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

Antibodies against the mammalian KDEL receptor (p23) were raised in rabbits by injecting synthetic peptide (conjugated to keyhole limpet hemocyanin) corresponding to the 2 I-residue sequence of the COOH terminus (CDFFYLYITK'VLKGKKLSLPAcOOH) of p23 . Specific antibody was affinity-purified from positive antisera with the same peptide conjugated to a solid support. Since this COOH terminal sequence is identical between the two closely related mammalian KDEL receptors (Hsu et al., 1992) , this antibody will detect both forms of the receptor, and our localization studies here represent a survey of total KDEL receptors. The peptide serum against the COOH-terminal epitope of M protein of MI-IV, as well as the monoclonal antibody against the NH2 terminus have been described before (Krijnse-Locker et al., 1994) . Monocloual and polyclonal antibodies against the tail (P5D4 epitope) of the VSV-G protein were kindly provided by Thomas Kreis (Department of Biological Sciences, University of Geneva, Geneva, Switzerland) (Kreis, 1986) . In a few experiments using the HeLa cells, a three-step labeling protocol was used whereby the primary antibody was amplified by a pig anti-rabbit antibody that was then detected by protein A gold (see Fig. 4 A). The antibody against rabl was made against the purified rablA protein, as described by Saraste, J., and B. Goud (manuscript submitted for publication). This antibody also crossreacts with rablB (Goud, B. and J. Saraste, manuscript submitted for publication). Antibodies against calreticulin (CaBP3), CaBP1, and CaBP2 purified from rat were raised in rabbits. The antigens were purified as described by Nguyen Van et al. (1989 and Rupp et al. (1994) . Antibodies against the KDEL-COOH terminus peptide were raised as described by Vanx et al. (1990) . A peptide was synthesized as follows, -K-X-X-X-X-X-K-D-E-L, where X is an equimolar mixture of A, D, H, Q, L, Y, and K. The peptide was coupled with the carbodimide EDC to keyhole limpet hemocyanin and used for immunization of rabbits.