Journal of Neuroscience, Vol 11, 1163-1172, Copyright © 1991 by Society for Neuroscience
Stable clathrin: uncoating protein (hsc70) complexes in intact neurons and their axonal transport
MM Black, MH Chestnut, IT Pleasure and JH Keen
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
We have studied the organization of clathrin during its transport in axons.
Using immunoprecipitation techniques we have confirmed earlier findings
that clathrin is transported as part of slow component b, but we also
detect small amounts of clathrin in fast component. As fast component is
known to correspond to the transport of membraneous material, including
coated vesicle membrane components, our findings suggest that some clathrin
in axons undergoes transport in the form of coated membranes and that a
portion of the clathrin delivered to axons and axon terminals arrives by
way of fast component. The organizational form of clathrin in slow
component b (SCb) was examined in more detail, as it is thought to
represent a non-membrane-associated species, is relatively long-lived, and
at any instant represents the major transport species in axons. We used
nondenaturing immunoprecipitation methods with stringent wash procedures to
identify other SCb proteins that interact with clathrin. The
immunoprecipitates contained major labeled bands that corresponded to
clathrin heavy and light chains, along with a prominent 70-kDa band and
several minor bands that ranged in apparent Mr from 70,000 to 150,000; the
70-kDa band was shown to be the ATP-dependent uncoating protein by
two-dimensional gel electrophoresis. A very similar profile of polypeptides
was also immunoprecipitated from extracts of cultured neurons. The results
from a variety of control immunoprecipitations, including the use of
antisera preadsorbed with purified clathrin trimers or clathrin light
chains, indicate that coprecipitation of clathrin and uncoating protein
with the other 70,000-150,000-Da polypeptides from SCb reflects specific
interactions. Including exogenous uncoating protein in the lysis buffer had
no detectable effect on the levels of endogenous uncoating protein
recovered in the immunoprecipitates, indicating that complexes of clathrin,
uncoating protein, and the other coimmunoprecipitating SCb protein existed
in the intact neurons prior to lysis. Finally, a specific and functional
association is further supported by the release of uncoating protein, but
not the other 70,000- 150,000-Da polypeptides, from the immunoprecipitated
complexes on the addition of ATP. Collectively, these observations provide
the first direct evidence of interaction between clathrin and uncoating
protein in intact cells, lend strong support to the concept that uncoating
protein plays an intimate role in clathrin dynamics within cells, and
reveal a family of 70,000-150,000-Da polypeptides that form a stable
nonmembranous association with clathrin in intact cells.
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