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

Preliminary experiments using the immunoperoxidase approach has revealed the presence of the KDEL-R in the ER and in Golgi cisternae . Because of the limitations of this technique, conclusive and quantitative results were not obtained. In this report, we describe our detailed immunogold labeling experiments using an affinitypurified antibody against the cytoplasmic COOH-terminal sequence of the KDEL-R in thawed cryosections of tissues and cultured cells, and particularly in the mouse L cell model system using MHV and in HeLa cells stably expressing a trans-Golgi network (TGN) marker, a construct of the o~-2,6-sialyltransferase tagged on its lumenal domain with 10 amino acids from the cytoplasmic domain of the G protein of vesicular stomatitis virus (VSV) (Rabouille, C., E Hunte, R. Kiekbusch, E. Berger, G. Warren, and T. Nilsson, manuscript submitted for publication). Our results demonstrate that the bulk of the KDEL-R is normally localized to both the Golgi stack and the intermediate compartment, and that increased concentrations reach the TGN when the cells are stressed either by virus infection or by extremes of temperature. medium with 10% serum. The MHV A59 was propagated in sac(-) ceils and was used to infect L cells for the localization studies, with or without streptolysin O treatment, as described (Krijnse-Loeker et al., 1994) . The conditions for infecting L cells with VSV-ts045 and for accumulating the G protein at 20"C were as desoribed earlier (Griffiths et al., 1985) . For infecting cells with the WR strain of vaccinia virus see Sodeik et al: (1993) . The use of the vaccinia recombinant expressing the M protein of MHV is described earlier (Krijnse-Locker et al., 1992) . The SA:48 cells are a HeLa cell line stably overexpressing the human a-2,6-sialyltransferase having a 10--amino acid peptide tag (P5IM epitope) from the cytoplasmic domain of the vesicular stomatitis virus G protein at the lumenal (COOH) terminus (see Rabouille, C., E Hunte, R. Kieksbusch, E. Berger, G. Warren, and T. Nilsson, manuscript submitted for publication). The characterization of the PSD4 epitope was done according to Soldati and Perriard (1991) . FOr the 20"C "blocks the ceils were incubated in "air medium" (Matlin and Simons, 1983) for 2 h at 20"C; for the heat shock treatment, they were put in the same medium for 4 h at 43°C. FOr the treatment of the SA:48 cells with bafilomycin AI (Bowman et al., 1984; obtained from the Kamiya Medical Co., Thousand Oaks, CA), the cells were allowed to internalize BSAgold (16 run) for 3 h (pulse) followed by an 8-h chase in the absence of gold and in the presence of 500 nM bafilomycin A1. The cells were given BSAgold (5 urn) for 10 rain before fixation to label early endosomes. To be sure that the bafilomycin treatment effectively neutralized low pH compartment parallel experiments were done at the light microscopy level using acridine orange (6 ttg/ml).