M. Benghezal et al., The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants, PL CELL, 12(7), 2000, pp. 1179-1201
The tomato Cf-9 disease resistance gene encodes a type I membrane protein c
arrying a cytosolic dilysine motif. In mammals and yeast, this motif promot
es the retrieval of type I membrane proteins from the Golgi apparatus to th
e 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 pla
nts, 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 mut
ation 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. Trip
le hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER i
n tobacco cells, and cleavage was also observed for the NNXX mutant protein
, suggesting an endoprotease recognition site located within the Cf-9 lumen
al 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 m
ammals and yeast and that Cf-9 is a resident protein of the ER.