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 Stenbeck ~, Rupert Schreiner", Doris Herrmann ~, Sylvia Auerbach ~', Friedrich Lottspeichb, James E. Rothman ~ and Felix T. Wieland ~ 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. INTRODUCTION In eukaryotic cells, constitutive secretory protein transport occurs from the endoplasmic reticulum (ER) via the various stations of the Golgi apparatus to the plasma membrane. Individual steps of this transport have been reconstituted in vitro (see for example [4-6]), and a variety of its biochemical parameters have been elucidated (for a review see [7]). Newly synthesized pro- teins appear to be transported through the Golgi stack in non.clathrin-eoated vesicles, which have been iso- lated and characterized [2]. They contain a set of coat proteins (COPs) (~-, ,6'-, 2,-, ~-, ~- and ~'-COP with mo- lecttlar weights of' 160, 107, 98, 61, 36, and 20 kDa, respectively). These proteins show similarity in molecu- lar weight but are immunologically unrelated to the subunits of the clathrin coat involved in endocytotic membrane traffic, fl-COP has been characterized at a molecular level and shows some homology to fl- adaptin, a protein involved in the clathrin system. By peptide sequence comparison [2,8], fl-COP was proven to represent a component of both the non-clathrin- coated transport vesicles and a cytosolic complex, the coatomer. Coatomer consist~ of subunits of molecular weights identical to ct-, fl-, 7'-, ~-, a- and ~'-COPs, indi- cating that it is a preorganized assembly of the coat of non-clathrin-coated vesicles. We have isolated the indi- Corresponcience addreaa. F.T. Wi¢land, Iantltut fill Btoeltufme I tier Universitat Heidelberg, ln~ Neuenhmmer Feld 328, D-6900 l-Ici- cle|berg, Germany. Fa~: (49) (6221) 564 366. vidual COPs from Golgi-derived non-clathrin-eoated vesicles, as well as their counter parts from coatomer. Here we report that 7-COP is a constituent of both the coatomer and the vesicles, and that this Golgi vesicle- derived coat subunit is related to Sec21p, a protein encoded by a gene required for vesicle budding in ER to Golgi transport [9,10]. This finding provides strong evidence that the coatomer is required for vesicular transport in vivo, and in addition that COP-coated ves- icles mediate transport both from the ER to the Golgi, as well as within the Golgi stack. 2. MATERIALS AND METHODS Preparation of non-clathrm-coated Ool~-derived transport vesi- cles, 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, cor- responding 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 dode- capeptide, 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"d to I tool of carrier. This product 'w~ injected into rabbits, and the antiserum obtained was depleted of antibodies directed against the carrier pro- tern 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 Pubhshed by Elsevier Science Publishers 17 V 195