Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.

Abstract

Abstract. The Saccharomyces cerevisiae Wbpl protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, 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 SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbpl fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial or,6 mannosyltransferase (Ochlp). Subcellular fractionation revealed that >90% of the al,6 mannose-modified fusion protein colocalized with the ER (Wbplp) and not with the Golgi Ochlp-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK--,QK, KQ, or QQ) did not change the kinetics of initial oil,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating otl,6 and al,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolyrically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Ochlp-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important feature of this sorting determinant.

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

“…These fractions were analyzed by immunoblotting with several antibodies. Previous studies have shown that ER and vacuolar membranes distribute mainly in the P13 fraction, whereas Golgi membranes are recovered in both P13 and P100 fractions (Nakano et al, 1988;Nishikawa and Nakano, 1991;Gaynor et al, 1994). In this experiment, Secl2p (ER marker) and Pho8p (vacuolar marker) were mostly detected in the P13 fraction as expected.…”

“…We refer to lighter (0.2 M sorbitol/1.2 M sucrose interface) and denser (1.2 M/1.5 M sucrose interface) bands as band 1 and band 2, respectively ( Figure 13A). Consistent with the report by Gaynor et al (1994), ER cells) were separated on an SDS gel and subjected to immunoblotting using an anti-BiP antibody.…”

“…In the case of Ochlp, the al ->6 mannosyltransferase localized to the cis Golgi, a slightly larger amount was recovered in P13 than in P100. This appears to be characteristic of membrane proteins that are residing in early Golgi compartments (Nakayama et al, 1992;Gaynor et al, 1994). Rerl-3HAp, the protein in question, showed similar behavior to Ochlp.…”

“…Subcellular fractionation was performed essentially as described by Gaynor et al (1994) with some modifications. Cells were grown to 1 X i07 cells/ml in MVD medium and converted to spheroplasts as described previously .…”

A New Functional Domain of Guanine Nucleotide Dissociation Inhibitor (α‐GDI) Involved in Rab Recycling

“…For subcellular fractionation studies, spheroplasts were labeled and processed as described (4,44). After clearing extracts of unbroken cells, lysates were centrifuged at 100000 ×g for 1 h to yield P100 particulate (membrane) and S100 soluble (cytosolic) fractions.…”