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Background:The substrates and regulation of DHHC protein palmitoyl acyltransferases (PATs) are largely unknown. Results: Flotillin-2 palmitoylation is abolished in DHHC5 gene-targeted neural stem cells, and neuronal differentiation induces DHHC5 turnover. Conclusion: Flotillin-2 is a substrate for DHHC5, which is regulated at the protein level. Significance: The paper describes an approach to PAT substrate identification and a new PAT regulation mechanism.

Cytosolic HSP90 requires multiple cochaperones in folding client proteins. However, the function of gp96 (HSP90b1, grp94), an HSP90 paralogue in the endoplasmic reticulum (ER), is believed to be independent of cochaperones. Here, we demonstrate that gp96 chaperones multiple Toll-like receptors (TLRs), but not TLR3, in a manner that is dependent on another ER luminal protein, CNPY3. gp96 directly interacts with CNPY3, and the complex dissociates in the presence of adenosine triphosphate (ATP). Genetic disruption of gp96–CNPY3 interaction completely abolishes their TLR chaperone function. Moreover, we demonstrate that TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other. We suggest that CNPY3 interacts with the ATP-sensitive conformation of gp96 to promote substrate loading. Our study has thus established CNPY3 as a TLR-specific cochaperone for gp96.