Abstract
Molecular chaperones are crucial machines that maintain protein homeostasis in the cell. In the ER, the two major chaperones performing this function are the Hsp70 BiP and Hsp90 Grp94. While BiP interacts with every nascent polypeptide targeted to the ER and has a specific client binding motif, less is known about how Grp94 chooses its clients. Here, I explore how BiP and Grp94 can work together and set the groundwork for studies exploring a shared client between BiP and Grp94.
Chapter 2 showcases how BiP and Grp94 interact only in a specific and nucleotide-dictated ADP-bound conformation of BiP. This is also the BiP conformation that binds clients with high affinity, giving BiP the opportunity to transfer a client to Grp94 in a ternary complex. This work sets some of the foundation for studying client transfer between BiP and Grp94. Chapter 3 shows research on how BiP and Grp94 work separately on the same client, a pro-protein of insulin-like growth factor 2. But, they have contrasting effects on proIGF2 and a negligible influence on the folding of proIGF2: BiP decreases the size of proIGF2 oligomers and Grp94 increases their size. Chapter 3 also describes how proIGF2 independently forms large oligomers, thought to aid in packaging the pro-protein for transportation to the Golgi for further processing.
There have been numerous examples of Hsp70 family chaperones binding oligomeric clients with both high affinity and low affinity. However, no mechanism has been discovered for how Hsp70s can bind oligomers with high affinity and why Hsp70 is only able to target a subset of these clients with high affinity. In Chapter 4, I discover how BiP utilizes a novel electrostatic steering mechanism to target the binding of oligomers over monomers using the client proIGF2. I find that negatively-charged BiP interacts with high affinity to positively-charged oligomers of proIGF2’s E-peptide. I also find that the binding affinity and the association rate are dependent on salt concentration, key features of electrostatic steering. Finally, I show that this mechanism can be applied to other known Hsp70 family members binding oligomeric species with high affinity if they are positively-charged.