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.

Deletion of the N-terminal dirigent domain in maize beta-glucosidase aggregating factor and its homolog sorghum lectin dramatically alters the sugar-specificities of their lectin domains.

Maize beta-glucosidase aggregating factor (BGAF) and its homolog Sorghum Lectin (SL) are modular proteins consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin (JRL) domain. BGAF is a polyspecific lectin with a monosaccharide preference for galactose, whereas SL displays preference for GalNAc. Here, we report that deletion of the N-terminal dirigent domain in the above lectins dramatically changes their sugar-specificities. Deletions in the N-terminal region of the dirigent domain of BGAF abolished binding to galactose/lactose, but binding to mannose was unaffected. Glucose, which was a poor inhibitor of hemagglutinating activity of BGAF, displayed higher inhibitory effect on the hemagglutinating activity of deletion mutants. Deletion of the dirigent domain in SL abolished binding to GalNAc, but binding to mannose was not affected. Surprisingly, fructose, an extremely poor inhibitor (minimum inhibitory concentration (MIC) = 125 mM) of SL hemagglutinating activity, was found to be a very potent inhibitor (MIC = 1 mM) of hemagglutinating activity of its JRL domain. These results indicate that the dirigent domain in this class of modular lectins, at least in the case of maize BGAF and SL, influences sugar specificity.

Determination of beta-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize beta-glucosidase isozyme Glu1.

Beta-glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called beta-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu(50)-Val(145)) and the C-terminal (Phe(466)-Ala(512)) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum beta-glucosidases (dhurrinases, which do not bind to BGAF) and maize beta-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric beta-glucosidases. The results showed that a region spanning 11 amino acids (Ile(72)-Thr(82)) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser(1)-Thr(29), together with C-terminal region Phe(466)-Ala(512), affects the size of Glu1-BGAF complexes. The dissociation constants (K(d)) of chimeric beta-glucosidase-BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile(72)-Thr(82) on Glu1 for BGAF binding, we constructed a chimeric sorghum beta-glucosidase, Dhr2 (C-11, Dhr2 whose Val(72)-Glu(82) region was replaced with the Ile(72)-Thr(82) region of Glu1). C-11 binds to BGAF, indicating that the Ile(72)-Thr(82) region is indeed a major interaction site on Glu1 involved in BGAF binding.

Lysine-81 and threonine-82 on maize beta-glucosidase isozyme Glu1 are the key amino acids involved in beta-glucosidase aggregating factor binding.

In certain maize genotypes (nulls), beta-glucosidase specifically interacts with a chimeric lectin called beta-glucosidase aggregating factor (BGAF), resulting in high molecular weight complexes. Previously, we showed that three regions (S1-T29, E50-N127, and F466-A512) on the maize beta-glucosidase isozyme Glu1 are involved in interaction and aggregation with BGAF. Recently, we found that the peptide span I72-T82 within E50-N127 is essential and sufficient for BGAF binding, whereas the S1-T29 and F466-A512 regions are required for formation of large complexes. To define the contribution of individual amino acids in the above three regions to BGAF binding, we constructed mutant beta-glucosidases based on sequence differences between maize beta-glucosidase and sorghum beta-glucosidase (dhurrinase 2, Dhr2), which does not bind BGAF. Binding was evaluated by gel-shift assay and affinity by frontal affinity chromatography (FAC). In the gel-shift assay, Glu1 mutants K81E and T82Y failed to bind BGAF, and their FAC profiles were essentially similar to that of Dhr2, indicating that these two amino acids within the I72-T82 region are important for BGAF binding. Substitution of N481 with E (as in Dhr2) lowered affinity for BGAF, whereas none of the mutations in the S1-T29 region showed any effect on BGAF binding. To further confirm the importance of K81 and T82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four amino acids (I72, N75, K81, and T82) that differ between Glu1 and Dhr2 in the peptide span I72-T82 are required to impart BGAF-binding ability to Dhr2.

Homolog of the maize beta-glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity.

Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and beta-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum beta-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein-protein interactions.

Maize beta-glucosidase-aggregating factor is a polyspecific jacalin-related chimeric lectin, and its lectin domain is responsible for beta-glucosidase aggregation.

In certain maize genotypes, called "null," beta-glucosidase does not enter gels and therefore cannot be detected on zymograms after electrophoresis. Such genotypes were originally thought to be homozygous for a null allele at the glu1 gene and thus devoid of enzyme. We have shown that a beta-glucosidase-aggregating factor (BGAF) is responsible for the "null" phenotype. BGAF is a chimeric protein consisting of two distinct domains: the disease response or "dirigent" domain and the jacalin-related lectin (JRL) domain. First, it was not known whether the lectin domain in BGAF is functional. Second, it was not known which of the two BGAF domains is involved in beta-glucosidase binding and aggregation. To this end, we purified BGAF to homogeneity from a maize null inbred line called H95. The purified protein gave a single band on SDS-PAGE, and the native protein was a homodimer of 32-kDa monomers. Native and recombinant BGAF (produced in Escherichia coli) agglutinated rabbit erythrocytes, and various carbohydrates and glycoproteins inhibited their hemagglutination activity. Sugars did not have any effect on the binding of BGAF to the beta-glucosidase isozyme 1 (Glu1), and the BGAF-Glu1 complex could still bind lactosyl-agarose, indicating that the sugar-binding site of BGAF is distinct from the beta-glucosidase-binding site. Neither the dirigent nor the JRL domains alone (produced separately in E. coli) produced aggregates of Glu1 based on results from pull-down assays. However, gel shift and competitive binding assays indicated that the JRL domain binds beta-glucosidase without causing it to aggregate. These results with those from deletion mutagenesis and replacement of the JRL domain of a BGAF homolog from sorghum, which does not bind Glu1, with that from maize allowed us to conclude that the JRL domain of BGAF is responsible for its lectin and beta-glucosidase binding and aggregating activities.