alpha-COP can discriminate between distinct, functional di-lysine signals in vitro and regulates access into retrograde transport

Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to E R transport di-lysine signal in its cytoplasmic tail. Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization. In contrast to ether proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant alpha-COP subunit of coato mer, Here we demonstrate by sucrose gradient fractionation and immunofluore scence analysis that recycling of Emp47p was unimpaired in ret1-1, Furtherm ore we have characterized three new alleles of ret1 and showed that Golgi l ocalization of Emp47p was intact in cells with those mutant alleles, We cou ld correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vi tro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fu sion protein. As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for alpha -COP in the differential binding to distinct di-lysine signals. In contrast to results with alpha-COP mutants, we found that Emp47p was mislocalised t o the vacuole in mutants affecting beta'-, gamma-, delta-, and zeta-COP sub units of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde trans port of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of alpha-COP for a particular transport step i n vivo.