Function of the KKXX motif in endoplasmic reticulum retrieval of a transmembrane protein depends on the length and structure of the cytoplasmic domain.

Transmembrane glycoproteins with type 1 topology can be retrieved to the endoplasmic reticulum (ER) by a retrieval signal containing a di-lysine (KK) motif near the C terminus. To investigate the structural requirements for ER retrieval, we have constructed mutants of the simian immunodeficiency virus (SIV) envelope (Env) protein with cytoplasmic tails of different lengths and containing a KK motif at the -3 and -4 positions. Such proteins were found to be retained intracellularly when the signal was located 18 amino acids or more away from the membrane spanning domain. The retrieval signal was found to be functional even when placed at the distal end of the wild-type SIV Env protein with 164 amino acids in the cytoplasmic tail, as shown by the lack of proteolytic processing and lack of cell surface expression of the mutant proteins. However, proteins with a cytoplasmic tail length of 13 amino acids or less having the di-lysine motif at the -3 and -4 positions were not retrieved to the ER since they were found to be processed and transported to the cell surface. The surface-expressed proteins were found to be functional in inducing cell fusion, whereas the proteins retained intracellularly were defective in fusion activity. We also found that the KK motif introduced near an amphipathic helical region in the cytoplasmic tail was not functional. These results demonstrate that the ability of the KK motif to cause protein retrieval and retention in the endoplasmic reticulum depends on the length and structure of the cytoplasmic domain. The ER retrieval of the mutant proteins was found to correlate with increased intracellular binding to beta COP proteins.