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Review
. 2010 Aug;11(8):579-92.
doi: 10.1038/nrm2941.

The HSP70 chaperone machinery: J proteins as drivers of functional specificity

Harm H Kampinga  1 , Elizabeth A Craig
Affiliations
Review

The HSP70 chaperone machinery: J proteins as drivers of functional specificity

Harm H Kampinga et al. Nat Rev Mol Cell Biol. 2010 Aug.

Erratum in

  • Nat Rev Mol Cell Biol. 2010 Oct;11(10):750

Abstract

Heat shock 70 kDa proteins (HSP70s) are ubiquitous molecular chaperones that function in a myriad of biological processes, modulating polypeptide folding, degradation and translocation across membranes, and protein-protein interactions. This multitude of roles is not easily reconciled with the universality of the activity of HSP70s in ATP-dependent client protein-binding and release cycles. Much of the functional diversity of the HSP70s is driven by a diverse class of cofactors: J proteins. Often, multiple J proteins function with a single HSP70. Some target HSP70 activity to clients at precise locations in cells and others bind client proteins directly, thereby delivering specific clients to HSP70 and directly determining their fate.

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Figures

Figure 1
Figure 1. Protein folding and degradation through client protein–chaperone binding and release cycle
Chaperones were originally defined as “proteins that prevent improper interactions between potentially interactive surfaces and disrupt any improper liaisons that may occur”. The Heat Shock Proteins (HSPs) family constitutes a large group of chaperones that interact with a wide variety of non-native proteins, facilitating acquisition of their native conformation, without being associated with their final functional structure. However, recent evidence indicates that chaperone function of HSPs is not restricted to assisting protein folding and assembly. It is also needed to facilitate client degradation via both proteasomal and autophagasomal pathways, as well as to stabilize or destabilize interactions between mature, folded proteins. In iterative cycles of client binding to and release from HSPs, which are often driven in an adenine nucleotide dependent manner, client aggregation is prevented (purple lines), productive folding occurs through a series of steps (bleu lines), and HSPs are re-cycled for client binding (black lines). If folding fails or in the case of non-foldable clients re-binding to the chaperone may occur, which in a stochastic manner, maintains client soluble for subsequent (proteasomal) degradation (red lines). Besides this passive, stochastic support of degradation, some chaperones can also ‘actively’ direct clients towards degradation (green line). In addition, chaperones can bind folded proteins (bleu circle and grey square) and induce conformational changes, thereby regulating protein:protein interactions and functionality of protein complexes.
Figure 2
Figure 2. Canonical model of mode of action of core Hsp70 machinery in protein folding and Hsp70 structure
a|Canonical mode of functioning of the Hsp70 core machine based on in vitro refolding studies of denatured proteins. [1] J-protein binds to client protein via its peptide-binding domain and [2] interacts with Hsp70(ATP) via its J-domain (J). [3] The client rapidly, but transiently, interacts with the “open” peptide binding site of Hsp70. ATP hydrolysis is stimulated by both the J-domain and client causing a conformational change in Hsp70 closing the helical lid over the cleft, stabilizing client interaction. J-protein leaves the complex. [4] Nucleotide exchange factor (NEF), which has a higher affinity for Hsp70(ADP) than Hsp70(ATP), binds Hsp70; [5] ADP dissociates through distortion of the ATP binding domain, after which [6] ATP binds to Hsp70. [7] Client is released because of its low affinity for Hsp70(ATP). ATP-binding to Hsp70 is favored since cellular ATP concentrations are typically much higher than those of ADP. If the native state of the client is not attained upon release, J-protein rebinds to exposed hydrophobic regions and the cycle begins again. Typically such reiterative binding is required. b| Hsp70 structure with ADP bound to the nucleotide binding domain (PDB code #2KHO). The ATPase domain and peptide-binding domain are connected via a short flexible linker. These domains dock when in the ATP-bound state, which is also thought to displace the lid, allowing easy access and egress of the client protein from the cleft , .
Figure 3
Figure 3. Diversity in domain architecture of J-proteins from the yeast S. cerevisiae and H. sapiens
Members are clustered according to known or presumed client binding ability. Functional orthologs are connected by lines. For clarity, not all known domains are indicated and some differences between yeast and human orthologs are not indicated. For more detailed information see Supplementary Tables I, II, where information about the presumed localization and function of all members is also provided. Regions of Class I and II J-proteins: G/F regions, of which the functional relevance is disputed (see main text) are defined as segments containing more than 5 glycines or/and phenylanines in the first 25 amino acids C-terminal to J-domain. C-terminal domain 1 (CTD1) - canonical type I members have Zinc finger like region (ZFLR); type II members lack a ZFLR. However, type II often have cysteine rich stretches and/or binding site for Histone Deacetylases (HADCs). The indicated dimerization domain (DD) has been firmly established only for a few type I and II (Ydj1, DNAJA1, Scj1, Sis1); for the others this domain is presumed for simplicity. “X” in DNAJB13 indicates the lack of the canonical HPD motif in the J-domain. For reasons of space, some members are not drawn at full size (indicated by numbers); //..// indicate stretches not shown.
Figure 4
Figure 4. J-domain and client protein binding domain structures
a| J-domains contain four alpha-helices, with the central ones forming a coiled-coil motif around a hydrophobic core. The invariant HPD tripeptide located in the loop between helices II and III is critical for ATPase stimulation and in vivo function. Residues in helix II and within the neighboring loop, including the HPD, form an Hsp70 interaction face. b| Class I Ydj1 and Class II Sis1 have similar client protein binding domains, the “DnaJ-type”. The structure of amino acids 102-350 of Ydj1, and amino acids 180-343 of Sis1 are shown. Both have J-domains at their N-termini, followed by a glycine/phenyalanine-rich (G/F) region. No full-length structure of a Class I or Class II J-protein has been obtained, presumably because of the flexibility of the G/F regions. Both Ydj1 and Sis1 are dimers, with the dimerization domain at their C-termini (C). Each monomer of Ydj1 and Sis1 has two adjacent domains that are similar structure, being predominantly composed of beta-sheets. These are often referred to as “C-terminal domain” (CTD1) and CTD2. Ydj1 (like DnaJ) has two zinc fingers (Zn1 and Zn2), which extend out from CTD1. In addition, Ydj1 has a CAAX motif for farnesylation, a modification important for membrane localization and binding of some client proteins . c|Jac1 (called HscB in E. coli), which is critical for Fe-S cluster biogenesis, has a specialized client protein binding domain that has neither sequence nor structural similarities to a DnaJ-type; amino acids 63-171 of HscB are shown. The face pointing outwards interacts with the Fe-S cluster scaffold Isu.
Figure 5
Figure 5. J-protein function with or without client binding
J-proteins can act without binding to clients, either untethered (a) or tethered to a particular site in the cell (b). In the case of J-proteins having client protein binding domains, clients may bind either bind the J-protein before interaction with Hsp70 or only serve to stimulate Hsp70’s ATPase activity (c) a| The simplest J-protein function is the action of a J-domain in the absence of a client protein binding domain to stimulate the ATPase activity, allowing it to capture a client protein that has transiently entered the open peptide-binding cleft of Hsp70(ATP) (i), by binding Hsp70 (ii) and stimulating ATP hydrolysis (iii). In such cases, Hsp70 is the driving force of client protein interaction, as there is no facilitation by the J-protein, either through direct binding or by subcellular localization. The in vivo function of a J-domain lacking other sequences has been demonstrated experimentally. b|J-domains either lacking (shown) or having (not shown) client protein binding domains are often tethered to a site rich in client proteins. In this way, upon initiation of client protein binding by Hsp70(ATP) (1), a high concentration of J-domains is present (2) facilitating ATP hydrolysis and thus client capturing by Hsp70 (3). c|In the case of J-proteins having client protein binding domains, two modes of J-protein function can occur: top: as in the canonical model of J-protein:Hsp70 function J-protein binds client first (x) and targets it to Hsp70 (y,z); bottom: binding occurs directly to Hsp70 (x’) as described in a); in such cases J-proteins stimulate Hsp70 ATPase activity only even though a client binding domain is present (y’,z). Evidence for such an alternative pathway has been found in the mitochondrial Fe-S cluster biogenesis pathway in yeast with the specialized J-protein Jac1 and Hsp70 Ssq1. In all cases, release of client is facilitated by NEFs (iv).
Figure 6
Figure 6. Example of tethering of J-proteins to site of action
Ribosome associated chaperones, an example of tethering to a site within a cellular compartment. All eukaryotes, illustrated by budding yeast (left) and humans (right), have a ribosome associated J-protein that binds near the exit site of the 60S subunit, regardless of whether translation is occurring or not (i). Fungi, though not other eukaryotes, also have a specialized ribosome-associated Hsp70 (Ssb) that independently associates with the 60S subunit. Zuo1 and Ssb function as a J-protein:Hsp70 pair upon emergence of a nascent polypeptide from the ribosome (ii). Later events in polypeptide folding, include binding of soluble J-proteins (iii) and recruitment of soluble Hsp70 (iv) prior to completion of translation and after nascent chain release (v). Herefore, yeast utilizes the abundant soluble J-protein (Ydj1) and Hsp70 (Ssa). In humans, the ribosome-associated J-protein (DNAJC2) recruits HSPA8 as a partner (ii), which partners with the soluble DNAJB1 in downstream folding events (iii-iv). Note, both Zuo and DNAJC2 form a stable heterodimer with the unusual Hsp70 (scSsz1 or hsHSP70A14) (gray oval). The function of this Hsp70, which is not known to have client protein binding activity, beyond being important for the ability of Zuo1 to stimulate the ATPase activity of its Hsp70 partner Ssb is not known.
Figure 7
Figure 7. Examples of J-protein function beyond protein refolding
a| J-protein targeted degradation. The ubiquitin-interacting motifs (UIMs) in DNAJB2 (HSJ1) recognizes clients that contain a mono- or poly-ubiquitin moiety (indicated by small yellow circle) (i). After transfer of the client to Hsp70 (ii), E3 ligases (such as CHIP) and the Ubc ubiquitin conjugation machinery can associate with the Hsp70/DNAJB2 complex (the precise manner and specificity of this associations remains to be understood) leading to further ubiquitination of the bound client (iii; iv). After the canonical ATP hydrolysis step (iv) and NEF-mediated nucleotide exchange (v), the poly-ubiquitinated client released from Hsp70 (iii,iv) is transferred to the proteasome for degradation . b| J-protein mediated modulation of protein:protein interactions. Alteration of interactions between mature, folded proteins typically are part of complex biological processes. Illustrated here is the role of the J-protein Jjj1 in the biogenesis of the 60S ribosome subunit, the destabilization of the biogenesis factor Arx1 in the biogenesis of 60S-. Arx1 is loaded on the pre60S subunit (beige) in the nucleus (i). Jjj1 binds directly to the ribosome, as does Rei1, another cytosolic factor required for Arx1 destabilization, and with which Jjj1 interacts (ii). Jjj1 partners with soluble Ssa (another example of targeting of Hsp70 by J-protein localization, see Figures 5c and 6). Once the Arx1:pre60S subunit interaction is destabilized, a step needed to generate the mature subunit (red), Arx1 is transported back into the nucleus (iv), where it engages in another cycle of subunit biogenesis.
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