Protein analyses

1D Gel electrophoresis

Seventy micrograms of protein was loaded onto a discontinuous sodium dodecyl sulphate bis-acrylamide gel. The SDS-PAGE gel consisted of a 4% polyacrylamide stacking gel (in

55 125 mM Tris-HCl, pH 6.8, 0.1% SDS) casted over a 12% resolving polyacrylamide gel (in 375 mM Tris-HCl, pH 8.8, 0.1% SDS). Classic Laemmli buffer (4% SDS, 20% Glycerol, 10%

2-mercaptoethanol, 0.004% Bromophenol blue and 0.125 M Tris-HCl, pH approximately 6.8) was used as sample buffer. The electrode chambers were filled with Tris-Glycine buffer, pH 8.3, containing 50 mM Tris, 200 mM glycine, 0.1% SDS. Electrophoresis was conducted at 120 V until the dye front reached the bottom of the gels. Staining was carried out in the microwave with Coomassie brilliant blue R250 in 50% [v/v] ethanol, 10% [v/v] acetic acid and destained with 12.5% [v/v] isopropanol and 12% [v/v] acetic acid according to the protocol described by de Beer et al. [37]. SDS-PAGE gel lanes were excised and sent to the proteomics laboratory at the Central Analytical Facility of Stellenbosch University (South Africa) for mass fingerprint analysis.

In gel digestion

Gel lanes were cut into smaller pieces and washed with water followed by 50% (v/v) acetonitrile and 50 mM ammonium bicarbonate. The gel pieces were incubated in acetonitrile until the gel pieces turned white and then dried in vacuo. Proteins were reduced with 10 mM dithioerythritol (DTT) followed by a wash step in ammonium bicarbonate, and then acetonitrile before being alkylated by 55 mM iodoacetamide. Following alkylation the gel pieces were washed with ammonium bicarbonate followed by acetonitrile before being dried in vacuo. The gel pieces were digested with 100 µl of 10 ng/µl trypsin solution overnight. The resulting peptides were extracted twice with 70% acetonitrile in 0.1%

trifluoroacetic acid and then in 100% acetonitrile and then dried. The dried peptides were dissolved in 5% acetonitrile in 0.1% formic acid.

Mass spectrometry

Peptide analysis was carried out on a Thermo Scientific EASY nLC II connected to a LTQ Orbitrap Velos mass spectrometer. (Thermo Scientific, Bremen, Germany) equipped with a nano-electrospray source. A total of 10 µl of trypsin digested sample was injected in a capillary chromatography system. Peptide mixtures were separated on an EASY-column (2 cm, ID 100 µm, 5 µm, C18) pre column followed by XBridge BEH130 Nanoease column (15 cm, ID 75 µm, 3.5 µm, C18) with a constant flow rate of 300 nl/min. Peptides were eluted with a solvent gradient from 5-17% B in 5 min, 17-25% B in 90 min, 25-60% B in 10 min, 60-80% B in 5 min and kept at 60-80% B for 10 min. Solvent A was 100% water in 0.1% formic acid and solvent B was 100% acetonitrile in 0.1% formic acid.

The mass spectrometer was operated in data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Data were acquired using the Xcaliber software package. The precursor ion scan MS spectra (m/z 400-2000) were acquired in the

56 Orbitrap with resolution R = 60000 with the number of accumulated ions being 1 x 10⁶. The 20 most intense ions were isolated and fragmented in linear ion trap (number of accumulated ions 1.5 x 10⁴) using collision induced dissociation. The lock mass option (polydimethylcyclosiloxane; m/z 445.120025) enabled accurate mass measurement in both the MS and MS/MS modes. In data-dependent LC-MS/MS experiments, dynamic exclusion was used with 60 s exclusion duration. Mass spectrometry conditions were 1.8 kV, capillary temperature of 250°C, with no sheath and auxiliary gas flow. The ion selection threshold was 500 counts for MS/MS and an activation Q-value of 0.25 and activation time of 10 ms.

Data analyses

Post run the proteins were identified using Thermo Proteome Discoverer 1.3 (Thermo Scientific, Bremen, Germany); the tandem mass spectra were submitted to the Mascot search algorithm (Matrix science, London, UK) and searched against the NCBI and Swissprot Saccharomyces cerevisiae, Uniprot Clavispora, Candida, Yarrowia and Lachancea thermotolerans databases, using a fixed modification of carbamidomethyl cysteine and variable modifications of oxidized methionine, N-acetylation and deamidation.

Precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.8 Da. Two missed tryptic cleavages were allowed. Proteins were considered positively identified with at least 2 unique tryptic peptides per protein and a Mascot score threshold of 20. Peptide validation was performed with Percolator with a maximum delta Cn of 0.5 and decoy database searches with a FDR of 0.02 and 0.05 with validation based on the q-value.

2D Gel electrophoresis

2D-PAGE was performed as previously reported [38] with minor modifications. Samples containing about 300 µg of protein were solubilised in 2D rehydration buffer (8 M Urea, 2%

CHAPS, 50 mM DTT, 0.2% Bio-Lyte^® 3/10 ampholyte, 0.002% bromophenol blue; Bio-Rad) and applied onto linear IPG strips (pH 3-10, 17 cm, Bio-Rad,) where it was allowed to rehydrate passively for 16 h. The first dimension was carried out on an IEF Cell (Bio-Rad) at 20°C using the following run parameters: 250 V for 15 min, linearly increasing the voltage to 10000 V during 3 h, focusing was finalized at for a total of 40 kvh. Immobilized pH gradient strips were reduced (2% DTT) and then alkylated (2% iodoacetamide) in equilibration buffer (6 M urea, 50 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS). The second dimension was carried out on homogeneous SDS-12% acrylamide gel. Electrophoresis was conducted at 16 mA/gel constant current for 30 min and 24 mA/gel constant current for four and a half hours in a Protean II cell (Bio-Rad). After electrophoresis gels were stained with a silver nitrate protocol adapted from Blum et al. [39]. Gels were briefly fixed in 50% methanol, 10% acetic acid solution for 30 min and 5% methanol for 15 min. Thereafter, they were rinsed 3 times in

57 mQ H₂O (Millipore, Billerica, MA), each rinse 5 min, before sensitizing in 0.2 g/l sodium thiosulphate solution for 2 min. The excess sodium thiosulphate was again rinsed off with mQ H₂O and the gels then incubated in cold 2% silver nitrate solution for 10 min. Gels were developed in developer solution (3% (w/v) sodium carbonate, 0.002% (w/v) sodium thiosulphate and 0.0185% (v/v) formaldehyde) for 10 min, and the developing reaction stopped by incubating gels in di-sodium ethylene diamine tetra-acetic acid (14 g/l, Na₂ -EDTA). All gels were stored in mQ H₂O until imaging could be done. Gel images were obtained using the Molecular Imager® PharosFX™ system (Bio-Rad).