Type II chaperonins, prefoldin, and the tubulin-specific chaperones.
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Animals↗
Biomedical subjects
N J Cowan
Publications and source records attributed to N J Cowan.
ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin.
The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the alpha/beta- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a beta-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.
Fungal Proteins↗
Tubulin folding cofactors as GTPase-activating proteins. GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer.
In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized alpha- and beta-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. alpha- and beta-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by beta-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein beta-tubulin to hydrolyze its GTP.
Binding Sites↗
Prefoldin-nascent chain complexes in the folding of cytoskeletal proteins.
In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first approximately 145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that alpha- and beta-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly.
Actins↗
A chaperone with a hydrophilic surface.
The folding of native tubulin involves at least seven different chaperone proteins: prefoldin, the cytosolic chaperonin CCT and five tubulin-specific chaperone proteins named cofactors A-E. The structure of the yeast homolog of cofactor A, Rbl2p, shows it to be a dimer with largely hydrophilic surfaces, reflecting the fact that it interacts with quasi-native, not unfolded, beta-tubulin.
Animals↗
A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo.
Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.
Chaperonin 10↗
Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin.
We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.
Actins↗
Mammalian cytosolic chaperonin.
Cytosolic chaperonin, the eukaryotic cytosolic homolog of GroEL, has certain unusual features that make it uniquely useful for studying the mechanism of chaperonin action. It is of particular interest as an essential component in the generation of native actin and tubulin in vivo. We describe a method for the purification of mammalian c-cpn from rabbit reticulocyte lysate via a three-step procedure involving ion-exchange chromatography, affinity selection on ATP-agarose, and gel filtration. We also describe a sensitive in vitro-folding assay for the activity of c-cpn and other chaperone proteins, and a simple nondenaturing gel assay for the analysis of folding reaction products.
Actins↗
A single ring is sufficient for productive chaperonin-mediated folding in vivo.
Facilitated protein folding by the double toroidal bacterial chaperonin, GroEL/GroES, proceeds by a "two-stroke engine" mechanism in which an allosteric interaction between the two rings synchronizes the reaction cycle by controlling the binding and release of cochaperonin. Using chimeric chaperonin molecules assembled by fusing equatorial and apical domains derived from GroEL and its mammalian mitochondrial homolog, Hsp60, we show that productive folding by Hsp60 and its cognate cochaperonin, Hsp10, proceeds in vitro and in vivo without the formation of a two-ring structure. This simpler "one-stroke" engine works because Hsp60 has a different mechanism for the release of its cochaperonin cap and bound target protein.
Adenosine Triphosphatases↗
Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors.
The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.
Amino Acid Sequence↗
Pathway leading to correctly folded beta-tubulin.
We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function.
Animals↗
GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination.
To investigate the structural role of glial fibrillary acidic protein (GFAP) in vivo, mice carrying a null mutation in GFAP were generated. In 7/14 mutant animals older than 18 months of age, hydrocephalus associated with white matter loss was detected. Mutant mice displayed abnormal myelination including the presence of actively myelinating oligodendrocytes in adults, nonmyelinated axons in optic nerve, and reduced myelin thickness in spinal cord. White matter was poorly vascularized and the blood-brain barrier was structurally and functionally impaired. Astrocytic structure and function were abnormal, consisting of shortened astrocytic cell processes, decreased septation of white matter, and increased CNS extracellular space. Thus, GFAP expression is essential for normal white matter architecture and blood-brain barrier integrity, and its absence leads to late-onset CNS dysmyelination.
Aging↗
Chaperonin-mediated folding of actin and tubulin.
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Actins↗
Quasi-native chaperonin-bound intermediates in facilitated protein folding.
Chaperonins are known to facilitate protein folding, but their mechanism of action is not well understood. The fact that target proteins are released from and rebind to different chaperonin molecules ("cycling") during a folding reaction suggests that chaperonins function by unfolding aberrantly folded molecules, allowing them multiple opportunities to reach the native state in bulk solution. Here we show that the cycling of alpha-tubulin by cytosolic chaperonin (c-cpn) can be uncoupled from the action of cofactors required to complete the folding reaction. This results in the accumulation of folding intermediates which are chaperonin-bound, stable, and quasi-native in that they bind GTP nonexchangeably. We present evidence that these intermediates can be generated without the target protein leaving c-cpn. These data show that, in contrast to prevailing models, target proteins can maintain, and possibly acquire, significant native-like structure while chaperonin-bound.
Animals↗
Specificity in chaperonin-mediated protein folding.
Chaperonins are ubiquitous multisubunit toroidal complexes that aid protein folding in an ATP-dependent manner. Current models of folding by the bacterial chaperonin GroEL depict its role as unfolding and releasing molecules that have misfolded, so that they can return to a potentially productive folding pathway in solution. Accordingly, a given target polypeptide might require several cycles of binding and ATP-driven release from different chaperonin complexes before reaching the native state. Surprisingly, cycling of a target protein does not guarantee its folding, and we report here that unfolded beta-actin or alpha-tubulin both form tight complexes when presented to either GroEL or its mitochondrial homologue, and both undergo cycles of release and rebinding upon incubation with ATP, but no native protein is produced. We conclude that different chaperonins produce distinctive spectra of folding intermediates.
Actins↗
Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells.
There is indirect, contradictory evidence both for increased and reduced synthesis of the endothelium-derived vasodilator, nitric oxide, in the pulmonary circulation during acute hypoxia. Therefore, we decided to directly measure the effect of acute hypoxia on nitric oxide production by cultured pulmonary endothelium. Because increases in the intracellular free calcium concentration are known to initiate nitric oxide synthesis, we also studied cytosolic calcium levels. We measured the accumulation of the stable nitric oxide metabolite, nitrite, in the fluid used to superfuse the cultured bovine pulmonary artery endothelial cells at an oxygen tension (PO2) of either 20.3 (normoxia) or 4.9 kPa (hypoxia) (152 or 37 mmHg). Intracellular calcium levels were measured with dual-excitation microfluorimetry after loading the cells with the fluorescent calcium indicator, fura 2. Basal NO synthesis, measured as nitrite accumulation over 10 min, was significantly higher under hypoxic than normoxic conditions (8.3 +/- 2.2 versus 4.6 +/- 0.8 nM). Hypoxia transiently increased cytosolic calcium concentration (from 113 +/- 10 to 231 +/- 45 nM). Ryanodine and thapsigargin (which deplete intracellular calcium stores), but not the removal of extracellular calcium, inhibited the hypoxic increase in cytosolic calcium, indicating that it resulted primarily from release of intracellular calcium. Bradykinin-elicited NO synthesis was potentiated by hypoxia. Bradykinin-induced increase in cytosolic calcium was not inhibited by hypoxia. We conclude that hypoxia acutely increases cytosolic calcium levels and basal and bradykinin-stimulated nitric oxide synthesis in pulmonary artery endothelium.
Animals↗
A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.
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.
Adenosine Triphosphatases↗