FKBP8 connects the Hsp70-Hsp90 chaperone machinery to the folding of membrane proteins.

Abstract

The folding of membrane protein cytoplasmic domains on the endoplasmic reticulum (ER) surface, and their coordination with transmembrane and exoplasmic regions, remains poorly understood. Through a genome-wide CRISPR-Cas9 screen, we identified the ER-anchored FK506 binding protein 8 (FKBP8) as a chaperone essential for membrane protein folding and assembly. Using ABC transporters as model substrates, we show that FKBP8 cooperates with Hsp70-Hsp90 machinery to remodel nascent or misfolded cytosolic domains into their native conformations. Cryo-EM analysis reveals that FKBP8 employs a conserved hydrophobic ϕ⁹⁴ϕ⁹⁶ϕ⁹⁷/ϕ¹⁵⁸ϕ¹⁶² cluster to help form a large client-binding cavity within the FKBP8-Hsp90 complex that captures folding intermediates. FKBP8 deficiency, disruption of this cluster, or disease-associated mutations within FKBP8 abolish substrate maturation, leading to ER retention and degradation. Reconstitution with purified components demonstrates that FKBP8 and Hsp40-Hsp70-HOP-Hsp90 constitute a minimal machinery capable of restoring the native structure of a misfolded ABC transporter. These findings uncover a dedicated folding module at the ER-cytosol interface that bridges cytosolic chaperones with membrane protein quality control, suggesting a broad role for FKBP8 in safeguarding the biogenesis of complex membrane proteins.