Protein and membrane trafficking depends on choreographed reactions which deform membranes and sequentially assemble and disassemble protein complexes. Hsp70s--the ubiquitous chaperones involved in protein folding and many cellular protein processing reactions--function as motors in trafficking processes such as translocation of proteins into ER and mitochondria, and disassembly of the clathrin coats that form around nascent endocytic vesicles. The ubiquitous role of Hsp70s suggests a common mechanism is being harnessed in these seemingly disparate reactions, but while we understand how Hsp70s bind and release protein substrates, our understanding of how Hsp70s generate force to move proteins or take apart protein complexes is limited. Clathrin coat disassembly provides an exceptional system to study these force generation mechanisms: unlike Hsp70 mediated processes such as protein translocation or dissociation of heterogeneous aggregates, coat disassembly can be precisely monitored in real time, structures of all players and reaction snapshots are known, coat stability can be easily controlled, and the single Hsc70 binding site in clathrin required for disassembly is known. By exploiting these features and our development of protocols for producing functional clathrin in bacteria, we have obtained evidence that neither previously proposed power- stroke nor Brownian ratchet/steric wedge models can explain how Hsc70 disassembles coats. Instead, our data indicate that coats are disassembled through pressure generated by collisions between coat walls and Hsc70s bound to flexible tethers in close apposition to these walls. We also discovered that self-association, characteristic of all Hsp70s, amplifies this force, thus providing a biological function for a ubiquitous phenomenon that has never had one. In our proposed work we will test and refine this collision pressure mechanism of Hsp70 force generation (Aim 1). Hsc70 cooperates with other chaperones, among them Hsp110, which acts as an Hsp70 nucleotide exchange factor (NEF). We determined the structure of the Hsc70:Hsp110 complex, and used the structural information to design experiments which revealed that Hsp110 regulates Hsc70 chaperoning of clathrin during synaptic vesicle recycling. The latter experiments used the giant lamprey reticulospinal synapse, which represents the in vivo experimental complement to our in vitro clathrin/Hsc70 system. Because neuronal synapses are compartments devoted primarily to membrane trafficking, they provide an exceptional system for addressing fundamental biological questions in trafficking and the role of chaperones in these processes. In our proposed studies we will exploit both the in vitro clathrin/Hsc70 system and the in vivo, lamprey RS synapse to further define the nucleotide exchange mechanism of Hsp110 (Aim 2), and the mechanism by which it regulates Hsc70 and clathrin availability during membrane trafficking (Aim 3). These studies will advance our understanding of how Hsp70s generate the forces by which they translocate proteins and remodel protein complexes, and of how their activities are regulated. They will also elucidate fundamental mechanisms underlying synaptic transmission and clathrin mediated vesicular trafficking.
This work is focused on understanding the roles and mechanisms of molecular chaperones in neuronal trafficking. Chaperones are involved in synaptic transmission, the process used by neurons to communicate with each other. A common feature of many neurological disorders is aberrant synaptic transmission, so this work will be important for understanding and developing therapeutic strategies that will enable clinical intervention. Moreover, the chaperone proteins that we will study are also involved in many diseases that are a consequence of the accumulation of damaged, aggregated proteins (Alzheimer's, ALS, Parkinson's, Huntington's, and others), so this work will also be relevant to the fight against neurodegenerative disorders.
|Farsi, Zohreh; Gowrisankaran, Sindhuja; Krunic, Matija et al. (2018) Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity. Elife 7:|
|Bianchi-Smiraglia, Anna; Rana, Mitra S; Foley, Colleen E et al. (2017) Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution. Nat Methods 14:1003-1009|
|Snead, Wilton T; Hayden, Carl C; Gadok, Avinash K et al. (2017) Membrane fission by protein crowding. Proc Natl Acad Sci U S A 114:E3258-E3267|
|Sousa, Rui; Liao, Hsien-Shun; Cuéllar, Jorge et al. (2016) Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation. Nat Struct Mol Biol 23:821-9|