The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

Abstract

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...

Genes Encoding Proteins of the Cation Diffusion Facilitator Family That Confer Manganese Tolerance

“…The pattern was unlike those described for the plasma membrane (Panek et al, 2000), the tonoplast (Suriapranta et al, 2000), or the Golgi (Ton et al, 2002) but was similar to that shown for the endoplasmic reticulum (Benghezal et al, 2000). Although no discrete signal was apparent at the plasma membrane or the vacuolar membrane, the notion that a small proportion of the protein was incorporated into these membranes could not be discounted.…”

“…The resulting cassette was removed as a NotI fragment, inserted into the NotI site of pPLEX502, and transferred to Agrobacterium as described above. A similar construct was prepared for the expression of a ShMTP1:GFP fusion protein in yeast except that the GFP was derived from the plasmid pCBJ4 (Benghezal et al, 2000), because the GFP version optimized for plant expression showed poor expression in yeast. The ShMTP1 cDNA that lacked a stop codon was inserted into the EcoRI site of pBC SK (Stratagene) with its 3Ј end adjacent to the PstI site.…”

Genomic organization, promoter activity, and expression of the human choline transporter-like protein 1

“…On the other hand, the COOH-terminal L 651 KKR 654 of the CTL1b protein has long been recognized as a classical dilysine (KKXX) motif for endoplasmic reticulum (ER) retrieval of type I transmembrane proteins (27). The KKXX motif is cytosolic and interacts directly with the coat protein I (COPI) coat during retrograde protein transport (7,29,65). It is present in the tails of numerous type I transmembrane proteins including ion channels, transporters, and receptors (32,43,52).…”

An Arabidopsis Prenylated Rab Acceptor 1 Isoform, AtPRA1.B6, Displays Differential Inhibitory Effects on Anterograde Trafficking of Proteins at the Endoplasmic Reticulum

“…In contrast, in MG132-treated protoplasts, the HA:AtPRA1.B6 levels continuously increased until the 24-h time point. Furthermore, the HA:AtPRA1.B6 levels were much higher in MG132-treated protoplasts than in DMSO-treated protoplasts at both 12 and 24 h. As a control, the level of ERlocalized GKX, a chimeric fusion protein containing the KKXX motif for ER retrieval from the Golgi complex, was examined as a cargo protein marker (Benghezal et al, 2000;Lee et al, 2009). The GKX level was not affected by MG132 treatment, indicating that 26S proteasome-dependent proteolysis is specific to HA:AtPRA1.B6.…”