A network of chaperones prevents and detects failures in membrane protein lipid bilayer integration

Abstract

A fundamental step in membrane protein biogenesis is their integration into the lipid bilayer with a defined orientation of each transmembrane segment. Despite this, it remains unclear how cells detect and handle failures in this process. Here we show that single point mutations in the membrane protein connexin 32 (Cx32), which cause Charcot-Marie-Tooth disease, can cause failures in membrane integration. This leads to Cx32 transport defects and rapid degradation. Our data show that multiple chaperones detect and remedy this aberrant behavior: the ER–membrane complex (EMC) aids in membrane integration of low-hydrophobicity transmembrane segments. If they fail to integrate, these are recognized by the ER–lumenal chaperone BiP. Ultimately, the E3 ligase gp78 ubiquitinates Cx32 proteins, targeting them for degradation. Thus, cells use a coordinated system of chaperones for the complex task of membrane protein biogenesis, which can be compromised by single point mutations, causing human disease.

Squaring the EMC – how promoting membrane protein biogenesis impacts cellular functions and organismal homeostasis

“…For some time, the EMC has been implicated in polytopic membrane protein biogenesis (Louie et al, 2012;Richard et al, 2013;Satoh et al, 2015;Shurtleff et al, 2018;Chitwood et al, 2018;Coelho et al, 2019;Volkmar et al, 2019); a role potentially distinct from that of a TA insertase. The loss of apparently unrelated polytopic membrane proteins producing various phenotypes had coincided with different EMC subunit mutations and deletions.…”

“…Such factors could promote folding of complex lumenal or cytoplasmic domains within EMC clients or triage misfolded forms to degradation pathways. Multiple lines of evidence implicate the EMC as a 'quality control hub' for membrane proteins, functioning as a context-dependent binding partner for both general and specialised molecular chaperones (Bagchi et al, 2016;Coelho et al, 2019;Kudze et al, 2018;Richard et al, 2013;Shurtleff et al, 2018; summarised in Fig. 2A).…”

“…The glycoprotein chaperone calnexin (CANX) engages the EMC through EMC1 (Christianson et al, 2012;Coelho et al, 2019;Satoh et al, 2015), while also recognising EMC clients themselves, e.g. rhodopsin-1 and the gap junction beta-1/connexin-32 protein (GJB1, hereafter referred to as Cx32) in humans (Coelho et al, 2019). Because interaction with nascent chains is cotranslational (Chen et al, 1995), CANX and the EMC might be envisaged to cooperate in folding of selected polytopic glycoproteins.…”

“…The ER-resident Hsp70 family A member 5 (HSPA5, hereafter referred to as BiP) also appears to work together with the EMC to deal with improperly inserted clients. Mutation of TMDs in Cx32 is associated with Charcot-Marie-Tooth disease, and leads to binding of these mutants by BiP, followed by ubiquitylation and degradation instead of correct Cx32 membrane insertion aided by the EMC (Coelho et al, 2019). Moreover, faulty TMD integration into the lipid bilayer and lumenal exposure of hydrophobic domains that result in BiP engagement might predict a link to ER stress (discussed below).…”

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

“…Consistent with this, we identify several positive charges in STT3A positioned on the interface of the catalytic subunit and the lipid carbonyl region that may therefore be recognized by RHBDL4. In general, ERAD-M degrons appear to be characterized by metastable TM segments that are subject to several different quality control mechanisms including but not limited to recognition by the ERAD-M E3 ubiquitin ligase Hrd1 (Sato et al, 2009), intramembrane proteolysis by SPP (Yucel et al, 2019), an assembly-dependent membrane integration of nascent TM segments at the Sec61 channel and the ER membrane protein complex (Chitwood et al, 2018;Coelho et al, 2019;Feige and Hendershot, 2013;Tector and Hartl, 1999). Additionally, rhomboid family proteins, with their ability to specifically recognize TM segments, emerge as important safeguards of the membrane proteome (Lemberg, 2013).…”