In a groundbreaking study published in Nature, researchers have unveiled the intricate structural mechanisms that regulate N-glycosylation at the secretory translocon, shedding light on a pivotal step of protein maturation within the endoplasmic reticulum (ER). This discovery offers unprecedented insight into how molecular machines delicately coordinate to ensure fidelity during protein synthesis and post-translational modification, and it unravels new dimensions in our understanding of cellular quality control systems.
Central to this research is the elucidation of the spatial and functional dynamics of the ER chaperone GRP94, particularly its pre-N and N-domains, in association with the translocon during translation. The team utilized state-of-the-art cryo-electron microscopy (cryo-EM) techniques to observe that these domains remain tethered to the translocon throughout translation, effectively preventing premature conformational changes or folding events. Notably, the structurally invisible M- and C-domains, which constitute the majority of the mature GRP94 protein, are sterically hindered from adopting their native conformations until full-length synthesis is complete. This spatial constraint averts aberrant intermolecular clashes, such as with CCDC134 and specific subunits of the oligosaccharyltransferase complex OST-A, highlighting a finely tuned folding landscape within the ER lumen.
Moreover, the study reveals a remarkable repositioning of the translocon-associated protein (TRAP) complex compared to earlier translocon models. Unlike previous structures where TRAP’s transmembrane domains interface proximally with the SEC61 complex’s components, the current structure displays a shift translating the TRAP complex away from the tethered GRP94 N-domain. This displacement uncovers a novel steric niche previously occluded by TRAPα’s transmembrane domain. In this vacated space, the researchers were able to identify a four-helix bundle characteristic of KCP2, the DC2-binding subunit of OST-A, an essential player in N-glycosylation. The DC2 subunit extends unusually long N-terminal regions, exceeding 20 Å, to bind KCP2 tightly, anchoring the OST-A complex closely to the translocon and tethered chaperones.
An intriguing feature identified in the lumenal architecture is the substantial mobility of TRAP’s lumenal domains. Their repositioning engenders a considerable lumenal vestibule lined by TRAP, OST-A, CCDC134, and the nascent GRP94 N-domain near the SEC61 channel exit site. This spatial organization creates a secluded microenvironment that facilitates the early folding steps of the GRP94 N-domain while strategically segregating it from OST-B, a paralogous oligosaccharyltransferase complex. This segregation is critical to prevent premature or inappropriate cotranslational glycosylation of the M-domain by OST-B, which acts in trans, thus preserving glycosylation specificity and efficiency.
The researchers also detailed the nuanced interactions within this vestibule, including a newly formed interface between RPN2 and reoriented TRAPδ and TRAPβ lumenal domains, which adjusts the local molecular landscape near the translocon exit tunnel. The repositioning of TRAP not only facilitates this interface but may also play a regulatory role in orchestrating the timing and specificity of glycosylation events. This interplay underscores the intricate structural choreography that governs co- and post-translational modifications fundamental to protein quality control.
Crucially, this study challenges previous assumptions about static translocon organization during protein synthesis. It depicts a dynamic assembly in which structural elements flexibly adapt to synthesis stages and client protein folding states. This flexibility ensures that critical domains, such as those of GRP94 and the multi-subunit OST complexes, are optimally positioned to selectively engage substrates and enzymatic effectors. The controlled exposure of domains at precise time points safeguards against misfolding and misglycosylation, which are hallmarks of numerous pathologies.
From a technical standpoint, the integration of high-resolution cryo-ET and advanced modeling allowed for visualization of these transient and otherwise elusive interfaces in situ. The researchers leveraged various structural references, including full-length GRP94 monomers and previously resolved translocon complexes, to interpret their novel observations. This comparative approach was instrumental in discerning domain displacements and in proposing functional models for the sequential folding and glycosylation process.
One of the most captivating aspects of the findings is the concept of a “lumenal vestibule” acting as a spatial regulator, sequestering partially folded client proteins like GRP94 from premature glycosylation or interference by OST-B. By providing a physical and molecular barrier, this vestibule effectively delineates the functional territories of OST paralogs, ensuring correct modification timing. This spatial separation could be a crucial determinant in the fidelity of protein processing in the secretory pathway.
Additionally, the involvement of accessory factors such as CCDC134 in this microenvironment suggests that co-chaperones and regulatory subunits play indispensable roles in maintaining the structural integrity and function of the translocon complex. These auxiliary components may act as molecular scaffolds or gatekeepers, further refining the quality control processes within the ER lumen.
The implications of these findings extend to a broader understanding of disorders related to ER stress and protein misfolding, such as neurodegenerative diseases, diabetes, and certain cancers. By discerning the molecular mechanisms that govern protein glycosylation fidelity and folding within the secretory pathway, novel therapeutic targets can potentially be identified. Modulating the interactions between GRP94, TRAP, and OST complexes may open avenues for correcting aberrant folding or glycosylation patterns linked to disease states.
Summarily, this landmark study delineates a sophisticated regulatory architecture within the ER translocon, revealing how spatial arrangement and molecular reorganization dynamically govern N-glycosylation during protein biosynthesis. Its insights redefine our understanding of co-translational maturation and underscore the elegant complexity by which cellular machinery sustains proteostasis.
These revelations represent a significant leap forward in molecular cell biology, inviting future exploration into the temporal orchestration of translocon components and their client proteins. As structural biology techniques continue to evolve, more intricate snapshots of these molecular processes are anticipated, further enriching our conceptual frameworks and therapeutic strategies.
Ultimately, the research by Yamsek, Ma, Jha, and colleagues sets a new benchmark for the structural and functional dissection of the secretory pathway, revealing molecular intricacies that underpin the fidelity of protein maturation—a cornerstone of cellular life.
Subject of Research:
Mechanisms regulating N-glycosylation at the secretory translocon, focusing on the structural interactions among GRP94, TRAP complex, OST-A, and associated cofactors within the ER lumen.
Article Title:
Structural basis of regulated N-glycosylation at the secretory translocon.
Article References:
Yamsek, M., Ma, M., Jha, R. et al. Structural basis of regulated N-glycosylation at the secretory translocon. Nature (2025). https://doi.org/10.1038/s41586-025-09756-8
Image Credits:
AI Generated
DOI:
https://doi.org/10.1038/s41586-025-09756-8
Keywords:
N-glycosylation, secretory translocon, GRP94, OST-A, TRAP complex, endoplasmic reticulum, protein folding, cryo-EM, molecular chaperones, proteostasis.
