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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.
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PMID:A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin. 791 Aug 27

Plastid chaperonin-60 protein was purified to apparent homogeneity from Brassica napus using a novel protocol. The purified protein, which migrated as a single species by nondenaturing polyacrylamide gel electrophoresis, contained four polypeptides: three variants of p60cpn60 alpha and p60cpn60 beta. Partial amino acid sequence determination demonstrated that each variant of p60cpn60 alpha is a distinct translation product. During this study, additional chaperonin-60 proteins were purified. These proteins, which were free from contaminating plastid chaperonin-60, were separated into at least two high molecular weight species that were resolved only by nondenaturing polyacrylamide gel electrophoresis. These proteins contained three 60-kD polypeptides. Two of these polypeptides were recognized by existing antisera, whereas the third was not. Partial amino acid sequence data revealed that each of these, including the immunologically distinct polypeptide, is a chaperonin-60 subunit of putative mitochondrial origin. The behavior of chaperonin-60 proteins during blue A Dyematrex chromatography suggests that this matrix may be generally useful for the identification of chaperonin-60 proteins.
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PMID:Brassica napus plastid and mitochondrial chaperonin-60 proteins contain multiple distinct polypeptides. 791 38

Although chaperonin-assisted protein folding has been studied in vitro by a number of investigators, the feature(s) of the unfolded polypeptide that are recognized and bound by chaperonins is not known. We have addressed this question using the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) as a substrate for GroEL. The protein was expressed in Escherichia coli as a C-terminal fusion to protein A. Protein A-pS and any associated cellular proteins were then purified by affinity chromatography. GroEL could be eluted from the fusion protein by incubation with ATP and either GroES or casein, consistent with results of in vitro folding assays. At least half of the transit sequence of pS is required to maintain this high stoichiometry of chaperonin binding. Using deletion mutagenesis from the C terminus of pS, we defined the smallest truncation of pS, PAxpS90T, that binds GroEL with high avidity (dissociation constant = 53 nM). A series of site-specific mutations targeting the C-terminal 15-20 amino acids of PAxpS90T was constructed and analyzed for the ability to bind GroEL. Our results show that complex formation between GroEL and pS is not dependent upon any single feature such as overall hydrophobicity, a net positive charge, or secondary structure but may be dependent upon a combination of these features.
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PMID:Identification of a chaperonin binding site in a chloroplast precursor protein. 791 91

The chaperonin GroEL is a ribosome-sized double-ring structure that assists in folding a diverse set of polypeptides. We have examined the fate of a polypeptide during a chaperonin-mediated folding reaction. Strikingly, we find that, upon addition of ATP and the cochaperonin GroES, polypeptide is released rapidly from GroEL in a predominantly nonnative conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound rapidly by other GroEL molecules. Folding appears to occur in an all-or-none manner, as proteolysis and tryptophan fluorescence indicate that after rebinding, polypeptide has the same structure as in the original complex. These observations suggest that GroEL functions by carrying out multiple rounds of binding aggregation-prone or kinetically trapped intermediates, maintaining them in an unfolded state, and releasing them to attempt to fold in solution.
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PMID:GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms. 791 1

Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.
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PMID:Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. 791 27

Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.
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PMID:Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone. 791 32

Chaperonins are ring-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a variety of compartments. Recent studies suggest that they function through multiple rounds of binding and release of non-native proteins: with each round of ATP-driven release into the bulk solution, a substrate protein kinetically partitions between folding to the native state or rebinding to another chaperonin molecule. To gain further insight into the mechanism of polypeptide binding and release by the chaperonin GroEL from Escherichia coli, we have undertaken a mutational analysis that relates the functional properties of GroEL to its crystal structure. Our functional tests identify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydrophobic residues. These same residues are essential for binding of the co-chaperonin GroES, which is required for productive polypeptide release. A highly conserved residue, Asp 87, positioned within a putative nucleotide-binding pocket in the top of the equatorial domain, is essential for ATP hydrolysis and polypeptide release.
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PMID:Residues in chaperonin GroEL required for polypeptide binding and release. 793 86

Rhodanese is a small (33 kDa) monomeric sulfurtransferase which is synthesized on cytoplasmic ribosomes and imported into the mitochondrial matrix without cleavage of its amino terminus. When we transfected mammalian cell lines with rhodanese cDNA under the control of an efficient viral promoter, up to 40% of the overexpressed protein was secreted into the medium. This secretion was not the result of cell lysis, did not occur via the endoplasmic reticulum, and did not require the amino-terminal mitochondrial import signal. Addition of a carboxyl-terminal peptide extension did not block secretion, nor did a number of inhibitors of cellular sorting processes. Rhodanese polypeptide is known to associate with chaperonin proteins. In the absence of available mitochondrial import sites, such a complex in the cytoplasm of transfected cells could deliver unfolded rhodanese to export pores on the inner surface of the plasma membrane. This mechanism could contribute to the nonclassical secretion of cytoplasmically synthesized interleukins, growth factors, and lectins.
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PMID:Rapid secretion by a nonclassical pathway of overexpressed mammalian mitochondrial rhodanese. 796 79

The protein-protein interactions during GroE-mediated protein refolding are of crucial importance for understanding how the assisted refolding of non-native proteins is achieved. Since GroEL seems to be a rather promiscuous polypeptide-binding protein it is not surprising that conditions for efficient dissociation from GroEL are promiscuous as well. To understand assisted protein refolding it is necessary to elucidate the underlying principles of the different partial steps of the functional cycle. Here we show a correlation between the overall stability of the complex between GroEL and ligand protein and the conditions for functional release from the chaperonin. As a model system, differently denatured species of an antibody Fab fragment were used. While weakly bound Fab fragments are functionally released in the absence of GroES, stably associated non-native forms of the same protein are dependent on the presence of the co-chaperonin for optimal GroE-mediated reactivation, suggesting that complex stability determines the release requirement. However, the observed overall stability of the complex between GroEL and substrate protein may be regarded as the net product of constant binding and rebinding of the ligand protein, once associated with GroEL, as shown by competition experiments.
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PMID:Correlation between the stability of the GroEL-protein ligand complex and the release mechanism. 796 29

Coagulation factors V and VIII are homologous glycosylated plasma proteins that provide essential functions for hemostasis. Factor V is secreted as a single chain polypeptide, whereas factor VIII is processed intracellularly to yield a metal-ion-associated heterodimer that is stabilized through interaction with von Willebrand factor. In transfected mammalian cells, factor V is more efficiently secreted than factor VIII. To provide insight into the different secretion efficiencies, we compared the post-translational processing requirements for factor V and factor VIII expressed in mammalian cells. In contrast to factor VIII, factor V was not detected in association with the immunoglobulin-binding protein (BiP), a chaperonin protein of the endoplasmic reticulum (ER). Depletion of intracellular ATP levels by treatment of cells with low concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), protonophore that uncouples oxidative phosphorylation, inhibited secretion of factor VIII but had no effect on the secretion of factor V. Inhibition of N-linked oligosaccharide addition by treatment with tunicamycin prevented secretion of both factor V and factor VIII, whereas treatment with an inhibitor of complex oligosaccharide addition, deoxymannojirimycin, did not affect secretion, although the specific activities of both factor V and factor VIII were slightly increased. Thus, complex oligosaccharide addition was not required for secretion or functional activity of either factor V or factor VIII. Depletion of intralumenal calcium with the ionophore A23187 did not affect secretion of either factor V or factor VIII. In the presence of A23187, the secreted factor V was fully functional, whereas the factor VIII heavy and light chains were not associated and the secreted molecule was inactive. In addition, A23187 treatment inhibited addition of serine/threonine (O)-linked oligosaccharides to factor V and factor VIII. The differences between factor V and factor VIII were further evaluated by characterization of a single chain mutant factor VIII. The single chain factor VIII was secreted with an efficiency similar to wild-type factor VIII and also required von Willebrand factor for stabilization. In addition, the activity of single chain factor VIII was also inhibited by A23187 treatment, suggesting a unique metal-ion requirement within the secretory pathway for functional factor VIII folding. The differences identified in BiP association, ATP requirements, and metal-ion dependence for effective functional secretion of these two molecules may underlie mechanisms accounting for their different secretion efficiencies.
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PMID:Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells. 800 42


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