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Single molecule dynamics of the TRiC chaperonin system in vivo

ABSTRACT: The essential chaperonin TRiC/CCT mediates protein folding in cooperation with the co-chaperone prefoldin (PFD). As shown in vitro, the cylindrical TRiC complex facilitates folding through ATP-regulated client protein encapsulation. However, the functional dynamics of the chaperonin system in vivo remain unexplored. Here, we developed single-particle tracking in human cells to monitor the interactions of TRiC/PFD with newly synthesized proteins. Both chaperones engaged nascent polypeptides repeatedly in brief probing events, typically lasting around one second, with PFD recruiting TRiC. As shown with the chaperonin client actin, the co-translational interactions of PFD and TRiC increased in frequency and lifetime during chain elongation. Close to translation termination, PFD bound for several seconds, facilitating TRiC recruitment for post-translational folding involving multiple reaction cycles of ~2.5 s. Notably, the lifetimes of TRiC interactions with a folding-defective actin mutant were markedly prolonged, indicating that client conformational properties modulate TRiC function. Mutant actin continued cycling on TRiC until targeted for degradation. Surprisingly, TRiC often remained confined near its client protein between successive binding cycles, suggesting that the chaperonin machinery operates within a localized ‘protective zone’ where free diffusion is restricted. Together, these findings offer detailed insight into the single-molecule dynamics and supramolecular organization of the chaperonin system in the cellular environment.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Barbara Steigenberger

LAB HEAD: F. Ulrich

PROVIDER: PXD066622 | Pride | 2025-11-26

REPOSITORIES: Pride

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			Action	DRS
	20200818_HF_RL_20679.raw	Raw
	20200818_HF_RL_20680.raw	Raw
	20200818_HF_RL_20681.raw	Raw
	20200818_HF_RL_20685.raw	Raw
	20200818_HF_RL_20686.raw	Raw

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Dynamics of chaperonin function in live human cells

Project description:The eukaryotic chaperonin TRiC/CCT plays an essential role in protein folding1-4. As shown in vitro, the cylindrical TRiC complex captures unfolded client proteins and facilitates their folding through ATP-regulated encapsulation5-8. However, the functional dynamics of the chaperonin system within the cellular environment remain largely unexplored. In this study, we developed single-particle tracking in live human cells to monitor the interactions of TRiC with newly synthesized proteins, as well as its interplay with the co-chaperone PFD. We found that PFD and TRiC engage nascent protein chains in repeated, brief probing events—typically lasting around one second—with PFD acting to recruit TRiC. Using actin as an obligate chaperonin client7, the co-translational interactions of PFD and TRiC increased in both frequency and lifetime during nascent chain elongation. Near the point of translation termination, PFD interactions lasted for ~6 seconds, enabling TRiC recruitment for post-translational completion of folding via multiple reaction cycles of ~2.5 seconds. Notably, the lifetime of TRiC cycles was 3-4 times longer when a folding-defective actin mutant was analyzed, indicating that the conformational state of the client protein modulates TRiC function. Mutant actin continued cycling on TRiC until being targeted for proteasomal degradation. Unexpectedly, TRiC remained confined near its client protein between successive binding cycles, suggesting that folding occurs within a localized ‘protective zone.’ Together, these findings offer the first detailed insights into the dynamic behavior and supramolecular organization of the chaperonin system in living cells. The methodology developed in this study offers a framework for systematically investigating protein folding within the intact cellular environment.

2025-11-26 | GSE304022 | GEO

Folding of beta-tubulin by the chaperonin TRiC/CCT studied by chemical cross-linking and mass spectrometry

Project description:Chemical cross-linking coupled to mass spectrometry was used to study the folding of the client protein, beta-tubulin, by the chaperonin TRiC/CCT. Different complexes containing TRiC/CCT and/or the chaperone prefoldin were cross-linked in absence or presence of nucleotides with the homobifunctional, noncleavable reagent, disuccinimidyl suberate (DSS).

2022-12-08 | PXD030590 | Pride

TRiC/CCT Chaperonin Regulates RNA Polymerase II in the Nucleus [DNase-Seq]

Project description:The molecular chaperonin TRiC/CCT is a large hetero-oligomeric structure that serves an essential role in eukaryotic cells by minimally supporting protein homeostasis including the folding of nascent polypeptides and the assembly/disassembly of protein complexes. TRiC/CCT is typically considered a strict cytosolic machine. Here, we investigated the influence of TRiC/CCT on nuclear features including epigenetic marks, chromatin accessibility, and transcription. Despite being linked to several chromatin modifiers, our work indicates TRiC/CCT does not have a sustained role with these factors. TRiC/CCT did actively contribute to transcription. Inactivation of TRiC/CCT resulted in a significant increase in the production of RNA leading to an accumulation of noncoding transcripts. Our data support a direct role for TRiC/CCT with RNA polymerase II as the chaperonin modulated nascent RNA production both in vivo and in vitro. Overall, our studies reveal a new avenue by which TRiC/CCT contributes to cell homeostasis by regulating the activity of nuclear RNA polymerase II.

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TRiC/CCT Chaperonin Regulates RNA Polymerase II in the Nucleus [4tU-Seq]

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TRiC/CCT Chaperonin Regulates RNA Polymerase II in the Nucleus [RNA-seq]

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Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT

Project description:Folding newly synthesized proteins relies on the ribosome intricately coordinating mRNA translation with a network of ribosome-associated machinery. The principles that drive the coordination of this diverse machinery remain poorly understood. Here, we use selective ribosome profiling to determine how the essential chaperonin TRiC/CCT and the Hsp70 Ssb are recruited to ribosome-nascent chain complexes to mediate cotranslational protein folding. Whereas substrate localization and nascent chain sequence are the major determinants of cotranslational recruitment of Ssb, we found that temporal and structural elements drive TRiC engagement. For both chaperones, however, local slowdowns in translation enhance chaperone enrichment. This work helps define the principles that dictate the coordinated activity of ribosome-associated factors to perform their critical role in maintaining a properly folded nascent proteome.

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1-deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding

Project description:Ischemic heart disease and stroke are the most common causes of death world-wide. In both pathologies anoxia, the lack of oxygen, triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that injury is caused by the accumulation of the non-canonical sphingolipid, 1-deoxydihydroceramide (DoxDHCer). We found that anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-deoxysphinganine is incorporated into DoxDHCer which impairs actin folding via the cytosolic chaperonin TRiC/CCT, leading to growth arrest in yeast and increased cell death upon ischemia-reperfusion injury in worms and mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

2019-08-20 | PXD014573 | Pride

The TRiC Chaperonin and Prefoldin Cooperate to Fold and Assemble Viral Capsid Oligomers

Project description:Chemical cross-linking coupled to mass spectrometry was used to study the folding of the reovirus sigma3 protein by the chaperonin, TRiC/CCT. Cross-linking was performed using the homobifunctional, noncleavable reagent, disuccinimidyl suberate (DSS).

2021-02-04 | PXD019751 | Pride

Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan

Project description:Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington’s disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.

2017-03-23 | PXD005123 | Pride

The chaperonin GroESL supports the functions of the Z-ring regulators FtsA and FzlA

Project description:The highly conserved chaperonin GroESL performs a crucial role in protein folding, however the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events such as DNA replication and chromosome segregation are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.

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