33
Supplemental material Figure legends
Figure S1. IPCs of 2∆.Lass5 and 4∆.Lass5 mostly contain C16:0 and C18:0 fatty acids by ESI-MS. A, YPK9 wt and 4∆.Lass5 were grown exponentially in LM (met-),
glycerophospholipids and IPCs were extracted using CHCl3-CH3OH (1:1), and the desalted lipid extract was analyzed by ESI-MS on a Bruker 4.7 T BioAPEX II FT/ICR mass
spectrometer in the negative ion mode. For sphingolipids, two consecutive numbers separated by a dash indicate the number of C atoms in the fatty acid moiety (based on the assumption that the LCB contains the usual 18 C atoms) and the number of hydroxyls in the entire ceramide moiety. For glycerophospholipids the sum of C atoms and double bonds in the two fatty acids is indicated in parenthesis. The same results were obtained when the samples were analyzed on a Bruker Daltonics mass spectrometer. B, 2∆.Lass5 cells were grown and lipids were analyzed as for panel A, either before or after deacylation with NaOH. The
concentration of lipids in the NaOH treated sample was 10 fold higher than in the control sample. The figure shows one of several concordant experiments, in which deacylated as well as crude lipid extracts were analyzed. In experiments of the type shown here, the DHS-based IPCs (having only 2 hydroxyl groups) were much more abundant than IPCs with 3 or 4 hydroxyls, quite in contrast to the experiment shown in Fig. 4A. The difference could be related to the extraction procedure or the different ESI-MS technology used.
Figure S2. Bud site selection is normal in 4∆.Lass5 and 4∆.Lass5 elo3∆ cells. Cells were maintained in exponential growth phase for at least ten cell divisions, stained with calcofluor white and viewed under the light microscope.
Cerantola et al., Figure 4
760.51 PS (34:1)
908.61
0.5 1.0 1.5 2.0
a.i. x 10-6
7008009001100m/z
WT (YPK9)
924.65 952.68 936.61
812.53 IPC16-4 824.56 IPC18-3 780.54 IPC16-2
3.0
700m/z
a.i x 10-6
4∆.Lass5
2.0 1.0
900
714.51 PE (34:2) 753.45 PI (28:0)
796.53 IPC16-3 807.50 PI (32:1)
800
835.53 (PI 34:1)
863.54 PI (36:1) PI 36:1 (863.56)
IPC24-3 IPC24-4
IPC26-4 IPC26-3
A
a.i. x 10-6
865.58 PI (36:0)
863.56 PI (36:1)
1.0 2.0 3.0 4.0 5.0
700800900m/z
714.51 PE (34:2)
807.50 PI (32:1)
835.53 PI (34:1) 780.53 IPC16-2
2.0 1.0 3.0 4.0 5.0
a.i. x 10-6
796.53 IPC16-3
700800900m/z
752.51 IPC14-2
812.53 IPC16-4 824.56 IPC18-3
840.57 IPC18-4
842.59 mannosyl-IPC16-2
B
2∆.Lass5, control 2∆.Lass5, NaOH treated
Cerantola et al., Figure S1
YPK9
4∆.Lass5
4∆.Lass5 elo3∆
elo3∆-2 (YPK11)
Cerantola et al., Figure S2
elo3∆-1 (BY4742)
