Thomas Ziegelhoffer

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups.

The translation machinery and 70 kd heat shock protein cooperate in protein synthesis

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome–nascent chain complex

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb–ribosome interaction was characterized. Incorporation of the aminoacyl‐tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross‐linked to nascent chains containing a modified lysine residue with a photoactivatable cross‐linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome–nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobacco.

The economical bioconversion of lignocellulosic biomass to ethanol is dependent on the availability of large quantities of inexpensive cellulase enzymes. One way to reduce the cost of such enzymes is to produce them in crop plants at high levels. In order to assess factors that limit recombinant cellulase expression in plants, we have introduced the gene encoding E1 endo-1,4-β-glucanase (cellulase) of Acidothermus cellulolyticus into tobacco (Nicotiana tabacum) plants. Both the holoenzyme (E1) and catalytic domain (E1cd) were targeted to three sub-cellular compartments; the cytosol, chloroplast and apoplast. Accumulation of both E1 and E1cd was greatest in the apoplast, with levels more than 100-fold higher than observed for cytosolic accumulation. In all three compartments, E1cd accumulated to higher levels than the full-length enzyme. By combining truncation and apoplastic localization, an increase in expression of more than 500-fold was achieved, compared to cytosolic full-length E1. This effect is primarily post-transcriptional, since E1cd mRNA levels are very similar despite the range of E1cd accumulation observed. Recombinant E1cd, expressed at up to 1.6% total soluble protein, is extremely stable in both crude leaf extracts and dried leaf material.

Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.)

The genes encoding thermostable cellulases E2 and E3 of Thermomonospora fusca were expressed in plants under the control of the constitutive, hybrid Mac promoter. For both E2 and E3, the genes were modified so as to remove the sequence encoding the bacterial leader peptide. Western blot analysis indicated that expression levels of recombinant cellulase in tobacco lines ranged up to about 0.1% (E2) and 0.02% of soluble protein (E3). No phenotypic effect of cellulase expression was noted. Recombinant E2 expressed in either tobacco or alfalfa was active and retained heat stability. These findings are an important first step in the development of crop plants as a production system for cellulases.

Expression of Acidothermus cellulolyticus E1 endo-β-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.

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. Apj1s 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.

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.

2 Cytosolic hsp70s of Saccharomyces cerevisiae : Roles in Protein Synthesis, Protein Translocation, Proteolysis, and Regulation

I. INTRODUCTION The 70-kD heat shock proteins, or hsp70s, are highly conserved in all organisms studied so far, from bacteria to yeast to humans. Eukaryotes, including the budding yeast Saccharomyces cerevisiae, encode multiple hsp70s in their genomes. These related proteins are localized to a variety of cellular compartments, including the cytosol, mitochondria, and endoplasmic reticulum (ER). Functionally, the organellar hsp70s are better understood, having major roles in protein translocation and folding. The functions of the cytosolic hsp70s have been more difficult to define, perhaps because these proteins are involved in multiple processes, including translation, protein translocation, protein folding, and regulation of the heat shock response. This chapter reviews evolutionary analyses and genetic data concerning the roles of these proteins in the cytosol. II. EVOLUTION OF THE HSP70 MULTIGENE FAMILY To gain a better understanding of the evolutionary relationships among hsp70s across the biological spectrum, a comparison of 36 hsp70s from 25 diverse genera was conducted (Boorstein et al. 1994). The analysis, carried out by both distance-matrix and character-state methods, showed that the eukaryotic hsp70s comprise four distinct clusters (see Fig. 1). These clusters correspond to the intracellular localization of the proteins: the cytosol, the ER, mitochondria, and chloroplasts. Analysis of the comparisons revealed that eukaryotic hsp70s appear to have evolved from ancestral genes by two types of mechanisms. Mitochondrial and chloroplast hsp70s appear to be derived from the establishment of an endosymbiotic relationship between a eukaryotic host and bacterial cells. Mitochondrial hsp70s are encoded in the nuclei, but they share...

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.