TRiC folds the giant ciliary protein IFT172 (~200 kDa) through a non-canonical mechanism in which TRiC and HSP70 simultaneously engage distinct domains (WD40 and TPR, respectively), specific TRiC subunits undergo pronounced conformational expansion to accommodate the oversized substrate, and productive folding of the WD40-1 domain occurs within the open ATP-bound chamber followed by substrate ejection upon ring closure rather than encapsulation—a fold-and-eject pathway that is essential for ciliary function in vivo and conserved across multiple IFT subunits with tandem WD40-TPR architecture.

The eukaryotic chaperonin TRiC/CCT is essential for folding complex proteins, yet how it folds substrates that exceed its closed chamber capacity remains a longstanding paradox. Here, we define the folding pathway of IFT172, the largest subunit (∼200 kDa) of the intraflagellar transport (IFT) machinery, and uncover a “divide-and-conquer” mechanism. TRiC and HSP70 engage IFT172 concurrently but on distinct domains: TRiC captures the N-terminal WD40 β-propellers within its chamber, whereas HSP70 independently stabilizes the C-terminal TPR domain in the cytosol. To accommodate this oversized client, specific TRiC subunits (CCT4, CCT2, and CCT7) undergo pronounced Z-shaped outward bending, thereby expanding the chamber. Unexpectedly, the first WD40 domain reaches a near-folded state within the open, ATP-bound chamber, and subsequent TRiC ring closure triggers substrate ejection rather than encapsulation. This non-canonical “fold-and-eject” mechanism challenges the classical view that the closed chamber is an obligate folding cage. We further demonstrate that this pathway is essential for ciliary functions in vivo, and reveal a conserved mode of chaperonin recognition among IFT components bearing tandem WD40-TPR architectures. Together, our findings establish a new paradigm for the folding of oversized, multi-domain proteins and identify TRiC as a central proteostasis hub in ciliary biogenesis, with direct implications for ciliopathy pathogenesis.