(A) In vivo labeling of C. neoformans WT, Δgcs1, and Δgcs1 + GCS1 using [³H]-DHS. The formation of GlcCer (boxed area) was examined by analysis of the extracted lipids onto a TLC. The soy GlcCer standard was visualized by iodine stain. (B) Analysis of purified GlcCer from the cultured strains using HPTLC. The lipids containing the sugar residues were visualized by staining with orcinol in 70% sulfuric acid, and the putative GlcCer is indicated. Plates were also stained with iodine to ensure equal lipid loading among the lanes (arrowhead). (C) Electrospray tandem mass spectrometric analysis of the glycosphingolipid analyzed on the HPTLC. The regions of the chromatogram indicated in B were extracted and analyzed by mass spectrometry as described in Methods. The indicated peaks are consistent with the [M+H]⁺ ions for monoexosylceramide with a methyl-sphingadienine backbone and a hydroxy-C18:1 fatty acid (the major species, m/z 756.7), a non–hydroxy-C18:0 fatty acid (m/z 740.7), and a hydroxy-C16:0 fatty acid (m/z 728.7). (D and E) ¹H-¹H–double quantum filtered correlation spectroscopy (D) and ¹H-¹³C heteronuclear single quantum correlation (E) NMR spectra of the monohexosylceramide WT extracted from the HPTLC. The solid connectivities in D among the 7 nonexchangeable hexose protons and their ¹H and ¹³C chemical shift characteristics (E) show that glucose was attached to the ceramide backbone, defining this glycosphingolipid as GlcCer. Dashed connectivities in D identify the exchangeable 6-OH in 100% dimethyl-sulfoxide-d6.