THE GOLGI-LOCALIZATION OF YEAST EMP47P DEPENDS ON ITS DI-LYSINE MOTIFBUT IS NOT AFFECTED BY THE RET1-1 MUTATION IN ALPHA-COP

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellul ar lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-termi nal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which confo rms with the consensus for di-lysine-based ER-localization signals. De spite the presence of this motif, Emp47p was shown to be a Golgi prote in at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER loca lization. Nevertheless, the di-lysine motif was required for Golgi-loc alization of Emp47p and showed the same charge-independent, position-d ependent characteristics of other di-lysine motifs. alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins . Consistent with this finding, the Ste2p-Emp47p hybrid protein was mi slocalized to the cell surface in the alpha-COP mutant, ret1-1. Surpri singly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we deve loped an assay to measure this step after block of forward transport i n a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recy cling occurred from a Golgi subcompartment containing alpha 1,3 mannos e-modified oligosaccharides suggesting that it originated from a media l- or later Golgi compartment. Thus Emp47p cycles between the Golgi ap paratus and the ER and requires a di-lysine motif for its alpha-COP-in dependent, steady state localization in the Golgi.