LOCALIZATION OF THE LYS, ASP, GLU, LEU TETRAPEPTIDE RECEPTOR TO THE GOLGI-COMPLEX AND THE INTERMEDIATE COMPARTMENT IN MAMMALIAN-CELLS

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related seq uence, is important for ER localization of both lumenal as well as typ e II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retr ieval occurs remains unresolved. With an affinity-purified antibody ag ainst the carboxyl-terminal sequence of the mammalian KDEL receptor, w e have investigated its subcellular localization using immunogold labe ling on thawed cryosections of different tissues, such as mouse sperma tids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartme nt, and we use this marker, as well as budding profiles of the mouse h epatitis virus (MHV) in cells infected with this virus, to identify th is compartment. Our results demonstrate that the KDEL receptor is conc entrated in the intermediate compartment, as well as in the Golgi stac k. Lower but significant labeling was detected in the rough ER. In gen eral, only small amounts of the receptor were detected on the trans si de of the Golgi stack, including the trans-Golgi network (TGN) of norm al cells and tissues. However, some stress conditions, such as infecti on with vaccinia virus or vesicular stomatitis virus, as well as 20 de grees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This s hift 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 GTP gamma S treatment of permeabili zed cells, the receptor was detected in the beta-COP-containing buds/v esicles that accumulate after this treatment, suggesting that these ve sicles may transport the receptor between compartments. We propose tha t retrieval of KDEL-containing proteins occurs at multiple post-ER com partments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments va ries according to physiological conditions.