The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

Abstract

Abstract. The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs.a-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 mislocalized to the cell surface in the oL-COP mutant, retl-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the retl-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a secl2 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing od,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its a-COP-independent, steady state localization in the Golgi.

The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

“…We tested wild-type cells, the ␤Ј-COP mutants sec27-95, sec27⌬1-285, and sec27-1, and the ␣-COP mutants ret1-1 and ret1⌬1-285 ( Figure 6A). Little difference in Emp47p levels between the 0-, 1-, and 2-h time points was found in wild-type and the ␣-COP mutant cells, consistent with previously published data (Schrö der et al, 1995;Schrö der-Kohne et al, 1998). Furthermore, the sec27-1 mutant also behaved similar to wild-type cells.…”

“…This type I integral membrane protein maintains its steady-state localization by continuous recycling between the ER and the Golgi complex. This recycling is dependent on its carboxy-terminal KTKLL signal (Schrö der et al, 1995;Schrö der-Kohne et al, 1998;Nakano, 2002, 2003). We tested the intracellular fate of Emp47p in ␣-and ␤Ј-COP mutants at a permissive temperature for growth of all mutants tested, to score primary trafficking defects caused by the mutations, rather than secondary defects that may arise upon temperature shift.…”

“…Emp47 and its close homologue Emp46p have been proposed to function as cargo receptors at the ER exit site Nakano, 2002, 2003). Wbp1p harbors a classical di-lysine motif, KKTN, and Emp47p presents a variant di-lysine motif, KTKLL Schrö der et al, 1995). A genetic selection for yeast mutants with specific defects in trafficking dependent on the di-lysine KKTN motif, as present on Wbp1p, yielded mutations in ␣-, ␥-, ␦-, and -COP, which provided strong evidence for a function of COP I in Golgi-to-ER retrieval .…”

“…In this study, we analyze in detail interactions of coatomer with variants of the di-lysine motifs as present on two well characterized proteins in yeast: Wbp1p, a subunit of the ER-localized oligosaccharyl transferase complex ; and Emp47p, a protein that achieves its steadystate Golgi localization by continuous recycling between the endoplasmic reticulum (ER) and the Golgi (Schrö der et al, 1995;Schrö der-Kohne et al, 1998). Emp47 and its close homologue Emp46p have been proposed to function as cargo receptors at the ER exit site Nakano, 2002, 2003).…”

“…Intriguingly, however, it was found that the recycling of Emp47p, which harbors the variant motif KTKLL, is unaffected in all ␣-COP mutants that strongly mislocalize KKTN-tagged proteins. Thus, ␣-COP cannot be the only receptor for di-lysine tagged COP I cargo proteins in yeast (Schrö der et al, 1995;Schrö der-Kohne et al, 1998). Both photo-cross-linking data (Harter et al, 1996;Harter and Wieland, 1998) and in vitro binding data (Wu et al, 2000) have suggested that a binding site for di-lysine motifs is present on mammalian ␥-COP.…”