Regulation of Gene Expression through a Transcriptional Repressor that Senses Acyl-Chain Length in Membrane Phospholipids

Summary Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

. Acc1 activity correlates with cellular inositol production, related to Figure 4 (A) Cells were grown overnight in YPD medium at 30°C and washed twice with sterile water. 5 µl of OD 600 = 1/ml aliquots were spotted on -I medium containing the indicated concentration of doxycycline (DC).
After 2 days of growth at 30°C, the plates were sprayed with a suspension of an inositol auxotrophic tester strain (AID) and its growth around colonies was scored after further incubation for 2 days. opi1 and cho2 mutants served as positive controls, wild type (wt) as a negative control. Overproduction and excretion of inositol (Opi phenotype) was determined by the appearance of halos around the spotted strains. (B) Model for the correlation between Acc1 activity and INO1 gene expression. A reduction of Acc1 activity in wild type (decreased ACC1 expression level in the tetO7-ACC1 strain in the presence of doxycycline or in wild type treated with SorA) leads to inositol overproduction (1), strains with Acc1 hyperactivity (acc1 S/A ,  1 H NMR spectra, (C) 13 C NMR spectra and (D) 31

In vitro mutagenesis of ACC1
The mutation generating the amino acid change from serine1157 to alanine (acc1 S/A mutant) was introduced by site directed mutagenesis as described (Ho et al., 1989). Two mismatch PCR reactions were performed using genomic DNA from a wild type strain as template in order to generate overlapping fragments at the region of the desired nucleotide exchange (forward primer 1: CAG AAT TTG AAG TGT   CGG TGA C and reverse primer 1: G AAC AGG GCT GTT GCT GTT TCA GAT; forward primer 2: GA   CAA ATC TGA AAC AGC AAC AGC CCT G and reverse primer 2: GAG AAA TCC AAA ACT GCC TTA CTC, changed nucleotides are in bold). Fragment sizes (686 bp and 987 bp, respectively) were checked on an agarose gel and both fragments were ligated in a subsequent PCR reaction without primers. The full length insert (1643 bp) was amplified (using forward primer 1 and reverse primer 2) and transformed into a wild type strain for genetic recombination. Cells were then plated on YPD-plates containing 1 μg/ml soraphen A for selection of mutants. The mutant generated in this fashion was back-crossed twice with a wild type strain. Correct nucleotide exchange was confirmed by sequencing.

Analysis of Acc1 by liquid chromatography/mass spectrometry
For qualitative analysis, Protein isolation of logarithmically growing cells was performed as previously described (Shirra et al., 2001). In brief, cells from wild type and the acc1 S/A mutant strains were mechanically disrupted with glass beads in a Merckenschlager disintegrator (B. Braun Biotech Inc., Allentown, PA, USA) for 3 min under CO 2 cooling. Acc1 protein was enriched using Pierce® Monomeric Avidin Kit (ThermoScientific, Rockford, IL, USA). The 250 kDa Acc1 protein bands of wild type and acc1 S/A mutant strains were excised from the gel and tryptically digested as described (Shevchenko et al., 1996).
Peptide extracts were dissolved in 0.1% formic acid and separated on a nano-HPLC system (Ultimate 3000™, Dionex, Amsterdam, Netherlands). 70 μl samples were injected and concentrated on the loading column (LC Packings C18 PepMap™, 5 μm particle size, 100 Å pore size, 300 μm ID x 1mm) for 5 min using 0.1% formic acid as isocratic solvent at a flow rate of 20 μl/min. The column was then switched to the nanoflow circuit, and the sample was loaded on the nanocolumn (LC-Packings C18 PepMap™, 75 μm inner diameter x 150 mm) at a flow rate of 300 nl/min and separated using the following gradient: solvent A: water, 0.3% formic acid, solvent B: acetonitrile/water 80/20 (v/v), 0.3% formic acid; 0 to 5 min: 4% B, after 40 min 55% B, then for 5 min 90% B and 47 min re-equilibration at 4% B. The sample was ionized in a Finnigan nano-ESI source equipped with NanoSpray tips (PicoTip™ Emitter, New Objective, Woburn, MA, USA) and analyzed in a Thermo-Finnigan LTQ linear ion trap mass spectrometer (Thermo, San Jose, CA, USA). The MS/MS data were analyzed by searching the NCBI non-redundant public database with SpectrumMill Rev. 03.03.084 SR4 (Agilent, Darmstadt, GER) software. Acceptance parameters were two or more identified distinct peptides according to Carr et al. (Carr et al., 2004). The MS/MS spectrum of the peptide confirming serine1157 as the Snf1 phosphorylation site is shown in Figure S1.

Quantitative analysis of Acc1 phosphorylation at position serine1157
For quantitative analysis, protein isolation of logarithmically growing cells was performed as described above but the avidin purification step was omitted due to possible degradation of Acc1 or loss of phosphorylation even in the presence of phosphatase inhibitors. The relative amount of Acc1 phosphorylation at position serine1157 from wild type, snf1 and acc1 S/A mutant cells was determined as follows:The internal standard peptide mixture used for quantitative analysis of Acc1 phosphorylation was generated as described (Holzmann et al., 2009). In brief, the EtEP-peptides (GVTASVAGARAVSVSDLSYVANSQSSPLR, GVTASVAGARAV(Sp)VSDLSYVANSQSSPLR), which were synthesized in house using solid-phase Fmoc chemistry, were purified and digested with trypsin. The resulting tryptic peptides were labeled with mTRAQ heavy (ABSCIEX, Foster City, CA) and quantified by nLC-MRM analysis on a 5500 QTRAP instrument (ABSCIEX, Foster City, CA) for the mTRAQ-heavy labeled equalizer peptide GVTASVAGAR. The individual EtEP-peptide digestions were then mixed in equimolar amounts, accordingly.
The mTRAQ-light labeled Acc1 peptides were then spiked with an equimolar mixture of the mTRAQ-heavy labeled internal standard peptides (100 fmol each) and were immediately separated on a Dionex Ultimate 3000 RSLCnano HPLC, fitted with a C18 PepMap column (75 μm x 150 mm, 3 μ particle size, 100 Å pore size) (Dionex, Amsterdam, The Netherlands), developing a binary gradient from 98% solvent A (2% acetonitrile, 0.1% formic acid) and 2% solvent B (80% acetonitrile, 10% TFE, 0.08% formic acid) to 40% solvent B over 60 minutes at a flow rate of 275 nl/min, followed by a gradient from 40% solvent B to 90% solvent B over 5 minutes at a flow rate of 275 nl/min. The eluting peptides were analyzed on a 5500 QTRAP (ABISCIEX, Foster City, CA), essentially as described in (Holzmann et al., 2011). In brief, the mass-spectrometer was operated in MRM mode, monitoring four MRM transitions per peptide, under previously optimized collision energy (CE)-and collision cell exit potential (CXP)-settings. The integration of the respective XIC peaks was performed manually, using the MultiQuant 1.2 software (ABISCIEX, Foster City, CA). The abundance of the sample-derived peptides, relative to the internal standard peptides, was calculated from the ratios of the respective light to heavy XIC peak areas. The degree of phosphorylation was then calculated from the ratio of the sample-derived phosphorylated peptide to the sample-derived non-phosphorylated peptide.
Protein and phosphor-peptide identification, as well as tryptic digestion quality control, was performed by Additionally, the relative amounts of Acc1 serine1157 phosphorylation was estimated from the XIC peak areas of the 2+ and 3+ charged ions of the peptides AVSVSDLSYVANSQSSPLR and AV(Sp)VSDLSYVANSQSSPLR, respectively. Manual data evaluation and integration of the XIC peaks was performed using the QualBrowser of the XCalibur 2.1 software (Thermo Fisher Scientific, Bremen, GER). The degree of phosphorylation was then calculated from the ratios of the peak areas of the phosphorylated to the non-phosphorylated peptide ions.

Overexpression of INO1
The INO1

Fluorescence microscopy
Microscopy was performed using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) with spectral detection and a Leica 100x immersion objective (NA=1.4). BODIPY® 493/503 fluorescence was excited at 488 nm. Fluorescence emission was detected between 500-550 nm. Nile Red fluorescence was excited at 543 nm. Fluorescence emission was detected between 550-570 nm. Opi1-GFP fluorescence was excited at 488 nm, GFP fluorescence emission was detected in the range between 500-550 nm. Transmission images were recorded using differential interference contrast (DIC) optics.

Opi1 overexpression and purification
The OPI1