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

Mouse seminiferous tubules, kidney, and liver were fixed with 0.1% glutaraldehyde and 4% formaldehyde in 200 mM Hepes buffer, pH 7.4, for the first 30 rain, followed by a subsequent overnight incubation in 4% formaldehyde alone. Pieces of fixed rat liver, pancreas, and kidney that bad been fixed in the above mixture were kindly provided by Dr. J. W. Slot (Department of Cell Biology, University of Utrecht, Utrecht, The Netherlands). Mouse L cells were infected with MHV and permeabilized with streptolysin O (SLO) as described previously (Krijnse-Locker et al., 1994) . The latter publication also describes the treatment of SLO-permeabilized cells with GTP3,S (50/~M). HeLa cells, as well as the SA:48-HeLa cell line (see above), were infected with the WR strain of vaccinia virus, as before (Sodeik et al., 1993) . Veto cells were infected with the ts 045 strain of VSV for the 20"C experiments, as described by Griffiths et al. (1985) . The cells grown in 6-cm dishes were rinsed with PBS and removed from the monolayer with either proteinase K on ice (20 t~g/ml in PBS for 2-3 min) or, in the case of L cells infected with MHV (without SLO), simply by squirting with PBS. A 16% solution of paraformaldehyde was added (final concentration '~4%) to these cell suspensions before centrifugation for 3 min at 1,000 g. The supernatant was removed and 4% para~rmaldehyde and 0.1% glutaraldehyde in 200 mM Hepes, pH 7.4, was carefully layered on the pellet. The cells were fixed for 4-24 h and centrifuged at 13,000 g for 5 rain. The SLO-trcated cells were fixed for 30 rain with 1% glutaraldehyde in 200 raM Hepes, pH 7.4, and scraped with a piece of Teflon. Pieces of these pellets were infused with sucrose, cryo-sectioned, and labeled with immunogold as described by Griffiths (1993) . Double labeling was done according to Slot et al. (1991) using a 1% glutaraldehyde step between the two sets of antibodies (see also Griffiths, 1993) .