The OST48 subunit of the oligosaccharyltransferase complex is a type I membrane protein containing three lysines in its cytosolic domain. The two lysines in positions 3 and 5 from the C-terminus are able to direct protein localisation within the endoplasmic reticulum. (ER) by COPI-mediated retrieval. Substitution of these lysines by arginine resulted in cell-surface expression of OST48, whereas ER residency was maintained when either Lys-5 or Lys-3 but not both was replaced with arginine. Localisation of OST48 was not affected by substitution of the two lysines by histidine, indicating that a His-Xaa-His sequence, in contrast to Arg-Xaa-Arg, contains Eft-specific targeting information. These differences show that simple charge interactions are not sufficient for ER retention and that other structural factors also play a role. The His-Yaa-His sequence could represent a new and independent signal for directing ER localisation differing from both the arginine motif in type H proteins and the lysine motif in type I proteins. Our data do not exclude, however, that the histidine sequence simply mimicks the lysine motif as a sorting signal, being recognised by and interacting with the same receptor subunit(s) in COP-1-coated vesicles. Conclusions arising from this assumption involving the conformation of lysine at the putative COP-1 binding site and the failure of Axg-Xaa-Arg to mediate ER localisation for type I proteins are discussed. (C) 2002 Elsevier Science (USA).