Clathrin-dependent vesicular transport is an important pathway in facilitating the movement of modified proteins (ie. post-phosphorylation or post-glycosylation) from the trans-golgi network to other cellular compartments, such as plasma membrane and endosome. Bocking and his group hypothesize that the accumulation of local conformational distortions on the clathrin lattice imposed by Hsc70 leads to the destabilization and uncoating of the clathrin coat. Previous reports have shown that auxilin recruits Hsc70 to bind to the QLMLT motif of clathrin triskelion, followed by uncoating of clathrin coat. However, the exact molecular mechanism of Hsc70 in aiding the uncoating is yet elucidated. As a significant amount of modified proteins are transported out of the golgi network, their work to elucidate the uncoating mechanism of clathrin coat is therefore highly significant.
To approach the question, Bocking group used three key approaches: (i) purified fluorescent clathrin and fluorescent Hsc70 proteins; (ii) formation of clathrin coat with fluorescent clathrin proteins; and (iii) single particle fluorescence imaging assay. Purified fluorescent proteins formed the foundation of their experiments. First, they purified Hsc70, wildtype clathrin and mutant clathrin that lacks the QLMLT motif. Next, they used maleimide to crosslink fluorophore AF568 to Hsc70 and fluorophore AF488 to clathrin proteins. The fluorescent products enabled the group to detect concentrations of each protein at different wavelengths. Second, they made fluorescent clathrin lattice by introducing the accessory protein, AP-2. They also made mixed clathrin coat using different concentration ratios of fluorescent wildtype clathrin to fluorescent mutant clathrin. Last, they designed a single particle fluorescence imaging assay, in which they put antibody against the light chain of clathrin to capture fluorescent clathrin lattice on coverslips. Then they added auxillin, followed by the addition of fluorescent Hsc70 to study the function of Hsc70 in disassembly. To control the disassembly of the coat, they also controlled the stability of lattice at pH6.0, and triggered disassembly at pH6.8.
Using the above approaches, Bocking and his group discovered that there are two major phases of Hsc70-dependent uncoating reaction, including accumulation of Hsc70 and unlocking of lattice, and rapid disassembly. The accumulation of Hsc70 is required prior to disassembly of the clathrin lattice, while the concentration of Hsc70 and the percent of uncoating shows a linear relationship. They also extrapolated that the uncoating is triggered when Hsc70 is accumulated to about 1 molecule for every 2 clathrin triskelions. Furthermore, without the QLMLT Hsc70-binding motif at the C terminal of clathrin, the clathrin coat does not undergo disassembly in the presence of Hsc70. Additionally, the percentage of uncoating depends on the number of wildtype clathrin but not the number of clathrin mutant. Bocking and his group also proposes an alternative function of auxilin. Aside from recruiting Hsc70 to the specific QLMLT domain, auxilin can also recruit Hsc70 to other nonspecific sites. With the presence of both unspecific binding and specific binding of Hsc70 to clathrin, it is estimated that there is one Hsc70 for each clathrin triskelion.
Bocking and his group have provided strong supporting results to validate their hypothesis. Their approach, in particular the design of a single particle fluorescence imaging assay, has effectively justified the contribution of Hsc70 in initiating and disassembling the clathrin coat. Bocking showed the importance of QLMLT motif in initiating the disassembly process by Hsc70 binding. What about the substrate binding domain of Hsc70 that is thought to bind to clathrin? It would be interesting to confirm the importance of Hsc70 by creating a Hsc70 mutant that carry an altered substrate binding domain. Hsc70 mutant can no longer recognize clathrin QLMLT mutant. Thus, Hsc70 mutant should not initiate disassembly, which supports and further confirms their findings. Furthermore, if Hsc70 can be recruited to various nonspecific sites during the accumulation phase, then what makes the specific site (QLMLT) on clathrin so favorable for (1) auxilin to recognize and bind, and (2) Hsc70 to bind? In other words, future research should look into how this specific QLMLT motif is more favorable than other nonspecific sites. This is important because nonspecific sites may also be a key factor in the disassembly reaction. Despite the initiation of disassembly requires Hsc70 to bind to the specific QLMLT motif, the binding to nonspecific sites might still be useful in catalyzing and maintaining a fast disassembly progress.