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It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6a activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6a abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6. [ABSTRACT FROM AUTHOR]

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b5: the endoplasmic reticulum-resident wild-type (b5wt) and a mutant thereof (b5ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyloleyl-phosphatidyicholine (POPC) or POPC mixed with either distearoyl-phosphatidylseri ne (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16- ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4°C. When present the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0°C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23°C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b5wt increased, whereas b5ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting. [ABSTRACT FROM AUTHOR]