In vivo assembly of coatomer, the COP-I coat precursor

Coatomer, a seven-subunit hetero-oligomeric complex, is the major component of the COP-I membrane coat of transport vesicles of the early secretory pathway, We have followed the assembly of this complex in vivo by pulse-chase experiments and immunoprecipitation of native coatomer subunits and found that it is an ordered process that takes 1-2 h to complete. During assembly, direct interactions between alpha-, beta'- and delta-COP, beta- and delta-COP, and gamma-, zeta-, and delta-COP occur. Coatomer, once it has assembled, is stable with a half-life of similar to 28 h. No significant amounts of partial coatomer complexes have been detected, The only subunit to exist at steady state out of the complex is zeta-COP, which has a similar half-life to coatomer subunits within the complex. Assembly is inhibited by brefeldin A, suggesting that it may be a regulated process. These results describe for the first time in vivo assembly of a coat protein complex involved in membrane traffic and extend our knowledge of how coatomer is structured.