Conformational rearrangements of tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC)-bound actin

The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC-actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.     

Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1 (CCT) subunits in maize seedlings, and the localization in coleoptiles

The cytosolic chaperonin containing TCP-1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the α- and ɛ- subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT-subunits, tubulins and actin decreased in the coleoptile in response to far-red light. In addition, independent from light treatment, the amount of CCTɛ abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCTɛ, no significant light regulation of CCTα was found in the mesocotyl. In two day old, light-grown rapidly elongating coleoptiles part of the CCTα subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT-subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far-red irradiated growth-arrested coleoptiles. CCTɛ was found to be co-localized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT-subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.     

Domain-specific chaperone-induced expansion is required for beta-actin folding: a comparison of beta-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)

Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native beta-actin, but is unable to guide beta-actin toward the native state. In this study we identify conformational rearrangements in beta-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of beta-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled beta-actin was characterized, and the structural rearrangement was compared to the case of the beta-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarstr?m, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the beta-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound beta-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the beta-actin molecule. It can be concluded that the specific and directed rearrangement of the beta-actin structure, seen in the natural beta-actin-TRiC system, is vital for guiding beta-actin to the native state.     

Group II chaperonins: new TRiC(k)s and turns of a protein folding machine.

In the past decade, the eubacterial group I chaperonin GroEL became the paradigm of a protein folding machine. More recently, electron microscopy and X-ray crystallography offered insights into the structure of the thermosome, the archetype of the group II chaperonins which also comprise the chaperonin from the eukaryotic cytosol TRiC. Some structural differences from GroEL were revealed, namely the existence of a built-in lid provided by the helical protrusions of the apical domains instead of a GroES-like co-chaperonin. These structural studies provide a framework for understanding the differences in the mode of action between the group II and the group I chaperonins. In vitro analyses of the folding of non-native substrates coupled to ATP binding and hydrolysis are progressing towards establishing a functional cycle for group II chaperonins. A protein complex called GimC/prefoldin has recently been found to cooperate with TRiC in vivo, and its characterization is under way.     

Piccolo, a presynaptic zinc finger protein structurally related to bassoon

Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.     

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.     

Characterization and reconstitution of Drosophila gamma-tubulin ring complex subunits

The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex (gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly and function, we first reconstituted gammaTuSC using the baculovirus expression system. Using the reconstituted gammaTuSC, we showed for the first time that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Next, we characterized two new gammaTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly.     

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.     

Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit complex (Gbetagamma) that has been recently shown to catalyze the formation of the Gbetagamma dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbetagamma dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbetagamma-assembly process. The results reported here delineate a mechanism for Gbetagamma assembly in which a stable ternary complex is formed between PhLP, the nascent Gbeta subunit, and CCT that does not include Ggamma. PhLP phosphorylation permits the release of a PhLP x Gbeta intermediate from CCT, allowing Ggamma to associate with Gbeta in this intermediate complex. Subsequent interaction of Gbetagamma with membranes releases PhLP for another round of assembly.     

O-GlcNAc transferase is in a functional complex with protein phosphatase 1 catalytic subunits

A hallmark of signal transduction is the dynamic and inducible post-translational modification of proteins. In addition to the well characterized phosphorylation of proteins, other modifications have been shown to be regulatory, including O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modifies serine and threonine residues on a myriad of nuclear and cytosolic proteins, and for several proteins there appears to be a reciprocal relationship between phosphorylation and O-GlcNAc modification. Here we report further evidence of this yin-yang relationship by demonstrating that O-GlcNAc transferase, the enzyme that adds O-GlcNAc to proteins, exists in stable and active complexes with the serine/threonine phosphatases PP1beta and PP1gamma, enzymes that remove phosphate from proteins. The existence of this complex highlights the importance of understanding the dynamic relationship between O-GlcNAc and phosphate in modulating protein function in many cellular processes and disease states such as Alzheimer's disease and type II diabetes.     

Hyperphosphorylation of N-60, a protein structurally and immunologically related to nucleolin after tumour-promoter treatment.

Okadaic acid, a non-TPA-type tumour promoter, induces hyperphosphorylation of a 60-kd protein in primary human fibroblasts. Treatment with TPA-type tumour promoters (e.g. TPA and teleocidin) did not cause this hyperphosphorylation. Phosphorylation of this protein was not seen at times earlier than 90 min after the addition of 75 ng/ml okadaic acid to the proliferating cell cultures. The presence of inhibitors such as actinomycin D and cycloheximide, did not significantly influence the level of hyperphosphorylation induced by okadaic acid treatment. By immunoblotting using an antibody anti-nucleolin, the 60-kd protein was identified as a fragment of nucleolar protein, nucleolin. Similarly, antibodies against the 60-kd protein cross-reacted with nucleolin. Furthermore peptide mapping, using staphylococcal V8 protease, showed that the 60-kd protein phosphorylated by casein kinase II in vitro and the okadaic-acid-induced hyperphosphorylated 60-kd protein exhibited identical phosphopeptide maps, indicating that there is also structural relatedness between N-60 and nucleolin. Hyperphosphorylation of the nucleolin fragment (N-60) was suppressed by anti-tumour promoter retinoic acid.     

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1–8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.     

Role of the chaperonin CCT/TRiC complex in G protein betagamma-dimer assembly

Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction.     

Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.     

Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.     

Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1

The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.     

PFA, a novel mollusk agglutinin, is structurally related to the ribosome-inactivating protein superfamily

The structural organization of PFA, a novel beta-galactose-specific agglutinin from the snail Pomacea flagellata, was partially characterized. Using mass spectrometry, the molecular weight of this glycoprotein was determined as 32,444 Da (7.4% carbohydrate). The agglutinin was found to form very large aggregates in solution, which were dissociated to monodisperse native-like dimers upon addition of polyethyleneglycol. The identity of the first 38 and the last 11 residues of the polypeptide chain was determined. It was found that PFA and the N-glycosidase subunit of ricin, a heterodimeric cytotoxin isolated from castor bean seeds, are homologous to each other in the N-terminal region. Furthermore, the far-UV circular dichroism spectra of these proteins were found to be nearly superimposable, evidencing that they share common general features in their secondary and tertiary structures. On the basis of these similarities, it can be concluded that PFA is structurally related to the ribosome-inactivating protein superfamily.