Intra-membrane client recognition potentiates the chaperone functions of Calnexin
One third of the human proteome are membrane proteins. They are particularly vulnerable to misfolding, often requiring assistance by molecular chaperones. Calnexin (CNX), one of the most abundant ER chaperones, plays an important role in membrane protein biogenesis and engages clients via its sugar-binding lectin domain. Using mass spectrometric analyses, we show that Calnexin (CNX) interacts with a large number of non-glycosylated membrane proteins, suggesting additional binding modes. We find that misfolded membrane proteins are preferentially bound by CNX and that CNX uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by CNX and identify sequence motifs within the CNX TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of CNX. Together, this study reveals a widespread role of CNX 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.