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 ER 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 other 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 coatomer. Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1. Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles. We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion 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 to the vacuole in mutants affecting beta'-, gamma-, delta-, and zeta-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of alpha-COP for a particular transport step in vivo.
Schröder-Köhne
S
S
Max-Planck-Institut für biophysikalische Chemie, D-37070 Göttingen, Germany.
Letourneur
F
F
Riezman
H
H
D000483:Alleles; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D020756:Coatomer Protein; D003600:Cytosol / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D006056:Golgi Apparatus / Q000378:metabolism; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014166:Transferases / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins
D020756:Coatomer Protein; D004151:Dipeptides; C096914:EMP47 protein, S cerevisiae; D005656:Fungal Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C024575:lysyllysine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase