Cell Stress and Chaperones (2016) 21:563–570 DOI 10.1007/s12192-016-0697-1

MINI REVIEW

J domain independent functions of J proteins Chetana Ajit Tamadaddi 1 & Chandan Sahi 1

Received: 4 March 2016 / Revised: 4 April 2016 / Accepted: 25 April 2016 / Published online: 4 May 2016 # Cell Stress Society International 2016

Abstract Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not Bcompletely^ rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes. Keywords J proteins . Hsp40 . Hsp70

Introduction Inside any living cell, many proteins are constantly at the risk of misfolding and aggregation, due to molecular crowding, mutations, or stress. To ameliorate this problem, every cell expresses a battery of very specialized proteins called molecular chaper* Chandan Sahi [email protected] 1

Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal, India

ones that assist in the re-folding of misfolded proteins and degradation of terminally misfolded proteins or aggregates. Heat shock proteins of 70 kDa (Hsp70s) along with heat shock proteins of 40 kDa (Hsp40s), also called J proteins constitute a versatile chaperone machinery that assist in diverse processes like nascent chain folding; protein transport across membranes; assembly and disassembly of protein complexes; degradation of unstable, misfolded, or aggregated proteins; and controlling the stability and activity of various proteins (Bukau and Horwich 1998; Kampinga and Craig 2010; Mayer et al. 2001; Mayer and Bukau 2005). J proteins have been often regarded as obligate partners of Hsp70s as neither Hsp70s, nor the J proteins can work without each other (Kampinga and Craig 2010). In all the organisms studied so far, the number of J proteins is always more than the number of Hsp70s. Since these two always partner with each other, it is evident that multiple J proteins function with a single Hsp70. Depending on the J protein an Hsp70 interacts with, it can perform either a general or a highly specialized function (Sahi and Craig 2007). Work done in Saccharomyces cerevisiae shows that generalized functions merely require indiscriminate stimulation of Hsp70’s ATPase activity by the signature J domain present in all J proteins (Sahi and Craig 2007). For more specialized functions, besides a functional J domain, specific client binding, protein; protein interactions or specific sub-cellular localization of the J protein is required. J domains are essential for the functionality of J proteins and mutations that render a J domain non-functional result into a completely null phenotype for that J protein. However, in last couple of years, several J proteins have been identified that do not require their J domain for performing all their functions, suggesting that J proteins can have J domain independent functions as well and not all functions of a J protein are Hsp70 dependent. Here we discuss specific examples of J proteins that have J domain independent functions and

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C. AjitTamadaddi, C. Sahi

Hsp70s by stimulating its ATPase activity (Craig et al. 2006; Laufen et al. 1999; Liberek et al. 1991; Misselwitz et al. 1998; Qiu et al. 2006). All J domains possess a characteristic tetrahelical structure with helix I, II, III, and IV. A well-conserved HPD tri-peptide motif between helices II and III is essential for its ability to accelerate the ATPase activity of Hsp70s (Fig. 2a) (Greene et al. 1998; Hennessy et al. 2005; Tsai and Douglas 1996). Although HPD is critical, in some cases mutations in helix II and III also result into complete loss of J domain function (Hennessy et al. 2005). Apart from the J domain, J proteins carry other domains and motifs that serve to categorize them into three classes (Fig. 2b) (Cheetham and Caplan 1998). Class I J proteins have all four of the structurally defined domains that are found in DnaJ of E. coli: a compact helical J domain that is linked by a glycine-rich region to a CXXCXGXG type cysteine-rich zinc-finger region where X is any amino acid. This is followed by a carboxyterminal substrate-binding domain. Class II J proteins are similar to class I except that they lack the zinc-finger domain. Class III J proteins, the most diverse among all, have only J domain and lack all other sequence features found in class I and II members of the family. Additionally, they can have other domains and motifs not found in DnaJ. Regions outside the J domain are often involved in binding and/or delivery of

summarize recent findings that are prodding for a re-evaluation of mechanisms of J protein function and evolution. J proteins: drivers of Hsp70 function Hsp70s are the core of the Hsp70: J protein machine. Hsp70s have an N-terminal ATPase domain, also called nucleotidebinding domain (NBD) that binds and hydrolyses ATP to ADP and a C-terminal substrate-binding domain (SBD) that transiently associates with client proteins. The SBD has a hydrophobic substrate-binding pocket guarded by an alpha helical lid which regulates its interaction with substrate. In the ATP bound state, the lid is open, and peptides bind and release relatively rapidly. Following ATP hydrolysis, the lid closes leading to tight binding of polypeptides to the substratebinding sub-domain (Mayer et al. 2001). Ironically, the intrinsic ATPase activity of Hsp70s is too weak and thus Hsp70s establish obligate partnership with their co-chaperone, J proteins, to carry out their cellular functions. J proteins stimulate the weak ATPase activity of their partner Hsp70s, thereby regulating its chaperone functions (Fig. 1a) (Jordan and McMacken 1995). J proteins are a diverse group of proteins; all having a signature J domain that regulates the chaperone activity of

RdRP subunits

a

b PA PolyQ Protein

J domain Regions other than J domain

J Hsp70

Influenza A virus replication in host

J protein Non -native protein

Pdr1 Prevention of polyQ aggregation

1

5

Clathrin Pleiotropic drug resistance

NEF

J

Substrates Clathrin disassembly

or

4

6

Flagellar beating

Native protein Substrates

J

7

Apoptosis and overcoming cell cycle arrest

RSP23

Inhibition of PKR

pRB

J

Prevention of client aggregation

Pi

Prevention of Rhodanese aggregation

NEF

RSP23

PKR

Substrates

2

Spliceosome disassembly

Hsp70

Client protein

Rhodanese

3

J domain dependent

J domain independent

Substrates

Ntr1

PRP43

Ntr2

Fig. 1 J domain dependent and independent functions of J proteins. a J domain dependent functions. Non-native client protein binds to substrate-binding domain (SBD) of Hsp70 and J domain of J protein stimulates ATP hydrolysis via interaction with nucleotide-binding domain (NBD) (1). Release of J protein and Pi leads to stabilized client interaction with Hsp70 via conformational changes induced in SBD in ADP bound state (2). Nucleotide exchange factor (NEF) binds Hsp70 and replaces ADP with ATP (3). This leads to low

affinity state of Hsp70 for client in ATP bound form. Client protein is released either in native functional form or non-native form which can go for further rounds of binding/release (4). Hsp70 in ATP bound state is recycled back to step 1 where it binds to J protein. b J domain independent functions. Domains other than J domain bind to substrates listed (5) by themselves (6) or carry them to Hsp70 to perform their respective functions without the involvement of a functional J domain (7)

J domain independent functions of J proteins Fig. 2 a J domain structure of E. coli DnaJ. J domain has four helices with HPD motif located in the loop between helix II and III. b Domain organization of J proteins

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b

a Helix I Helix IV

J domain

G/F

J domain

G/F

ZnF

C-term

Class I

C-term

Class II

Helix III J domain

Helix II

Class III

HPD

clients to Hsp70, mediating protein-protein interactions or specifying the sub-cellular localization of J proteins and thus determine the functionality, specificity, and diversity of the Hsp70 machine. While some J proteins guide Hsp70’s precise sub-cellular location for its action on clients, others present the clients themselves by directly binding to them (Kampinga and Craig 2010).

suggesting that many functions of Hsp70 chaperone machineries only require indiscriminate stimulation of Hsp70’s ATPase activity by the J domains of other J proteins in the same sub-cellular location or compartment. In such a case, dependency of an Hsp70 on a particular J protein for the stimulation of its ATPase activity can be further reduced if J proteins with highly similar and functionally redundant J domains populate the same cellular compartment.

Complexity of the Hsp70: J protein network J domain independent functions of J proteins J proteins have outnumbered Hsp70s in all the organisms studied. In humans, 11 Hsp70s and at least 41 J proteins have been identified. S. cerevisiae has 14 Hsp70s and 22 J proteins while Arabidopsis thaliana is reported to have 18 Hsp70s and 116 J proteins. Similarly, in C. elegans, there are 15 Hsp70s and 22 J proteins, and in Drosophila melanogaster, there are 14 Hsp70s and 22 J proteins (Jungkunz et al. 2011; Kampinga and Craig 2010; Qiu et al. 2006; Rajan and D’Silva 2009; Walsh et al. 2004). Thus, at least some Hsp70s must partner with more than one J protein. An example for this is Ssc1, the major Hsp70 of mitochondria in S. cerevisiae (Liu et al. 2001). Approximately 10 % of Ssc1 is tethered to protein translocation channel and functions with the J protein Pam18 in mitochondrial protein import (D’Silva et al. 2004; Mayer 2004). The remaining 90 % is in the matrix, where it partners with the class I J protein Mdj1, in mitochondrial protein folding and quality control processes (Liu et al. 2001; Wagner et al. 1994). This situation is even more complex in the yeast cytosol, where Ssa class of Hsp70s work with about a dozen different J proteins to perform an array of functions (Craig et al. 2006). Ydj1, the major cytosolic J protein in budding yeast, is a general chaperone, while all other J proteins are highly specialized with very little or no functional redundancy with other J proteins residing in the cytosol (Sahi and Craig 2007; Sahi et al. 2013). Deletion phenotypes of most of the cytosolic J proteins could be rescued only by the expression of the same protein. In contrast, the severe growth phenotype caused by the absence of Ydj1 is substantially rescued by expression of J domain containing fragments of many cytosolic J proteins

J domain is crucial for J protein’s functions but there are exceptions. Several J proteins, belonging to all the three classes have been shown to have J domain independent functions either in vitro or in vivo. While most of these are nonessential and accessory functions of a particular J protein, some are essential as well. In Table 1, we compile the available information on J proteins that have J domain independent functions.

Class I J proteins Class I J proteins are closely related to the ancestral J protein DnaJ of E.coli and members of this class contain all the domains found in DnaJ. Studies have shown that among the class I J proteins, only the C-terminal substrate-binding domain has evolved and become more diverse to bind to different peptide clients which contribute to the specificity of J protein function (Sahi et al. 2013). Ydj1, the most abundant cytosolic J protein in S. cerevisiae, is involved in general protein folding, protein transport, and degradation (Caplan et al. 1992; Caplan and Douglas 1991; Cyr 1995; Levy et al. 1995). The deletion of Ydj1 causes growth defects and H34Q mutation in the conserved HPD within the J domain results into a null phenotype. However, Ydj1H34Q can still bind unfolded polypeptides though it cannot stimulate the ATPase activity of Hsp70. The C-terminal fragment of Ydj1 (Ydj1179–384), completely lacking the J domain, GF, and part of the

566 Table 1

C. AjitTamadaddi, C. Sahi List of J proteins with J domain independent functions

J protein

Function

Ydj1

Suppression of rhodanese S. cerevisiae aggregation Assisting influenza H. sapiens A virus replication in host Remodeling of H. sapiens polyglutamine (polyQ) aggregates

DnaJA1 DnaJB6b and DnaJB8

ERdJ3 (DnaJB11) Binding and prevention of client aggregation Rsp16 Flagellar beating mediated by radial spokes Zuo1 Activation of Pdr1 P58IPK Swa2p

Organism

Mutation

Domain

Effect

References

Ydj1179–384 Ydj1H34Q DnaJA1201–397 DnaJA11–200

Lacking J domain J domain Lacking J domain J domain

Functional Functional Functional Non-functional

(Lu and Cyr 1998)

DnaJB6bH31Q and DnaJB8H31Q DnaJB6bΔ(1–69) and DnaJB8Δ(1–69) ERdJ3H53Q and ERdJ3D55N

J domain

J domain

Partially functional Partially functional Functional

C. reinhardtii Rsp16Δ(1–70)

Lacking J domain

Functional

S. cerevisiae

Zuo1365–433

C-terminal

Functional

P58IPKHPD→QPD P58IPKHPD→AAA

J domain

Functional

Swa2pHPD→AAA

J domain

(Xiao et al. 2006)

Swa2pG388R

TPR domain

Partially functional Partially functional Non-functional Functional Functional Functional Functional

(Sheng et al. 2000)

Functional Functional

(Sahi et al. 2010)

H. sapiens

Inhibition of PKR H. sapiens (interferon-induced protein kinase) Clathrin coat disassembly S. cerevisiae

Swa2pHPD→AAA Swa2pG388R Polyomavirus PyLTΔ(2–79) PyLTH42Q Simian virus SV40LT83–708 SV40LTH42Q 40

PyLT antigen

Apoptosis

SV40 LT antigen

Binding to pRB protein and overcoming cell cycle arrest Spliceosome disassembly S. cerevisiae

Cwc23

Cwc23Δ(1–80) Cwc23H50Q

Lacking J domain

J domain and TPR domain Lacking J domain J domain Lacking J domain J domain Lacking J domain J domain

(Cao et al. 2014) (Gillis et al. 2013) (Hageman et al. 2010) (Shen and Hendershot 2005) (Yang et al. 2008) (Ducett et al. 2013; Eisenman and Craig 2004) (Yan et al. 2002)

(Gjoerup et al. 2000)

Ydj1 and DnaJA1 are class I J proteins whereas DnaJB6b, DnaJB8, ERdj3, and Rsp16 belong to class II. The remaining J proteins fall under class III

cysteine-rich zinc-finger was capable of suppressing rhodanese aggregation in vitro with an efficiency similar to that of full-length protein (Lu and Cyr 1998). Suppression of protein aggregation is a classical chaperone function. DnaJA1, another class I J protein in mammals, acts as a host factor for influenza A virus replication (Cao et al. 2014). DnaJA1 associates with the PA and PB2 subunits of RNA-dependent RNA polymerase (RdRp) through its C-terminal substrate-binding domain and is imported to the nucleus where it enhances viral RNA synthesis both in vivo and in vitro (Cao et al. 2014). A DnaJA1 truncation mutant (DnaJA1201–397), which lacks J domain completely could associate with both PA and PB2 subunits, whereas DnaJA11–200, having an intact J domain but lacking the C-terminal client binding domain could not. The viral ribonucleoprotein complex (vRNP) of influenza A virus is the minimal functional unit for viral RNA transcription and replication in the nuclei of infected cells. The vRNP consists of an RNA-dependent RNA polymerase complex (RdRp), a viral RNA, and multiple copies of nucleoprotein (NP). A DnaJA1201–397 fragment could significantly increase the levels of NP inside the nuclei, an indication of viral replication. This was comparable to the full-length DnaJA1 activity. On the contrary, the DnaJA11–200 fragment, containing an intact J domain could not function to the similar levels suggesting that the role of DnaJA1 in stimulating influenza viral RNA polymerase activity is mainly dependent on its C-

terminal substrate-binding domain and completely independent of its J domain (Cao et al. 2014). It is possible that either this process does not require the J domain of DnaJA1 or J domains of other proteins can substitute for the DnaJA1 J domain in trans. Not all the processes regulated by DnaJA1 are J domain independent. In pancreatic cancer, interaction of DnaJA1 with DnaK requires J domain and this interaction mediates the suppression of apoptosis. DnaJA1, thus has a completely functional J domain that is essential for JNKinduced anti-apoptotic signaling pathway, but it is not required for viral replication (Cao et al. 2014; Stark et al. 2014).

Class II J proteins Class II J proteins are similar to class I, except that they lack the cysteine-rich zinc-finger domain that is important for client binding and delivery (Cheetham and Caplan 1998; Kota et al. 2009). One of the most important class II J proteins in S. cerevisiae is Sis1. Sis1 is essential and its J domain mutant does not support the viability of a sis1Δ yeast strain (Yan and Craig 1999). Along with the Hsp70 partner Ssa1 and Hsp104, Sis1 plays a critical role in the remodeling of prion aggregates in yeast in a J domain dependent manner (Lopez et al. 2003). DnaJB6b and DnaJB8 are mammalian orthologs of Sis1, where they have been linked to the remodeling of

J domain independent functions of J proteins

polyglutamine (polyQ) aggregates (Chai et al. 1999). The ability to prevent aggregation of polyQ proteins by DnaJB6b and DnaJB8 was only partially affected by mutations or deletion of their respective J domains (Gillis et al. 2013; Hageman et al. 2010). Mutating His to Glu (H31Q) in the HPD motif only partly impaired the ability of DnaJB6b and DnaJB8 to reduce polyQ aggregation in vivo. On the other hand, deletion of the serine-rich region (amino acids 155–194) within DnaJB6b and DnaJB8 abolished the anti-aggregation properties of these chaperones (Gillis et al. 2013). The J domain of DnaJB6b and DnaJB8 is known to interact with HSPA1A (Hsp70) and stimulate its ATPase activity, however it is less important for the anti-aggregation properties of these J proteins, suggesting a limited role of J domain in prevention of polyQ aggregation (Gillis et al. 2013). This Hsp70 independent functioning of J proteins in client binding and prevention of aggregation is similar to that of Ydj1 (Johnson and Craig 2001). ERdj3 (hsDnaJB11), an ER-localized class II J protein can bind the unfolded and mutant secretory pathway proteins even in the presence of J domain inactivating mutations H53Q and D55N (Shen and Hendershot 2005). Also, the Hsp70 (Bip) mediated immunoglobulin λ-light chain folding in the presence of J domain mutant of ERdj3 was not delayed, but the subsequent release of ERdj3 form Bip, which requires ATPase stimulation, is interrupted (Shen and Hendershot 2005). These results indicate that, J domain of ERdj3 is dispensable for client binding, however stimulation of Hsp70’s ATPase is required only for its own release from the Hsp70, which is J domain dependent (Jin et al. 2008). ERdj3 prevents client aggregation and presents the clients for Bip-mediated folding (Fig. 1b). This is evident by the loss of wild-type ERdj3 form Bip: substrate complex well before the completion of folding (Shen and Hendershot 2005). It is not clear if J domain of any other J protein is working with Bip to achieve proper folding of the substrate. J protein RSP16 of Chlamydomonas reinhardtii presents a rather extreme case. Dimeric RSP16 is incorporated during the assembly and maturation of the radial spokes at the flagellar tip and thus is important for flagellar-beating movement in C. reinhardtii (Yang et al. 2008). The J domain of RSP16, is not required for its role in flagellar-beating function as the complementation of rsp16Δ cells with the Rsp16ΔDnaJ, lacking the N-terminal 70 amino acid residues comprising the J domain, rescued flagellar-beating function almost like the wild-type RSP16 (Yang et al. 2008). The fact that the normally highly conserved HPD motif of the J domain, which is essential for interaction with Hsp70, is poorly conserved among the spoke RSP16 orthologs supports the idea that the J domain of Rsp16 is not absolutely important for its function. Orthologs of Rsp16 identified in C. reinhardtii, C. intestinalis, and sea urchin have intact HPD motif in their J domains. Most other orthologs, the ones in humans (hsDnaJB13), mouse (NP_705755), m osquito (XP_393200), zebrafish

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(XP_684040), cow (AAI02673), dog (XP_534013), chicken (XP_417251), and Xenopus (AAI08634) have the HPD motif in their J domains altered, raising the possibility of an increase in functional importance of domains other than J domain during evolution, with a decrease or gradual loss in J domain function (Yang et al. 2008). DnaJB13 is a radial spoke protein of the mouse B9+2^ axoneme which participates in spermiogenesis and the motility of mature spermatozoa (Guan et al. 2010; Li and Liu 2014).

Class III J proteins These are the most diverse group of J proteins. Besides the presence of a J domain, these do not share any similarity to class I and II J proteins. These are most unique and often perform very specialized functions. Some J proteins perform more than one function in the cell. A classic example is that of Zuo1 in S. cerevisiae. Zuo1 associates with the ribosome and is involved in nascent protein folding (Yan et al. 1998). When not associated with the ribosome, it plays a very unique role in imparting pleotropic drug resistance (PDR) to yeast cells (Hallstrom et al. 1998). While the nascent chain folding function of Zuo1 is Hsp70 dependent and requires an intact J domain, for its role in inducing PDR, the J domain, and hence the Hsp70 dependent activity of Zuo1 is completely dispensable (Eisenman and Craig 2004; Gautschi et al. 2002). A fragment containing the last 69 residues of Zuo1 (Zuo1365–433) which is dispensable for Zuo1’s chaperone function on ribosome is sufficient for inducing PDR in yeast cells (Ducett et al. 2013; Eisenman and Craig 2004). Similarly, the Simian virus 40 (SV40) large T antigen (LT) can immortalize and transform several cell types by associating and inactivating the two major tumor suppressors—p53 and the retinoblastoma protein, pRB. Retinoblastoma susceptibility gene (Rb) family is multifunctional and works both in E2F dependent and independent manner. While most effects of LT on pRB require a functional J domain (Sullivan et al. 2000), J domain is dispensable for overcoming p53 mediated growth suppression (Gjoerup et al. 2000). A J domain mutant, H42Q, as well as a complete J domain truncation (aa 83–708) was almost as active as wildtype LT in forming colonies at 32 °C and overcoming the p53 cell cycle block (Gjoerup et al. 2000). Similarly, the polyomavirus LT promotes cell cycle progression, immortalizes primary cells, and blocks cell differentiation via its effects on tumor suppressors of the Rb family. Besides the Rb binding site, a functional J domain is required for the activation of simple E2F-containing promoters and stimulation of cell cycle progression (Sheng et al. 2000). On the contrary, apoptosis caused by overexpression of LT in differentiating C2C12 myoblasts requires Rb binding but is completely J domain independent as an H42Q mutant or a complete deletion mutant lacking the J domain (Δ2-79) could also completely induce

568

cell death (Sheng et al. 2000). Thus, it is apparent that J protein-Rb interaction leading to E2F-mediated cell cycle progression under normal conditions and apoptosis under high levels of LT, do not require a functional J domain whereas, disruption of Rb-E2F complex and subsequent cell cycle progression is largely dependent on J domain (Fimia et al. 1998; Gjoerup et al. 2000; Sheng et al. 2000; Sullivan et al. 2000). Mammalian J protein inhibitor of protein kinase (P58IPK) is an inhibitor of interferon-induced, protein kinase, protein kinase R (PKR), which is a negative regulator of viral protein synthesis (Yan et al. 2002). P58IPK contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain. The human P58IPK J domain can substitute for the J domain of DnaJ and Ydj1 supporting the bacterial and yeast growth at 37 °C similar to wild-type protein. Introduction of point mutations in the HPD tri-peptide motif (H to Q or HPD to AAA) resulted in loss of function for both the chimeras, indicating that P58IPK has a classical J domain which dictates P58IPK’s interactions with its Hsp70 partner (Yan et al. 2002). However, inhibition of PKR activity is completely independent of this J domain as mutations within the conserved HPD tri-peptide motif could still inhibit the PKR activity and supported cell growth in a yeast rescue assay (Yan et al. 2002). Overexpression of the HPD mutants of P58IPK, similar to their wild-type counterpart, also stimulated viral mRNA translation in a mammalian cell system, suggesting that P58IPK has both J domain dependent as well as independent functions (Yan et al. 2002). Swa2p, the auxillin homologue in S. cerevisiae operates with Hsp70 in uncoating of clathrin-coated vesicles (CCV) (Pishvaee et al. 2000). It has three N-terminal clathrinbinding (CB) motifs, an ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J domain. Mutation in the invariant HPD motif to AAA within the J domain and point mutation G388R in adjacent TPR domain independently, affect Swa2p function only partially in vivo. HPD to AAA mutation in the J domain, instead of showing a null phenotype, could only lead to partial loss of function and could complement growth defects of swa2Δ strain. However, combination of the HPD to AAA and G388R hypomorphic mutations completely abolish Swa2p function by failing to complement swa2Δ in the growth, halo or synthetic lethal assays emphasizing on the importance of TPR domain along with J domain in Swa2p function. Further, a truncation fragment of Swa2p having just one CB domain was nearly as active in vivo as the full-length Swa2p protein (Xiao et al. 2006). Interestingly, the slow growth phenotype of swa2Δ strain was also partially complemented by the Cterminal TPR-J fragment of Swa2p as well as J domain containing fragments of multiple cytosolic J proteins (Sahi and Craig 2007; Xiao et al. 2006). It is previously reported that the TPR domain containing proteins Sti1p and Cns1p could stimulate Hsp70 ATPase activity even though these proteins do not contain a J domain. In this case, one can speculate that the

C. AjitTamadaddi, C. Sahi

ATPase stimulation by TPR domain might be sufficient for Hsp70’s activity (Hainzl et al. 2004; Wegele et al. 2003). Several scenarios are possible. First, Swa2p performs functions other than modulating the clathrin dynamics which are J domain independent. Second, J domain is dispensable for the uncoating of clathrin-coated vesicles, and the Hsp70 can be recruited by just a CB domain. Third, other J proteins or just TPR domain somehow stimulate the ATPase activity of Hsp70 for the release of the triskelia, and Swa2p merely functions in the client (clathrin)-binding and recruitment of Hsp70 to the clathrin lattices and Hsp70 itself might be sufficient to perform the clathrin-uncoating function (Fig. 2b). It has been shown that more abundant Hsp70s can themselves perform some of the chaperone functions without a significant involvement of J proteins in vitro (Mayer and Bukau 2005). It is reasoned that the basal ATPase activity of at least some Hsp70s is sufficient and the J protein partner just performs the function of delivery of a client or tethering of Hsp70 to a particular sub-cellular compartment or complex. Thus, ATPase stimulation might not be absolutely required for some functions under normal growth conditions and only under sub-optimal conditions or in the presence of destabilizing mutations, ATPase stimulation of Hsp70 is physiologically important. The sufficiency of weak basal ATPase activity of selected Hsp70s in carrying out some of their chaperone functions and the dispensability of J domains in some J proteins clearly indicates that not all processes requiring the Hsp70 J protein machine really depend on the stimulation of Hsp70’s ATPase activity by a J domain, thus making such partnerships less obligatory than previously thought. In all the cases mentioned above, regions outside the J domain are important for at least some of the J protein functions. In others, J proteins have been found to have some J domain dependent functions as well. Cwc23, an essential J protein of S. cerevisiae, presents the most extreme case. Cwc23 is essential and is believed to be involved in spliceosomal remodeling. It interacts with the spliceosomal protein, Ntr1 via its C-terminal domain, and this interaction is important for its function in vivo (Sahi et al. 2010). A Cterminal truncation (Cwc231–225) that does not interact with Ntr1 is slow growing, shows significant pre-mRNA splicing defects as well as accumulation of intron lariat (Sahi et al. 2010). Interestingly, Cwc23ΔJ cells did not exhibit any growth or splicing defects under any growth condition tested. This establishes that the J domain is not required for Cwc23’s essential function(s). The J domain became essential only in combination with mutations that destabilized physical interactions between the spliceosomal disassembly factors Ntr1 and Prp43. Even though Cwc23 has a J domain that is dispensable for cell viability, the J domain is completely functional (in budding yeast) and highly conserved in all Cwc23 orthologs identified in higher eukaryotes. This indicates that either Cwc23 has some other cellular function for which J domain

J domain independent functions of J proteins

is required or having a J domain provides some evolutionary or adaptive advantage, especially under stress conditions or in the presence of destabilizing mutations. Cwc23 provides an extreme example of J proteins whose biological role is defined more by their non-J domain regions.

Conclusions and perspectives There is no doubt that J domain is critical for J protein function, and most cellular functions carried out by J proteins require a functional J domain which enables them to operate with a partner Hsp70. Some J proteins however do not fall in this category and can have J domain independent functions. Accumulating data suggests that such J proteins are more common than previously thought. They can have essential or accessory functions in the cell that does not require a functional J domain. Nonetheless, it is premature to exclude the involvement of Hsp70 in the functions discussed above. It is speculated that other J proteins or co-chaperones might be recruited to perform chaperone-mediated functions. This supports two extreme possibilities of J protein evolution; J proteins became more complex by acquiring new domains, in some cases, these additional domains outran the J domain in its functional importance; or proteins that independently function in certain cellular processes gained a J domain, to recruit the Hsp70 system to fine-tune an existing chaperone activity. Reducing the dependency of a J protein on its J domain for at least some of its functions might render the system more versatile and robust. It is conceivable that the requirement of a J protein for recruiting Hsp70s to a particular intracellular localization can be reduced if the concentration of Hsp70 is increased in the cell. It is believed that all J proteins are descendants of the ancestral J protein DnaJ of E.coli. It is possible that in the course of evolution if regions other than the J domain became more important for J proteins’ functions, gradually the J domain became dispensable and thus acquired mutations, even in the critical HPD motif, giving rise to J-like proteins (JLPs). Collectively, (a) basal ATPase activity of Hsp70 being sufficient for a particular cellular process, (b) other J proteins/J domains/co-chaperones compensating for J domain dependent functions of a particular J protein, and (c) regions/domains or motifs other than the J domain having more important functions, indicate that a J protein might gradually acquire a J domain that is dispensable for some of its functions in vivo.

References Bukau B, Horwich AL (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92:351–366

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