NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.

The area surrounding the tunnel exit of the 60S ribosomal subunit is a hub for proteins involved in maturation and folding of emerging nascent polypeptide chains. How different factors vie for positioning at the tunnel exit in the complex cellular environment is not well understood. We used in vivo site-specific cross-linking to approach this question, focusing on two abundant factors-the nascent chain-associated complex (NAC) and the Hsp70 chaperone system that includes the J-domain protein co-chaperone Zuotin. We found that NAC and Zuotin can cross-link to each other at the ribosome, even when translation initiation is inhibited. Positions yielding NAC-Zuotin cross-links indicate that when both are present the central globular domain of NAC is modestly shifted from the mutually exclusive position observed in cryogenic electron microscopy analysis. Cross-linking results also suggest that, even in NAC's presence, Hsp70 can situate in a manner conducive for productive nascent chain interaction-with the peptide binding site at the tunnel exit and the J-domain of Zuotin appropriately positioned to drive stabilization of nascent chain binding. Overall, our results are consistent with the idea that, in vivo, the NAC and Hsp70 systems can productively position on the ribosome simultaneously.

Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Functionality of Class A and Class B J-protein co-chaperones with Hsp70.

At their C-termini, cytosolic Hsp70s have an EEVD tetrapeptide that interacts with J-protein co-chaperones of the B, but not A, class. This interaction is required for partnering with yeast B-type J-proteins in protein folding. Here we report conservation of this feature. Human B-type J-proteins also have a stringent EEVD requirement. Human A-type J-proteins function less well than their yeast orthologs with Hsp70ΔEEVD. Changes in the zinc binding domain, a domain absent in B-type J-proteins, overcomes this partial EEVD dependence. Our results suggest that the structurally similar A- and B-class J-proteins of the cytosol have evolved conserved, yet distinct, features that enhance specialized functionality of Hsp70 machinery.

Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.

Sequential duplications of an ancient member of the DnaJ-family expanded the functional chaperone network in the eukaryotic cytosol.

Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, and biochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinary functional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones to form a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all three kingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteins diverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversification and thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimental analyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, which emerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modeling revealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However, despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in the translocation of proteins from the cytosol into mitochondria. Apj1's specialized role is related to degradation of sumolyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specialized functions, allowing expansion of the chaperone functional network, while maintaining the overall structural organization of their parental gene.

Expression of Acidothermus cellulolyticus E1 endo-beta-1,4-glucanase catalytic domain in transplastomic tobacco.

As part of an effort to develop transgenic plants as a system for the production of lignocellulose-degrading enzymes, we evaluated the production of the endo-beta-1,4-glucanase E1 catalytic domain (E1cd) of Acidothermus cellulolyticus in transplastomic tobacco. In an attempt to increase the translation efficiency of the E1cd cassette, various lengths of the N-terminus of the psbA gene product were fused to the E1cd protein. The psbA gene of the plastid genome encodes the D1 polypeptide of photosystem II and is known to encode an efficiently translated mRNA. Experiments in an Escherichia coli expression system indicated that the fusion of short (10-22 amino acid) segments of D1 to E1cd resulted in modest increases in E1cd abundance and were compatible with E1cd activity. Plastid expression cassettes encoding unmodified E1cd and a 10-amino-acid D1 fusion (10nE1cd) were used to generate transplastomic tobacco plants. Expression of the E1cd open reading frame in transplastomic tobacco resulted in very low levels of the enzyme. The transplastomic plants accumulated a high level of E1cd mRNA, however, indicating that post-transcriptional processes were probably limiting the production of recombinant protein. The accumulation of 10nE1cd in transplastomic tobacco was approximately 200-fold higher than that of unmodified E1cd, yielding 10nE1cd in excess of 12% of total soluble protein in the extracts of the lower leaves. Most importantly, the active recombinant enzyme was recovered very easily and efficiently from dried plant material and constituted as much as 0.3% of the dry weight of leaf tissue.

Fungal phyA gene expressed in potato leaves produces active and stable phytase.

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 microM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.

Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.