Regulation of lipid saturation without sensing membrane fluidity

Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here, we have reconstituted the core machinery for sensing and regulating lipid saturation in baker’s yeast to directly characterize its response to defined membrane environments. Using spectroscopic techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains and provide unprecedented insight into the molecular rules of membrane adaptivity. Our data challenge the prevailing hypothesis that membrane viscosity serves as the measured variable for regulating lipid saturation. Rather, we show that the signaling output of Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which senses the lateral pressure and/or compressibility profile in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such membrane properties in the future.

reconstituted the core machinery for sensing and regulating lipid saturation in baker's yeast 23 to directly characterize its response to defined membrane environments. Using spectroscopic 24 techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional 25 regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl 26 chains and provide unprecedented insight into the molecular rules of membrane adaptivity. 27 Our data challenge the prevailing hypothesis that membrane viscosity serves as the 28 measured variable for regulating lipid saturation. Rather, we show that the signaling output of 29 Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which 30 senses the lateral pressure and/or compressibility profile in a defined region of the 31 membrane. Our findings suggest that membrane property sensors have evolved remarkable 32 sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the 33 way toward the development of genetically encoded reporters for such membrane properties 34 in the future. Introduction reconstituting this sense-and-response construct in liposomes with defined lipid 125 compositions, we demonstrate a remarkable sensitivity of Mga2 to specific changes in the 126 bilayer composition. We provide compelling evidence for functional coupling between the 127 TMH and the site of ubiquitylation using electron paramagnetic resonance (EPR) and 128 Förster-resonance energy transfer (FRET). Strikingly, our data rule out the hypothesis that 129 Mga2 acts as a sensor for membrane viscosity/fluidity. Instead, we propose based on our

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A minimal sense-and-response construct reports on membrane lipid saturation 139 We proposed that Mga2 uses a rotation-based mechanism to sense membrane lipid recruiting the E3 ubiquitin ligase Rsp5 41 , three lysine residues K 980 , K 983 and K 985 152 ubiquitylated in vivo 36 , and the disordered region linking these motifs to the TMH (Figure 2A). 153 The construct was recombinantly produced and isolated in the presence of Octyl-β-D-154 glucopyranoside (OG) using an amylose-coupled affinity matrix and size-exclusion 155 chromatography (SEC) ( Figure 2B, S1A). Expectedly, the N-terminal zipper stabilizes a 156 dimeric form of the sense-and-response construct and supports, at increased concentrations, reconstituted the sense-and-response construct in liposomes at molar protein-to-lipid ratios 160 between 1:5,000 -1:15,000 and detected no sign of protein aggregation in our preparations 161 using sucrose-density gradient centrifugations ( Figure S1D). 162 We then tested if the sense-and-response construct could be ubiquitylated in vitro and 163 adapted a strategy established for the ubiquitylation of substrates of the ER-associated 164 degradation (ERAD) machinery 42 . We incubated the proteoliposomes with an ATP-165 regenerating system, purified 8xHis ubiquitin, and yeast cytosol containing enzymes required to 166 mediate the ubiquitylation reaction ( Figure 2D). Subsequent immunoblot analyses revealed a 167 time-dependent ubiquitylation of the sense-and-response construct, which became apparent 168 as a ladder of MBP-positive signals ( Figure 2E). Control experiments validated the specificity 169 of the ubiquitylation reaction: No ubiquitylation was observed, when the Rsp5-binding site 170 (∆LPKY) was deleted from the sense-and-response construct ( Figure 2E). Furthermore, 171 despite the presence of 50 lysine residues in the entire construct, the substitution of the three 172 lysine residues (3KR) targeted by Rsp5 in vivo 36 was sufficient to prevent the ubiquitylation 173 ( Figure 2E). We conclude that the in vitro ubiquitylation assay is specific and that the 174 conformational dynamics in the juxtamembrane region is likely to reflect the structural 175 dynamics found in full-length Mga2 protein. Most importantly, this newly established in vitro 176 system also allowed us to test the hypothesis of functional coupling between the sensory 177 TMHs and protein ubiquitylation. 178 We reconstituted the sense-and-response construct in two distinct membrane environments 179 based on a phosphatidylcholine (PC) matrix but differing in their lipid acyl chain composition.

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One membrane environment contained 50% unsaturated 18:1 and 50% saturated 16:0 acyl 181 chains (100 mol% POPC(16:0/18:1)), while the other was less saturated and contained 75%  In order to detect changes of the conformational dynamics in the juxtamembrane region, we 193 established an in vitro FRET assay. We hypothesized that the average distance between the 194 binding site of the E3 ligase Rsp5 (LPKY) and a lysine residue targeted by Rsp5 may be 195 affected by changes in the membrane lipid environment. We thus generated a donor 196 construct labeled with Atto488 at the position of a target-lysine (K983 D ) and an acceptor 197 construct labeled with Atto590 within the Rsp5 recognition site (K969 A ) (Förster radius of 59 198 Å) ( Figure 3A). Notably, the required amino acid substitutions to cysteine at the positions of 199 labeling did not interfere with the activation of full-length Mga2 in vivo ( Figure S2A). The reporter was concentration-dependent in detergent solution ( Figure 3C), thereby suggesting 208 a dynamic equilibrium between monomeric and oligomeric species (presumably dimers) of 209 the labeled sense-and-response construct. To validate this interpretation and to rule out the 210 possibility that the FRET signal was predominantly caused by FRET between stable K983 D -211 K983 D and K969 A -K969 A dimers bumping into each other, we performed competition 212 experiments. We found that the ratiometric FRET efficiency of the K983 D +K969 A reporter 213 was substantially reduced upon titrating it with an unlabeled sense-and-response construct 214 containing an N-terminal leucine-zipper ( Figure 3D). However, it remained unaffected upon 215 titration with an unlabeled construct lacking a zipper ( Figure 3D). This indicates (i) that the 216 zipper centrally contributes to the stability of the dimer, (ii) that individual protomers readily 217 exchange in detergent solution, and (iii) that the FRET signal is mainly due to K983 D -K969 A 218 heterooligomers. In fact, additional titration experiments with the K969 A acceptor revealed 219 that the observed FRET efficiency is a linear function of the molar fraction of the acceptor 220 ( Figure S2C,D), thereby indicating that the FRET signal is indeed caused by dimers 45 .

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Next, we studied the structural dynamics of the sense-and-response construct in liposomes 222 using the FRET reporter. To this end, we reconstituted K983 D,only and the pre-mixed 223 K983 D +K969 A pair in liposomes of defined lipid compositions and recorded fluorescence 224 spectra ( Figure 3E-G). We used a low protein-to-lipid ratio of 1:8,000 in these experiments to 225 minimize the contribution of unspecific proximity FRET to the overall signal 45 . We observed a     Figure S4A). Expectedly, we find that 298 the 'kink' introduced by a cis double bond supports membrane fluidity ( Figure 5A, S4B) by 299 lowering both, lipid packing and membrane order ( Figure 5B). Importantly, ∆6-cis acyl chains 300 render the membrane more viscous than ∆9-cis acyl chains ( Figure 5A) with no detectable 301 impact on membrane order as studied by C-laurdan spectroscopy ( Figure 5B). In contrast, 302 ∆9-trans 18:1 acyl chains render the bilayer substantially more viscous ( Figure 5A) and allow 303 for a much tighter packing of lipids ( Figure 5B). Using these bilayer systems differing by the 304 position and configuration of the double bond in the unsaturated lipid acyl chains, we set out 305 to study their impact on various aspects of the structure and function of Mga2 in vitro. 306 First, we studied how the double bond position and configuration affects the structural 307 dynamics of Mga2's TMH region using EPR spectroscopy ( Figure 5C). A substantial 308 broadening of the continuous wave EPR spectra recorded at -115°C ( Figure 5C) and an 309 increased interspin proximity ( Figure 5D) were observed, when the sensor was placed in the 310 tightly packed membrane with ∆9-trans acyl chains. Much less spectral broadening was 311 observed in membrane environments with either ∆6-cis or ∆9-cis acyl chains ( Figure 5C).

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This indicates that ∆9-trans double bonds in lipid acyl chains -more than ∆9-cis and ∆6-cis ). This suggests that the position of the cis-double bond has a significant, but rather 328 modest impact on the average distance between K969 A and K983 D in the FRET reporter.

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The highest FRET efficiency was observed when the reporter was placed in a membrane 330 with tightly packing ∆9-trans 18:1 acyl chains ( Figure 5F). These findings demonstrate that In order to address the functional consequences of these structural changes, we performed 335 in vitro ubiquitylation assays with the sense-and-response construct ( ZIP-MBP Mga2 950-1062 ) 336 reconstituted in the three distinct membrane environments. While barely any ubiquitylation 337 above background was detectable, when the sense-and-response construct was 338 reconstituted in a loosely packed bilayer with ∆9-cis acyl chains, we observed a robust 339 ubiquitylation when the construct was situated in a bilayer with either ∆9-trans or ∆6-cis acyl 340 chains. Strikingly, the highest degree of ubiquitylation of the reporter was observed in the 341 membrane with ∆6-cis lipid acyl chains, followed by the more viscous and more tightly  In order to test this prediction, we have substituted W1042 to either tyrosine (Y),  Figure 6B). The only exception to this near-perfect correlation was the W1042Q mutation.

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Given that intra-membrane glutamines are known to mediate homotypic interactions 49 , we 375 speculate that the W1042Q mutation stabilizes a rotational conformation of the TMHs, where 376 the two Q1042 side chains face each other and interact, thereby stabilizing Mga2 in a 377 processing-competed configuration. Intriguingly, the phenotypic differences between the 378 W1042Q, W1042L and W1042A variants show that an aromatic character at the sensory 379 position is not absolutely required for sensing.

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Next, we studied the impact of these mutations on the proteolytic processing of full-length

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All plasmids and strains used in this study are listed in Table S1       Bacteriol. 106, 449-55 (1971 Table S1. Plasmids used in this study. Table S2. Strains used in this study.