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TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61β and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61β, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61β polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.

Highlights ► We analyzed the effect of Ca2+-calmodulin on membrane integration of tail-anchored proteins. ► We found that Ca2+-calmodulin inhibits membrane insertion of TA proteins. ► This inhibition was prevented by trifluoperazine, a CaM antagonist. ► The effect of Ca2+-CaM was also seen for a purified TA protein. Ca2+-CaM can be cross-linked to TA proteins., Cytosolic components and pathways have been identified that are involved in inserting tail-anchored (TA) membrane proteins into the yeast or mammalian endoplasmic reticulum (ER) membrane. Searching for regulatory mechanisms of TA protein biogenesis, we found that Ca2+-calmodulin (CaM) inhibits the insertion of TA proteins into mammalian ER membranes and that this inhibition is prevented by trifluoperazine, a CaM antagonist that interferes with substrate binding of Ca2+-CaM. The effects of Ca2+-CaM on cytochrome b5 and Synaptobrevin 2 suggest a direct interaction between Ca2+-CaM and TA proteins. Thus, CaM appears to regulate TA insertion into the ER membrane in a Ca2+ dependent manner.