Analysis of ceramides and IPCs in WT, 4∆ and 6∆ by LC-MS(A, B and E) or by direct infusion MS (C).

<p>A. Extracted ion chromatograms (XIC) in negative ion mode of LC-MS analysis in time window where ceramides elute. (Cer44:0;4 is also shown in positive ion mode in the last column to the right). Elution times of peak signals for each ceramide species in minutes are in grey, apexes were set to 100% and the intensity at the apex indicated in blue. Only major species are shown. B. For experiment of panel A, the average intensity across the width of the entire LC peak for each ceramide species was determined and these averages for the various ceramides having a given number of C atoms were summed up and plotted. C. In an independent experiment lipid extracts were analyzed by direct infusion-FTMS and after addition of Cer35:1;3 as an internal standard. D. WT, 4∆ and 6∆ cells were labeled with [³H]inositol for 2 h and lipid extracts before and after deacylation were analyzed by TLC and autoradiography. E. In the experiment of panels A and B, IPCs were calculated and plotted as was done for ceramides in panel B.</p

Effect of myriocin treatment on LCB levels.

<p>A. Cells growing exponentially in YPD were further incubated without (upper plot) or with 20 μg ml^-1 myriocin (lower plot) for 4 hrs and LCB levels in the lipid extracts were determined by ESI-MS. A similar pretreatment with 2 μg ml^-1 of myriocin repressed LCB levels of <i>ypr114w∆</i> and WT to the same degree, albeit less strongly (not shown). B. Plates containing myriocin with or without 20 mM NAC were incubated for 2 days (top sections W303 background, bottom sections BY4742 background). C. Strains in the W303 background were grown in YPD with or without myriocin (1.2 μg ml^-1) for 24 h and superoxide anions were detected by staining with DHE. After 24 h all strains had grown to the same density.</p

Disruption of YJR116w makes cells hypersensitive to copper.

<p>A. 10-fold serial dilutions of <i>yjr116w∆</i>, <i>ypr114w∆</i> and yy∆∆ mutants were plated on SC containing 2% glucose and indicated concentrations of CuSO₄. B. WT, <i>yjr116w∆</i> (116∆), or <i>ypr114w∆</i> (114∆) cells harboring either empty pYES2NT vector (ev) or the same expressing YJR116w or YPR114w (114 or 116, respectively), were plated on SC with 2% galactose (Gal). C. Superoxide anion (O₂^-) levels of three independent clones of the same strains grown for 15 h in presence or absence of 2 mM CuSO₄ were assessed by staining cells with DHE. * <i>yjr116w∆</i> cells showed a significantly higher percentage of DHE positive cells than WT (P = 0.0039). D. As A, but plates were incubated anaerobically and contained 5 mg ml^-1 Tween 80 and 20 μg ml^-1 ergosterol. The freckles are precipitates caused by these additions to the medium. In each plate section, in the upper and lower rows are WT and <i>yjr116w∆</i> cells, respectively. E. Copper sensitivity of indicated mutants was tested as in A. F. First and 3^rd columns as in A, the others supplemented additionally with 20 mM NAC. The 3^rd and 4^th columns show the growth behavior of ρ^<i>-</i> strains lacking the respiratory chain. G, growth on copper and higher concentrations of NAC.</p

Genes in focus.

<p>A. Pathways of sphingolipid biosynthesis and degradation in yeast. Gene names are in italics, for enzymes requiring complex formation, genes are underlain in grey. B. Cladogram of ceramide synthases and their TLC domain containing paralogs of yeast (orange) and humans (blue). C. Alignment of <i>LAG1</i>, <i>LAC1</i>, YJR116w and YPR114w. The TLC domain is shown in red, the TRAM domain in blue. D. Homologues of <i>LAG1</i>, <i>LAC1</i>, YJR116w and YPR114w in intensively studied fungal species were found using PSI-Blasts (<a href="http://blast.ncbi.nlm.nih.gov/" target="_blank">http://blast.ncbi.nlm.nih.gov/</a>) as described in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145831#pone.0145831.s001" target="_blank">S1 File</a>. The cladogram was generated by clustal W (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>).</p

Protein targeting and organellar structures.

<p>A. Western blotting of cell extracts. Cells were grown to an OD₆₀₀ of 0.8 either at 24°C or at 37°C. B and C. Vacuoles of cells growing in YPD were stained with FM4-64. Typical pictures are shown in B, the percentages of cells having 1 to >3 vacuoles are shown in C. >250 cells were inspected for each strain.</p

<i>Yjr116w∆</i> and <i>ypr114w∆</i> make lower amounts of superoxide anions in stationary phase than WT and have an increased CLS.

<p>A. Cells were stained with DHE for measurement of superoxide anion levels. Fluorescence and transillumination pictures were merged to illustrate the difference between positive and negative cells. B. Percentages of DHE positive cells amongst 200 cells were determined in three independent experiments and the difference between mutants and WT in the same condition was evaluated by student’s t-test with P< 0.05 = *, P< 0.005 = **. C. The chronological life spans were determined in diploid deletion strains by measuring the colony-forming units after different periods in water (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145831#sec002" target="_blank">Materials and Methods</a>). Data represent three biological replicates using different clones and the differences between mutants and WT were evaluated as above.</p

Molecular mass and scores of XLs found by pLink for Pma1p and BSA.

<p>Distributions of scores and calculated molecular masses of XLs generated by pLink by scanning through all mgf files from BS3 experiments (<b>A-D</b>) or EDC experiments (<b>E-H</b>) for Pma1p and BSA, a protein that is not present in the sample, are plotted as calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s007" target="_blank">S7 Table</a>. The cut off lines used to filter all pLink XLs (stippled red lines) indicate a minimal molecular mass of XL of 1’500 Da and scores of ≤ 7.5 x 10⁻⁴ for BS3-XLs, or ≤ 4 x 10⁻⁴ for EDC-XLs.</p

Experimental workflow and chemical structure and properties of the crosslinking agents employed.

<p><b>A,</b> schematic representation of the experimental workflow used for growing cells, preparing microsomes, carbonate wash of membranes, chemical crosslinking of proteins and preparation of peptides for MS analysis. <b>B,</b> chemical structure and reactivity of the crosslinkers employed BS3 (bis[sulfosuccinimidyl] suberate) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.</p

BS3 crosslinks mapped onto the topological model of Fks1p, the catalytic subunit of 1,3-beta-D-glucan synthase.

<p><b>A,</b> Arch model of Fks1p indicating the positions of BS3 (red) XLs visualized using xVis. <b>B,</b> topology model of Fks1p proposed using the crosslinking data to validate a topology prediction visualized by Protter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref019" target="_blank">19</a>].</p

Frequency of XLs found by pLink for a given protein is correlated with its copy number.

<p>The number of XLs for 18 arbitrary chosen MSPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s004" target="_blank">S4 Table</a> is plotted as a function of the copy number of the respective MSP (<b>A</b>) or its amino acid length (<b>B</b>). In panel <b>C</b>, the number of BS3- and EDC-generated XLs for these 18 proteins are plotted separately, as a function of copy number. In panel <b>D</b>, the distance in the primary sequence between crosslinked amino acids in 160 XLs found in 27 proteins of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s014" target="_blank">S14 Table</a> are plotted.</p