Intramembrane client recognition potentiates the chaperone functions of calnexin.

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome. Synopsis: Calnexin is a membrane‐integral chaperone that acts on glycosylated proteins. This study shows that its transmembrane domain is also involved in substrate binding, defines how intramembrane substrate recognition occurs, and reveals that this additional binding mode supports chaperoning. The mass‐spectrometric interactome of calnexin contains many non‐glycosylated membrane proteins.Calnexin can bind membrane protein substrates in the lipid bilayer via its own transmembrane domain.A systematic analysis reveals how calnexin recognizes particular features within its clients in the membrane.Intra‐membrane client recognition synergizes with sugar‐based recognition of calnexin clients to protect labile chaperone substrates. [ABSTRACT FROM AUTHOR]