UNIT 8.1C Subcellular Fractionation of Tissue Culture Cells The development of cell fractionation techniques over the last few decades has provided the means to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation is essential for the development of cell-free assays that reconstitute complicated cellular processes. These assays have provided new and important tools to understand the molecular mechanisms of complex cellular functions, permitting these functions to be studied in the test tube as a series of biochemical reactions. General methods for fractionating mammalian tissues are presented in UNIT 8.1B. The protocols in this unit describe fractionation of tissue culture cells to immunoisolate early and late endosomes under conditions where these compartments retain their capacity to support membrane transport in vitro. To prepare different populations of endosomes, monolayers of cells are exposed to a fluid-phase marker for endosomal compartments (horseradish peroxidase; HRP) either briefly to label early endosomes or with a chase period to label endosomal carrier vesicles or late endosomes (see Basic Protocol 1). For subsequent immunoisolation, many antibodies are available against cytoplasmically exposed epitopes present on early or late endosome proteins (Gruenberg, 2001). Anti- body-antigen couples do not, however, always work efficiently in immunoisolation experiments. An attractive alternative is to use a foreign antigen, against which appropri- ate antibodies are already available (e.g., an epitope added to an ectopically expressed protein). This protocol was used for the immunoisolation of recycling endosomes from MDCK cells expressing a myc-tagged version of the transferrin receptor (Gagescu et al., 2000). Alternatively, vesicular stomatitis virus (VSV) can be fused with the cells to add VSV G protein to the cell membranes (see Basic Protocol 2), and the cells are then exposed to HRP. Labeled cells from either protocol are then homogenized (see Basic Protocol 3), and the postnuclear supernatant (PNS) is fractionated by a flotation gradient (see Basic Protocol 4). Specific endosomal compartments are isolated using magnetic or polyacry- lamide beads coupled to an antibody specific for VSV G protein (see Basic Protocol 5). The Support Protocol describes growth conditions for preparing BHK-21 cells for isolation of endosomes. The Commentary discusses preparation of a “balance sheet” for the isolation procedure (see Background Information) and problems that may be encoun- tered in subcellular fractionation of tissue culture cells (see Critical Parameters as well as the Commentary in UNIT 8.1B). BASIC PROTOCOL 1 INTERNALIZATION OF FLUID-PHASE MARKER Several proteins, including Rab GTPases and receptors (Table 8.1C.1), can be used to follow early or late endosomes. In the absence of appropriate reagents, markers endocy- tosed in the fluid phase can allow convenient discrimination between the compartments (Table 8.1C.1). A word of caution is warranted, however, because solutes can efficiently be internalized by other routes in some cell types, in particular by macropinocytosis in cells with high ruffling activity. Horseradish peroxidase (HRP) is widely used as a marker for fluid-phase endocytosis. HRP internalized at a concentration of 0.5 to 10 mg/ml distributes uniformly within the lumen of endosomal compartments and is easily meas- ured using a colorimetric reaction. Internalization is carried out directly in the tissue culture dish for cells growing attached to the substratum. For suspension cells, the method is essentially identical, except that all changes of buffers and solutions must be accom- plished by centrifugation. Once labeled, the cells must be washed thoroughly to remove nonspecifically adsorbed marker. Washed cells may be incubated further in the absence of marker to label endosomal carrier vesicles and late endosomes. Supplement 57 Contributed by Fernando Aniento and Jean Gruenberg Current Protocols in Immunology (2003) 8.1C.1-8.1C.21 Copyright © 2003 by John Wiley & Sons, Inc. 8.1C.1 Isolation and Analysis of Proteins