Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles
Cell fractions remaining after differential and equilibrium density-gradient centrifugation usually contain more than one type of organelle. Monoclonal antibodies to various organelle-specific membrane proteins are a powerful tool for further purifying such fractions. One example is the purification of vesicles whose outer surface is covered with the protein clathrin; these coated vesicles are derived from coated pits at the plasma membrane during receptor-mediated endocytosis, a topic we will discuss in detail in Chapter 14. An antibody to clathrin, bound to a dead bacterial cell that expressed protein A on its surface, can selectively bind these vesicles in a crude preparation of membranes, and the whole antibody complex can then be isolated by low-speed centrifugation (Figure 4-38). A related technique uses tiny metallic beads coated with specific antibodies. Organelles that bind to the antibodies, and are thus linked to the metallic beads, are recovered from the preparation by adhesion to a small magnet on the side of the test tube.
FIGURE 4-38 Coated vesicles can be purified by binding to an antibody specific for a vesicle surface protein and linkage to bacterial cells. In this example, a suspension of membranes from rat liver is incubated with an antibody specific for clathrin, a protein that coats the outer surface of certain cytosolic vesicles. To this mixture is added a suspension of killed Staphylococcus aureus bacteria, whose surface membrane contains protein A, which binds to the constant (Fc) region of antibodies. (a) Interaction of protein A with antibodies bound to clathrin-coated vesicles links the vesicles to the bacterial cells. The vesicle-bacteria complexes can then be recovered by low-speed centrifugation. (b) A thin-section electron micrograph reveals clathrin-coated vesicles bound to an S. aureus cell. See E. Merisko et al., 1982, J. Cell Biol. 93:846.
[Micrograph courtesy of George Palade.]
All cells contain a dozen or more different types of small membrane-limited vesicles of about the same size (50–100 nm in diameter) and density, which makes them difficult to separate from one another by centrifugation techniques. Immunological techniques are particularly useful for purifying specific classes of such vesicles. Fat and muscle cells, for instance, contain a particular glucose transporter (GLUT4) that is localized to the membrane of one of these vesicle types. When insulin is added to the cells, these vesicles fuse with the plasma membrane and increase the number of glucose transporters able to take up glucose from the blood. As we will see in Chapter 15, this process is critical to maintaining the appropriate concentration of sugar in the blood. The GLUT4-containing vesicles can be purified by using an antibody that binds to a segment of the GLUT4 protein that faces the cytosol. Likewise, the various transport vesicles discussed in Chapter 14 are characterized by unique surface proteins that permit their separation with the aid of specific antibodies.
A variation of this technique is employed when no antibody specific for the organelle under study is available. A gene encoding an organelle-specific membrane protein is modified by the addition of a segment encoding an epitope tag; the tag is placed on a segment of the protein that faces the cytosol. Following stable expression of the recombinant protein in the cell under study, an anti-epitope monoclonal antibody (described above) can be used to purify the organelle.