Intramembrane client recognition potentiates the chaperone functions of calnexin

Abstract

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.

Characterization and Comparison Analysis of Milk Fat Globule Membrane Proteins between Human and Porcine Milk

“…CANX was upregulated 9.65-fold in HM compared to PM. As one of the most abundant ER chaperones, CANX performs a significant role in the biogenesis of membrane protein by interacting with client proteins through its sugar-binding lectin domain and a large number of nonglycosylated membrane proteins . The maximal clique centrality (MCC) method was used to rank the top 10 hub proteins (Figure B).…”

“…As one of the most abundant ER chaperones, CANX performs a significant role in the biogenesis of membrane protein by interacting with client proteins through its sugar-binding lectin domain and a large number of nonglycosylated membrane proteins. 45 The maximal clique centrality (MCC) method was used to rank the top 10 hub proteins (Figure 6B). Among these, the highest MCC score was peroxisomal acyl-coenzyme A oxidase 1 (ACOX1), followed by peroxisomal bifunctional enzyme (EHHADH), peroxisomal acyl-coenzyme A oxidase 2 (ACOX2), peroxisomal multifunctional enzyme type 2 (HSD17B4), enoyl-CoA delta isomerase 2 (ECI2), peroxisomal acyl-coenzyme A oxidase 3 (ACOX3), mitochondrial delta (3,5)-delta (2,4)dienoyl-CoA isomerase (ECH1), CAT, sterol carrier protein 2 (SCP2), and very long-chain acyl-CoA synthetase (SLC27A2).…”

Calnexin reveals a sugar‐free taste within the lipid bilayer

“…To gain insight into which region of calnexin is necessary and sufficient for this interaction, Bloemeke et al (2022) developed fluorescently tagged constructs containing the TMD of calnexin alone, the ER‐luminal lectin binding domain lacking its TMD, or the tagged fluorescent protein. The two soluble proteins contained a C‐terminal ER retention sequence to prevent their escape from the ER.…”

“…Rhodopsin was degraded more rapidly in cells expressing the calnexin Y/T/L mutant, suggesting that the intramembrane interaction was important for rhodopsin maturation and stability. Based on these results, Bloemeke et al (2022) propose a model in which calnexin interacts with transmembrane protein substrates through: (i) the calnexin TMD and a substrate TMD; (ii) an N‐glycan and the calnexin lectin domain; or (iii) using both modes (Fig 1). These results greatly expand the potential clientele of calnexin by including non‐glycosylated membrane proteins.…”

“…An earlier study identifying an interaction between calnexin and proteolipid protein (Swanton et al , 2003) had led to the proposal of a glycan‐independent role of the calnexin transmembrane domain (TMD) in binding clients within the membrane. The new study by the Feige group now further analyzed this carbohydrate‐independent role of the calnexin transmembrane region in binding and assisting the intramembrane domains of maturing membrane protein clients (Bloemeke et al , 2022).…”