Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells

Abstract. The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rabl is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20°C or 43°C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP3,S treatment of permeabilized cells, the receptor was detected in the/3-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions. p ROTEIN sorting/targeting along the exocytotic pathway is mediated by various types of targeting/sorting signals (Pelham, 1989; Hong and Tang, 1993) . The carboxyl-terminal Lys, Arp, Glu, Leu tetrapeptide (KDEL) ~ (HDEL in yeast) and related sequences have been shown to mediate ER localization of lumenal and membrane proteins that have their COOH terminus within the lumenal side of the membrane (Hardwick et al., 1992; Pelham, 1990; Sweet and Pelham, 1992; Tang et al., 1992a) . Recent studies argue strongly that KDEL or HDEL sequence functions as a retrieval signal at some post-ER compartment(s) Pelham, 1988 Pelham, , 1991 Jackson et al., 1993) .