SIGNAL-MEDIATED RETRIEVAL OF A MEMBRANE-PROTEIN FROM THE GOLGI TO THEER IN YEAST

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysi ne (KKXX) motif. This motif has previously been shown to direct Golgi- to-ER retrieval of type I membrane proteins in mammalian cells (Jackso n, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317 -333). To analyze the role of this motif in yeast, we constructed a SU C2-WBP1 chimera consisting of the coding sequence for the normally sec reted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytopla smic tail) of Wbp1p. Carbohydrate analysis of the invertase-Wbp1 fusio n protein using mannose linkage-specific antiserum demonstrated that t he fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revea led that >90% of the alpha 1,6 mannose-modified fusion protein colocal ized with the ER (Wbp1p) and not with the Golgi Och1p-containing compa rtment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatic ally increase the rate of modification by more distal Golgi (elongatin g alpha 1,6 and (alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-depend ent manner. While amino acids surrounding the dilysine motif played on ly a minor role in retention ability, mutations that altered the posit ion of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our res ults indicate that the KKXX motif does not simply retain proteins in t he ER but rather directs their rapid retrieval from a novel, Och1p-con taining early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate CO OH-terminal position which represents the most important feature of th is sorting determinant.