Abstract. Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] a-X), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine a-COP, the 135-kD subunit of coatomer as well as ~-COP, the 57-kD subunit and have identified a yeast homolog of 8-COP by cDNA sequence comparison and by NHz-terminal peptide sequencing. ~-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the 8-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between a-and ~-COPs and between 13-and ~-COPs. Moreover, the two-hybrid system indicates interactions between -,/-and E-COPs as well as between c~-and [Y-COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

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.

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a &tl 1 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N-terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.

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