Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval

Dilysine signals confer localization of type I membrane proteins to the end oplasmic reticulum (ER). According to the prevailing model these signals ta rget proteins to the ER by COP I-mediated retrieval from post-ER compartmen ts, whereas the actual retention mechanism in the ER is unknown. We express ed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constru cts, which had access to post-ER compartments, the KKAA chimeras were local ized to the ER by confocal microscopy and mere neither processed by cis-Gol gi-specific enzymes in vivo nor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are pe rmanently retained in the ER. The ER localization was nonsaturable and excl usively mediated by the dilysine signal because mutating the lysines to ala nines led to cell surface expression of the chimeras. Although the KKAA sig nal avidly binds COP I in vitro, the ER retention by this signal does not d epend on intact COP I in vivo because it was not affected in an epsilon-COP -deficient cell line. me propose that dilysine ER targeting signals can med iate ER retention in addition to retrieval.