Association of an Axonally Transported Polypeptide

Polypeptide H (mol wt 195,000) is axonally transported in rabbit retinal ganglion cells at a velocity of 0.7-1 .1 mm/d, i.e ., in the most slowly moving of the five transport groups described in these neurons . To identify the organelle with which H is associated, we purified H, prepared antibodies directed against it, and adsorbed the antibodies onto Formvar-coated electron microscope grids . When the resulting "immuno-affinity grids" were incubated with extracts of spinal cord and then examined in the electron microscope, they contained as many as 100 times more 100-A filaments than did grids coated similarly with nonimmune IgG. The ability of the anti-H IgG to specifically adsorb filaments to grids was completely blocked by incubating the IgG with polypeptide H. The 100 filaments adsorbed to anti-H immunoaffinity grids could be specifically decorated by incubating them with anti-H IgG . These observations demonstrate that H antigens (and most likely H itself) are associated with 100-A neurofilaments . In addition, they suggest that the use of immunoaffinity grids may be a useful approach for determining the organelle associations of polypeptides. The availability of high-resolution techniques (e .g ., SDS polyacrylamide gel electrophoresis [SDS-PAGE] and isoelectric focusing) for separating proteins has made it possible to study the synthesis, turnover, and transport of individual proteins without regard to their identity or function . However, once a protein with interesting behavior has been singled out by such techniques, it is often difficult to establish its identity and function . Here we outline a systematic approach for determining the organelle association of a polypeptide known only by its electrophoretic mobility . The strategy is as follows: An electron microJ . CELL BIOLOGY © The Rockefeller University Press " 0021-9525/80/06/0587/10 $1 .00 Volume 85 June 1980 587-596 scope grid is coated with an antibody directed against the protein of interest, and the resulting "immunoaffinity grid" is used to adsorb specifically organelles containing the antigen . The organelle is then identified by electron microscopy . This strategy may prove to be generally useful for determining the organelle association of some proteins. In this communication, we describe the use of immunoaffinity grids to show that polypeptide H, an axonally transported polypeptide (15, 16), is associated with the intermediate filaments of nervous tissue . Polypeptide H (mol wt 195,000) is 587 on Jne 7, 2017 D ow nladed fom Published June 1, 1980