The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP-and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER.
INTRODUCTIONProteins resident in the endoplasmic reticulum (ER) contain structural motifs responsible for their correct subcellular localization. Many soluble ER proteins, such as BiP or protein disulfide isomerase, carry a C-terminal tetrapeptide H/KDEL motif for localization in the ER (Munro and Pelham, 1987;Pelham et al., 1988;Mazzarella et al., 1990;Andres et al., 1991; Denecke et al., 1992 Denecke et al., , 1993Napier et al., 1992). A membrane-bound receptor encoded by the yeast ERD2 gene retrieves escaping HDEL-marked proteins from the Golgi apparatus or salvage compartment back to the ER Semenza et al., 1990). Homologs of the Erd2 receptor have been cloned from mammals Pelham, 1990, 1992; Tang et al., 1993) and plants ( Lee et al., 1993), highlighting conservation of the H/KDEL motif and retrieval mechanism.In mammals and yeast, the cytosolic dilysine motif is critical for ER localization of type I membrane proteins (Nilsson et al., 1989;Jackson et al., 1990). The two lysine residues need to be in either the Ϫ 3, Ϫ 4 (KKXX) or Ϫ 3, Ϫ 5 (KXKXX) positions relative to the C terminus, and no other basic amino acid can be substituted (Jackson et al., 1990(Jackson et al., , 1993. Mutation of lysine residues leads to expression of the reporter protein on the cell surface in mammals (Nilsson et al., 1989;Jackson et al., 1990) and to its vacuolar delivery in yeast (Gaynor et al., 1994). Studies of the post-translational modification kinetics of dilysine-tagged reporter proteins have demonstrated a constant cycling of the reporter from the ER to the Golgi apparatus and back to the ER (Jackson et al., 1993;Gaynor et al., 1994). The retrieval mechanism is mediated by vesicular Golgi apparatus-to-ER retrograde transport, involving the coat protein I (COPI) complex (Cosson and...