Cer1p functions as a molecular chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae.

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

The unique chaperone operon of Thermotoga maritima: cloning and initial characterization of a functional Hsp70 and small heat shock protein.

The hyperthermophilic eubacterium Thermotoga maritima possesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of T. maritima, one of the deepest-branching eubacteria known.

High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions.

Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution.

NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction.

The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments. The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins. The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues. A second hydrophobic binding site was identified using paramagnetically labeled peptides. It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.

Chromophore formation in green fluorescent protein.

The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria forms an intrinsic chromophore through cyclization and oxidation of an internal tripeptide motif [Prasher, D. C., et al. (1992) Gene 111, 229-233; Cody, C. E., et al. (1993) Biochemistry 32, 1212-1218]. We monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inclusion bodies in Escherichia coli lacks the mature chromophore, suggesting that protein destined for inclusion bodies aggregated prior to productive folding. This material was used to follow the steps leading to chromophore formation. The process of chromophore formation in S65T-GFP was determined to be an ordered reaction consisting of three distinct kinetic steps. Protein folding occurs fairly slowly (k(f) = 2.44 x 10(-3) s(-1)) and prior to any chromophore modification. Next, an intermediate step occurs that includes, but is not necessarily limited to, cyclization of the tripeptide chromophore motif (k(c) = 3.8 x 10(-3) s(-1)). The final and slow step (k(ox) = 1.51 x 10(-4) s(-1)) in chromophore formation involves oxidation of the cyclized chromophore. Since the chromophore forms de novo from purified denatured protein and is a first-order process, we conclude that GFP chromophore formation is an autocatalytic process.

Cer1p, a novel Hsp70-related protein required for posttranslational endoplasmic reticulum translocation in yeast.

Proteins enter the secretory pathway by translocation across the endoplasmic reticulum (ER) membrane. In Saccharomyces cerevisiae, import of proteins into the ER occurs both cotranslationally and posttranslationally. Presumably, the cotranslational targeting to the ER membrane is directed by the signal recognition particle, as demonstrated in other eukaryotic systems. The deletion of a gene, called CER1, inhibits the translocation of proteins that enter the ER posttranslationally, but not those that enter cotranslationally. This translocation defect is more pronounced at lower temperatures. A strain possessing a null mutation of CER1 in combination with a kar2 temperature-sensitive mutation displays synthetic growth defects, whereas overexpression of the ER DnaJ homolog Scj1p suppresses the translocation defect in cer1Delta strains. CER1 is predicted to encode a 100-kDa polypeptide, residing in the ER lumen that is related to the hsp70 family of molecular chaperones.

GroEL binds to and unfolds rhodanese posttranslationally.

The Escherichia coli chaperone GroEL is a member of a class of molecular chaperones that possesses a stacked double ring structure containing seven subunits per ring, with approximately 60-kDa subunits. It has been suggested that newly synthesized proteins may interact with a eukaryotic homolog of GroEL co-translationally, thereby sequestering the unfolded protein from other proteins in the cell. To test whether it is essential for GroEL to form a stable interaction with a nascent polypeptide co-translationally, we translated the well studied GroEL substrate rhodanese in bacterial and wheat germ translation extracts. We found that rhodanese formed stable complexes with GroEL solely posttranslationally. Upon binding to GroEL, the protease resistant N-terminal domain of rhodanese unfolds. This interaction with GroEL leads to productive folding of the full-length rhodanese. We conclude that GroEL is able to assist in the folding of newly synthesized proteins following release from the ribosome and that GroEL can unfold a trapped protein folding intermediate of rhodanese.

The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology.

Modern NMR methods were used to determine the secondary structure topology of the 18 kDa peptide binding domain of the chaperone protein Hsc70 in solution. This report constitutes the first experimental conformational information on this important domain of the class of Hsp70 proteins. The domain consists of two four-stranded antiparallel beta-sheets and a single alpha-helix. The topology does not resemble at all the topology observed in the human leukocyte antigen (HLA) proteins of the major histocompatibility complex. This is significant because such resemblance was predicted on the basis of limited amino acid homology, secondary structure prediction, and related function. Moreover, the exact meander-type beta-sheet topology identified in Hsc70 has to our best knowledge not been observed in any other known protein structure.

Stch encodes the 'ATPase core' of a microsomal stress 70 protein.

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.

Individual subunits of bacterial luciferase are molten globules and interact with molecular chaperones.

We have studied the assembly of a large heterodimeric protein, bacterial luciferase, by mixing purified subunits expressed separately in bacteria. The individual subunits alpha and beta contain much (66% and 50%, respectively) of the alpha-helix content of the native heterodimer as measured by circular dichroism, yet the alpha subunit lacks observable tertiary structure as measured by NMR. These results are consistent with the alpha subunit existing in a molten globule or collapsed form prior to assembly. The molecular chaperone GroEL binds reversibly to both subunits prior to assembly. Since these observations were obtained under physiological conditions, we propose that the molten globule exists as a stable form during folding or assembly in the cell. Either the molten globule form of the subunits is an authentic folding intermediate or it is in rapid equilibrium with one. GroEL may function by facilitating assembly through stabilization of these incompletely folded subunits.

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.

Reduction in microtubule dynamics in vitro by brain microtubule-associated proteins and by a microtubule-associated protein-2 second repeated sequence analogue.

Microtubule-associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP-depleted MTs occurs on a time scale characteristic of that observed with MAP-free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end-to-end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT-binding region of MAP-2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP-2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT-binding region.

Positive cooperativity in the functioning of molecular chaperone GroEL.

In the presence of its partner, GroES, the tetradecameric molecular chaperone GroEL binds 14 ATP molecules, half of which are hydrolyzed in a cooperative manner. Moreover GroEL can bind, with a positive cooperativity, more than two molecules of nonfolded protein rhodanese. The role of the cooperative mechanism in the functioning of GroEL is discussed.

Peptide-binding specificity of the molecular chaperone BiP.

Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins. This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site. The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length. The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.

Peptide binding and release by proteins implicated as catalysts of protein assembly.

Two members of the hsp70 family, termed hsc70 and BiP, have been implicated in promoting protein folding and assembly processes in the cytoplasm and the lumen of the endoplasmic reticulum, respectively. Short hydrophilic (8 to 25 residues) synthetic peptides have now been tested as possible mimics of polypeptide chain substrates to help define an enzymatic basis for these activities. Both BiP and hsc70 have specific peptide binding sites. Peptide binding elicits hydrolysis of adenosine triphosphate, with the subsequent release of bound peptide.

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast.

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.