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The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix., (Copyright 2000 Academic Press.)

We have developed a stably transfected CHO cell line (CHO24S) that expresses the three structural proteins of rubella virus (RV). RV proteins C (capsid), E2, and E1 are secreted from CHO24S cells in the form of RV-like particles (RLPs) which form by budding into the cisterna of the Golgi complex. RLPs resemble RV virions in their size and morphology and have an identical buoyant density when purified on sucrose gradients. Release of RLPs into the medium was found to be dependent upon the E1 cytoplasmic tail since deletion or substitution of this domain with the same region from vesicular stomatitis virus G protein abrogated release of RV proteins from transfected cells. These results indicate that the RV 40S genomic RNA is not required for efficient particle assembly. Therefore, RLPs may serve as a convenient source of RV antigen for use in diagnostic assays and as an alternative to live attenuated vaccine strains.

We have presented our detailed methods for localizing antigens in cultured cells and tissue sections by IP at the EM level. Immunoperoxidase cytochemistry is particularly well suited for the study of sparse antigens as a result of the enzymatic amplification afforded by the method, and of molecules confined within a membrane-enclosed compartment wherein the DAB reaction produce can accumulate. Although IP is commonly used to localize membrane-compartmentalized molecules, reliable qualitative information can also be obtained on cytoplasmic antigens as well (Anderson et al., 1978; Merisko et al., 1986; Rodman et al., 1984). For these and other reasons, it is likely that IP cytochemistry will continue to be an important tool for the cell biologist especially in the study of membrane traffic. Other inventive combinations of immunocytochemical methods will likely be forthcoming, for example, combining IP localization with postembedding labeling by colloidal-gold conjugates to provide triple EM labeling.

Multiple pathways of intracellular membrane traffic have been detected in various cell types. The major established routes are (a) the exocytosis pathway, utilized in secretory cells for the discharge of secretory products, and which is also believed to be used for delivery of intrinsic membrane glycoproteins in all cell types; (b) the plasmalemma to Golgi route, also highly developed in secretory cells, which is believed to be utilized for the recovery and recycling of the membranes of containers used in packaging of secretory products (i.e., secretory granules or vesicles); (c) the lysosomal pathway, which is available in all cells but is the major route utilized in phagocytic cells; (d) the transcellular route, which represents the major type of traffic encountered in nonfenestrated, capillary endothelial cells and also appears to be the preferred route for the transport of immunoglobulins (intact) across cells; and (e) the biosynthetic pathways used for transport of secretory products, lysosomal enzymes, and membrane proteins from the ER to the Golgi complex and for transport of lysosomal enzymes from the Golgi complex to lysosomes in all cell types. It has become clear that cells repeatedly reutilize or recycle the vesicular membranes involved in carrying out these various transport operations. Clathrin-coated vesicles have been found to be involved in transport along all the routes detected so far, suggesting that there are multiple populations of coated vesicles with different transport functions in every cell. It has become clear that considerable sorting of membrane constituents and ligands takes place at the plasmalemma (receptor-mediated uptake), in the Golgi complex, and in endosomes. The Golgi complex is the intracellular site where much of the biosynthetic and recycling membrane traffic converges and where products are sorted and directed to their correct destinations. In summary, we have become aware of the existence of multiple pathways of membrane traffic and of the extensive reutilization or recycling of membranes that occurs in cells. The basic pathways are similar in all cells except that some are emphasized or deemphasized according to the predominant function and organization of a given cell type. What now remains to be done is to determine how these transporting membranes and the membranes of the receiving compartments are constructed, how their specific interactions are controlled, and how individual cell types utilize these pathways to carry out their specific functions.(ABSTRACT TRUNCATED AT 400 WORDS)