A subset of UPR-induced transmembrane proteins are 5 prematurely degraded during lipid perturbation

58 Background: Phospholipid homeostasis in biological membranes is essential to maintain functions of 59 organelles such as the endoplasmic reticulum. Phospholipid perturbation has been associated to non- 60 alcoholic fatty liver disease, obesity and other metabolic disorders. However, in most cases, the 61 biological significance of lipid disequilibrium remains unclear. Previously, we reported that 62 Saccharomyces cerevisiae adapts to lipid disequilibrium by upregulating several protein quality 63 control pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and 64 the unfolded protein response (UPR). 65 Results: Surprisingly, we observed certain ER-resident transmembrane proteins (TPs), which form 66 part of the UPR programme, to be destabilised under lipid perturbation (LP). Among these, Sbh1 was 67 prematurely degraded by fatty acid remodelling and membrane stiffening of the ER. Moreover, the 68 protein translocon subunit Sbh1 is targeted for degradation through its transmembrane domain in an 69 unconventional Doa10-dependent manner. 70 Conclusion: Premature removal of key ER-resident TPs might be an underlying cause of chronic ER 71 stress in metabolic disorders. Δ these results integration into the membrane by PC depletion. To study the topology of these four proteins, we performed proteinase K (PK) digestion from isolated microsomes 2c). In WT cells, the C-termini HA tags of Cue1-HA, Emc4-HA and Nsg2-HA are oriented towards the cytosol. Thus, the HA tag will be cleaved off if the proper topology is preserved, while the detection of a HA-bearing peptide after PK digestion 185 indicates an inverted topology. The three proteins were found to be fully digested under LP and the predicted smaller protein fragments of 23.7, 8.53, and 5.8 kDa were not detected for Cue1-HA, Emc4- HA, and Nsg2-HA, respectively, in both WT and opi3 Δ . Sbh1-HA is a tail-anchored protein where the C-termini HA tag is found in the ER lumen. The predicted protein fragment of 10.5 kDa after PK digestion was detected in both WT and opi3 Δ strains, indicative of its correct membrane topology. Sbh2 opi3 Sbh1 Doa10 within the lipid-embedded Sbh1 α -helix [54]. further we used a stable Sbh2 mutant wherein the two non-conserved amino acids of the transmembrane domain of Sbh2 have been mutated from serine to proline and alanine at positions 61 306 and 68, respectively [Sbh2(S61P,S68A)]. These two point mutations drive Sbh2 native interaction 307 from the Ssh1 translocon to the Sec61 translocon. As previously reported, Sbh2(S61P,S68A) was stable in WT cells (Fig. 5f). Unexpectedly, Sbh2(S61P,S68A) was similarly stable in opi3 Δ cells, suggesting the Sec61 translocon maintains its ability to interact with the non-essential β subunit. these findings suggest that the Doa10 complex recognises the Sbh1 transmembrane degron LP in ER

PC in the liver, PEMT [9,73]. Concurrently, chronic ER stress and the activation of the UPR are both 3 2 3 associated with NAFLD pathologies [9,74,75]. Despite these connections, little is known on the effect 3 2 4 of phospholipid perturbation on pathways of the ER. Thus, we sought to better understand how the 3 2 5 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/178947 doi: bioRxiv preprint first posted online Aug. 21, 2017; ER fails to reach homeostasis under chronic PC depletion and how the protein quality control 3 2 6 machinery is implicated using our previously reported yeast model system [13]. 3 2 7 3 2 8 The proteostasis network undergoes extensive remodelling upon PC depletion in yeast [13]. Although 3 2 9 a large subset of proteins is increased in these stressed cells, we noticed that key proteins are rapidly 3 3 0 degraded and are indeed sensitive to phospholipid variations. Out of the 66 proteins which displayed 3 3 1 decreased protein abundance despite being genetically upregulated, 40% are transmembrane 3 3 2 proteins (TPs). As 30% of the proteome is predicted to be integral or peripheral membrane proteins 3 3 3 [52], it suggests that TPs are more sensitive to LP compared to other types of proteins. Among the 3 3 4 identified TPs, a large proportion are ER-resident proteins suggesting this organelle is more 3 3 5 vulnerable to the effects of LP, and that this in turn affects TP integrity in the ER. The virtual absence 3 3 6 of sterol at the ER, a key regulator of membrane fluidity, might contribute to its susceptibility to  the ER over whole cell suggests either the ER is more susceptible to LP due to the minimal presence 3 5 0 of ergosterol at the ER [77] or cells respond more aggressively to the ER membrane bilayer disruption 3 5 1 to alleviate ER stress. Accordingly, a rise in membrane lipid packing from elevated saturated fatty 3 5 2 acids will reduce the propensity to form curvatures. All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. proposed to sufficiently drive its Doa10-mediated degradation. Interestingly, none of the Sbh1 3 6 5 cytosolic lysine residues are required for its degradation through the Doa10 complex suggesting Sbh1   (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/178947 doi: bioRxiv preprint first posted online Aug. 21, 2017; response especially under prolonged LP (Fig. 6). ER stress induced from a temporary lipid 3 8 5 perturbation will result in the upregulation of UPR target genes and consequently ER homeostasis.
damage [90]. Thus, targeting phospholipid biosynthesis in combination with artemisinin might be an  Here, we report that a subset of transmembrane proteins, part of the UPR programme, are 4 0 7 prematurely degraded under LP. ER-resident proteins Cue1, Emc4, Nsg2, and Sbh1 topology and 4 0 8 integration into the ER are not affected by LP while they are prematurely degraded. By further 4 0 9 investigating the β subunit of Sec61 ER translocation complex, Sbh1, we proposed that it is 4 1 0 prematurely degraded by the Doa10 complex through the recognition of a specific transmembrane 4 1 1 degron. The proper association of Sbh1 with its interacting partners as well as the maintenance of 14 was previously described [14]. The plasmids pGT0179, pGT0181, pGT0183, and pGT0185, were   IRDye-conjugated secondary antibodies. Immunoreactive species were visualised using the NIR 5 0 3 fluorescence system (Odyssey CLx Imaging System).

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Louis, MO) and incubated at 80°C for 1 h to hydrolyse and esterify FAs into FA methyl esters (FAME).

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FAMEs were extracted three times with 1 ml of hexane and separated on a gas chromatography with

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The temperature was held 3 min at 160°C and increase to 180°C with 1.5°C/min increments and to 5 2 0 220°C with 4°C/min increments. Fluorescence recovery after photobleaching (FRAP) was carried out as previously described [45].

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Typically, early log phase cells expressing Sec63-sGFP were fixed on coverslips in Attofluor cell  All rights reserved. No reuse allowed without permission.
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hybrid screen uses the split ubiquitin two hybrid (N-terminus, N ub and C-terminus, C ub ). Briefly, MYTH [49]. Interactors that activate the reporter system in yeast carrying the negative control bait were 5 6 1 All rights reserved. No reuse allowed without permission.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.