Mechanism of Hsp70 specialized interactions in protein translocation and the unfolded protein response

Abstract

Hsp70 chaperones interact with substrate proteins in a coordinated fashion that is regulated by nucleotides and enhanced by assisting cochaperones. There are numerous homologues and isoforms of Hsp70 that participate in a wide variety of cellular functions. This diversity can facilitate adaption or specialization based on particular biological activity and location within the cell. In this review, we highlight two specialized binding partner proteins, Tim44 and IRE1, that interact with Hsp70 at the membrane in order to serve their respective roles in protein translocation and unfolded protein response signalling. Recent mechanistic data suggest analogy in the way the two Hsp70 homologues (BiP and mtHsp70) can bind and release from IRE1 and Tim44 upon substrate engagement. These shared mechanistic features may underlie how Hsp70 interacts with specialized binding partners and may extend our understanding of the mechanistic repertoire that Hsp70 chaperones possess.

Keywords: BiP; Hsp70 chaperones; IRE1; Tim44; UPR; protein translocation.

Figure 1.

Hsp70 chaperone substrate cycle. (a) Cartoon representation of Hsp70 NBD showing the organization of the four subdomains IA, IB, IIA, IIB, with ATP bound in the active site (PDB 4B9Q [9]). (b) The full-length structure of Hsp70 bound to ATP, illustrating the arrangement of the two domains NBD and SBD relative to one another. (c) The structure of SBD bound to substrate peptide [14]. The SBDα lid closes over the peptide binding site, which is present within the SBDβ subdomain, trapping the substrate. (d) A schematic diagram depicting Hsp70 conformations as regulated by nucleotides. When Hsp70 is bound to ATP, there is close association between NBD and SBD that results in the lid (SBDα) being positioned such that it exposes the peptide binding site within SBDβ. This favours fast substrate binding and release and works in synergy with J-protein cochaperone that facilitates substrates recruitment to Hsp70. The association of J-protein with substrate mediates ATP hydrolysis, which enables Hsp70 to transition to the closed conformation. In this state, the SBDα closes over the substrate. NEF mediates the exchange of ADP to ATP thus facilitating the transition to the open ATP-Hsp70 bound conformation, where substrate release can occur.

Figure 2.

Protein translocation. (a) Diagram depicting post-translational translocation into the ER. The polypeptide chain is fully synthesized and dissociated from the ribosome but kept in a partially folded state by cytosolic chaperones (not shown to scale). The nascent chain interacts with the membrane bound Sec61-62-63 complex which facilitates its translocation into ER lumen. The driving force for polypeptide insertion comes from BiP. Initially, BiP is attached to Sec63. As the polypeptide chain translocates through the channel, BiP binds to the nascent chain causing its release from Sec63. This serves to increase its entropy as it moves away from the constraints of the membrane. (b) Protein translocation into the mitochondrial matrix. A simplified diagram illustrating the pre-sequence pathway for protein insertion in the matrix. Newly synthesized polypeptides attach to cytosolic chaperones that interact firstly with the TOM translocase complex and subsequently with TIM23 complex. Integral membrane proteins usually engage a different translocase complex within the inner membrane (TIM22) (not shown). Also, TIM23 complex can transfer proteins laterally into the membrane depending on certain cues within the polypeptide sequence. The PAM motor helps drive the pre-sequence polypeptide into the matrix by entropic pulling of the nascent chain by mtHsp70. The key PAM components, mtHsp70 (yellow), Tim44 (pink) and Pam18 (light brown) are shown connected to the channel forming components Tim17-Tim23 (green - blue).

Figure 3.

BiP-dependent UPR activation models. (a) Allosteric model for ER stress sensing. BiP interacts with IRE1 via its NBD domain, preventing BiP association with its cochaperones, which switches BiP to an ER stress sensor role. ATP primes BiP to engage misfolded proteins. The binding of misfolded protein to BiP SDB triggers release of BiP NBD from IRE1 via a conformational change. IRE1 dimerizes or oligomerizes to activate UPR signalling. BiP is now able to associate with its cochaperones and refold the attached misfolded protein via its well characterized nucleotide dependent substrate cycle. (b) Competition model for UPR repression. BiP binds to IRE1 via its SBD as a chaperone substrate interaction. This is the same site that misfolded proteins bind to BiP. ERdj4 is required to recruit BiP to an IRE1 dimer in order to break the dimer and repress UPR signalling in a process that requires ATP hydrolysis. The ADP bound form of BiP causes release of ERdj4 from IRE1-BiP complex. Nucleotide exchange factors enable the exchange of ADP to ATP bound BiP. The ATP bound form of BiP results in the dissociation of BiP from IRE1 monomer. Now, the free IRE1 monomer can either spontaneously form an IRE1 dimer leading to UPR activation, or BiP could rebind IRE1 via ERdj4 active targeted recruitment and keep UPR repressed. In high ER stress, BiP and ERdj4 are occupied binding to misfolded protein so the number of ATP bound BiP and ERdj4 is sufficiently low that there is none available to rebind IRE1 leading to UPR activation. In low ER stress, there is a preponderance of BiP and ERdj4 enabling UPR repression.

Figure 4.

A graphical summary illustrating the key mechanistic features of specialized interactions involved in IRE1-BiP and Tim44-mtHsp70 systems, and their differing biological outputs.