C-terminal tail length guides insertion and assembly of membrane proteins

Abstract

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ~60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ~80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

Molecular basis for the evolved instability of a human G-protein coupled receptor

“…Our results provide additional evidence that the truncation of the C-terminal tail results in a striking reduction in PME ( Figure 2A ) ( Lin et al, 1998 ). Consistent with recent observations in other helical membrane proteins ( Sun and Mariappan, 2020 ), we show that this truncation compromises the translocon-mediated membrane integration of TM7 ( Figure 2C ). Additionally, we find that the TM domains of mammalian GnRHRs are more polar than their non-mammalian counterparts ( Figures 3 and 4 ), and this compromises the translocon-mediated membrane integration of two other TM domains ( Figure 5 ).…”

“…In contrast, truncation of the C-terminal tail generates three bands that correspond to the untargeted/unglycosylated protein (band VI), a higher weight band bearing the native glycans (band V), and a band bearing an additional glycan resulting from the misintegration of TM7 (band IV) ( Figure 2C ). Consistent with the findings of Sun and Mariappan (2020) , the appearance of an additional high weight band upon truncation of the C-terminal tail suggests this natural modification indirectly compromises the topology of nascent GnRHR. Taken together, our results suggest the evolutionary truncation of the C terminus of mammalian GnRHRs compromises the fidelity of topogenesis in a manner that coincides with a sizable decrease in PME.…”

“…Such variations should alter the propensity of the receptor to misfold during translocon-mediated cotranslational folding (stage I) and/or post-translational folding (stage II) ( Popot and Engelman, 1990 ). Interestingly, Sun and Mariappan (2020) recently found that translocon-mediated membrane integration of C-terminal TM domains, which is the final step of stage I folding, becomes inefficient when the C-terminal loop is shorter than ~60 amino acids. In this case, translation terminates before the final TM domain can reach the translocon.…”

A Molecular Mechanism for Turning Off IRE1α Signaling during Endoplasmic Reticulum Stress

“…HEK293-Flp-In T-Rex cells and derivative cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 mg/mL streptomycin at 5% CO2. IRE1aÀ/À or Sec63À/À HEK293 cells created by CRISPR/Cas9 were previously described (Plumb et al, 2015;Sun and Mariappan, 2020). Sec62À/À and 2xStrep-Sec61a HEK293 cells were described in method details.…”

Membrane Protein Biogenesis: PAT Complex Pats Membrane Proteins into Shape

“…Alternatively, the Sec61 translocon itself may function as a holdase for semi-hydrophilic TMDs to prevent non-productive interactions. Recent evidence suggests that semihydrophilic carboxy-terminal TMDs are post-translationally retained by the Sec61 translocon to facilitate assembly with the TMD from a partner protein [12].…”