Stable clathrin: uncoating protein (hsc70) complexes in intact neurons and their axonal transport

Abstract

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.

Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

“…In addition, comparisons between the H2 and Hi lanes in Figure 3B indicate In contrast to synaptophysin, radiolabeled 180-kDa clathrin heavy chain was significantly labeled in optic nerve only at the 4-d SCb time point (Figure 4,A and B), consistent with previous reports . A small portion of axonal clathrin (s10% of that present in SCb) may be present in FC (Black et al, 1991), but levels of FC clathrin were below the level of detectability. As with synaptophysin, clathrin immunoprecipitates from the 4-d time point contain several faint radioactive bands corresponding to SCb proteins that are immunoprecipitated nonspecifically.…”

“…As with synaptophysin, clathrin immunoprecipitates from the 4-d time point contain several faint radioactive bands corresponding to SCb proteins that are immunoprecipitated nonspecifically. However, some additional radiolabeled bands appear that may represent polypeptides which interact with clathrin heavy chains including clathrin light chains (Black et al, 1991) and the HSC70/clathrin uncoating ATPase (de Waegh and Brady, 1989). Previous studies have shown that radiolabeled tublin and neurofilaments are present in rat optic nerve at 21 d post-labeling (Brady and Black, 1986;Oblinger et al, 1987) Total nerve kinesin (the sum of axonal and nonneuronal kinesin) was measured to permit estimates of kinesin specific activity in these experiments.…”

Cytoskeletal Requirements in Axonal Transport of Slow Component-b

“…First, radioisotopic labeling studies have provided data suggesting that many of the SCb proteins move together as if they comprised discrete structures that are transported in axons (Willard et al, 1974(Willard et al, , 1979Black and Lasek, 1980;Tytell et al, 1981;Lasek, 1981, 1982). This possibility was reinforced by studies in which large subsets of SCb proteins were isolated by subcellular fractionation (Lorenz and Willard, 1978) or nondenaturing immunoprecipitation (Black et al, 1991). Finally, a review of the literature reveals that many other SCb proteins interact with the three proteins that are cotransported in our system.…”

“…Furthermore, interactions of synapsin with the SCb proteins spectrin and calmodulin have been reported (Baines and Bennett, 1985;Nicol et al, 1997), and the SCb protein MAP-1B was shown to bind to GAPDH, spectrin, and clathrin, all SCb proteins (Cueille et al, 2007). It is also known that hsc70 exists as a complex with clathrin and a variety of other SCb proteins (Black et al, 1991). Given our data showing that ␣-synuclein, synapsin-I, and GAPDH are cotransported as a complex and the above described interactions between the three proteins that we studied and other known SCb members, we infer that all these proteins are a part of a transported SCb complex that depends on microtubules rather than on the actin network.…”

Cytoplasmic dynein is associated with slow axonal transport.

“…Because phosphatases and kinases were included in the extraction buffer to prevent changes in the phosphorylation state of the dynein that might alter its functional properties (14). It is interesting to note that when brain is homogenized, many proteins in the SCb transport complex are apparently extracted, including metabolic enzymes (2,25,26) and dynein (Fig. 3).…”