYFYDIPSLESKLSSYISSNTDSFATAFDVNQVRKFTED~MCJ~IN618 y-cop 225 LKSVPLATAPLAEQRIESTPVTAAKQPEKVAATR .............. Q 259 tl : I I I ill l see21 619 LKRKQEQIFNQKSETTLDTTPEAESVPEKRADANSFAGPNLDDHQEDLLA~ y-cop 26¢ EIFQEQL/V~VPEFQGLGPLFKSSPEPVALTESETEYVIRCTKHTFTOPdvIV3 ~ •

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sec21 669 TKYADELLSIEQIKPFGQLVNSSR,AZSLTEPEAEFVVRGV~ILFKDNVV717 y-cop 31¢ FQFDCTNTLHDQTLENVTVo.Q~EPgEAYEVLCYVP/d~SLPYNQPGTEYT35? II: Illl I I'll I I l: I: secZ1 71~ LQFt~ZTNTLTDZALDNVSVVCTPEISDEAELEELFTLQVDRLLPSEEAAC76? (-cop 358 LVALPKEDPTAVACTFSCVMRFTVKDEDPTTGEAoDDEGYEDEYVLEDL4¢5 I;! I 1 : : : I I 1. :1 t I : :111;,111 : : : see21 768 YVAFKKLDEZVMEGFLNNLTFTTKEIHPDTNEPFDGDEGFQDEYEIDgZ 616 y-cop 406 EVTIADHZQKVHKLNFEAAWDEVGDEFQREETFTLSTIKTLEEAVGNIVK455 : :1 : . II I : 1 1 " I I : : 1 I, I: see21 BI? FLHAGDYVKSSFTG~FSA~FDELPCE..EVAVFNIQEDLSZQEWDKIIL$6~ y-cop 456 FLGMHPCERSDKVPDNKNTHTLLLAG,,VFRGGHDILVRSRLLLLDTVTMS03 : I I : I : I III I I : l: I : : : see21 ~65 NSSCLPVESTQFAPSDSNSHTLKLFGKSALTGSKVALQIYJ~IK55KGLAL914 y-cop 504 QVTARgSEELPVDIVLASV522 I:.::1 :::° sot21 9!5 KVHCKGEDSLLC~DL~NGL G33 Fig. 3. Comparison of the partial eDNA-derived amino acid sequence of F-COP with Sc¢21 p. Identical amino acids are indicated by a Yerticol dash; conserved amino acids are indicated by a colon.

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transport in rive. See21 p has been characterized as one o f several temperature-sensitive yeast mutants defective in protein transport, and results from synthetic lethality studies indicate a role for Sec21p in budding of transport vesicles from the ER [10]. The close homology o f a coat subunit isolated from Golgi-derived transport vesicles, and a protein required for ER-to-Golgi transport would certainly be consistent with the simple idea that transport vesicles from both the ER and the Golgi apparatus recruit their coats from a common pool of, cytosolic precursor, the coatomer, and that the coat is required for vesicle budding. We would predict that Sec21p, like ?'-COP will exist in a similar coatomer complex in yeast. In summary, the mechanisms underlying vesicle budding from the ER and from the Golgi are likely to be very similar, if not identical, in animals and in yeast, as has been found earlier for the process o f membrane fusion [17]. Results from yeast cell-free ERto-Golgi transport assays have revealed evidence for a vesicular carrier but no coat has been described to date [18,19]. In light o f our findings, it appears likely that a transient COP-coated vesicle intermediate has been missed. Acknowledgentents. We thank Cordula Hatter For discussions, Ruiner Frank for peptide synthesis, Theme5 Kr¢i~ for kindly provadmg the monoclonal antibody, M3AS, and Irene Speckhard for her help with the preparation of the manuscript. Thin work wa~ ~upported b2¢ the Deutsche Forsdlangsgememschaft (SFB 352, C2) and the Human Frontiers Science Program to J.E.R. and F.T.W.

REFERENCES [I] Malhotra, V., Serafim, T., Orci, L., Shepherd, J.C. and Kothman, J.E. (1989) Cell 58, 329-336 [2] Serafini, T., Stenbeek, G., Brecht, A., Lottspeich, F., Orci, L., Rothman, J.E. and Wleland, F.T. (1991) Nature 349, 215-220. [3] Waters, G , Serafim, T. and Rothmaa, J.E. (1991) Natarc 349, 248-250. [4] Balch W.E., Dunphy, W.G., Braell, W.A. and Rothman, J.E. (1989) Cell 39, 405--416. [5] Rothman, J E 0987) J. Biol. Chem. 26, 12502-12510. [6] Becker~, CJ.M., Keller, D.S. and Balch, W.E. (1987) Cell 50, 523-534. [7] Rothman, J.E. and Orci, L. (1992) Nature 355, 409-415 [8] Daden, R , Grlffiths, G., Frank, R., Argos, P. and Kreis, T.E. (1991) Ceil 64, 649-665. [9] 1N'evik, P., Field, C. and Schekman, R. (1980) Ceil 21,205-215. [10] Kaisel, C.A. and Seheckman, R. (1990) Cell 61, 723-733. [l II Samokhin, G P. and F,hmonov, l N. (1985) Anal. Biochem 145, 311-314. [12] Tahan, J.C., Olmsted, J.B. and Goldman, R.D. (1983) J. Cell. Biol. 97, 1277-1282. [13] Frohman,~, M . A , Dush, M.K. and Martin, G.R. 0988) Proc Natl. Acad. Set. USA 85, 8998-9002 [14] Genetics Computer Group Sequence Analysts Software Package 1.1984) Nucleic Acids Re~. 12 387-395 [15] Shiota, S. and Nakayama, H. 0989) Mol. Gen. Genet. 217, 332340. [16] H0sobuclu, M.M. and $checkman, R. (1991) Protein Sequence Database pir3, accession number A33151; (1991) Gene Sequence Database eml'un, accession number M59708. [17] Wdson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.J., Henzel W.J., Block, M.R., Ulrich, A. and Rothman, J E 0.98V) Nature 339, 355-359. [18] Groesch, M., Ruohola, H., Bacon, R. and Ferro-Novlck, S (1990) J. Cell. Biol. 111, 45-53. [19] Rexach, M.F. and Sehekman, R.W. (1991) J. Cell. Btol 114, 219-320.

NOTE A D D E D IN PROOF Independent work by R. fichekman and co-workers (Nature, in press) leads to conclusions similar to our own

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