A. Diagram of Sre1 in the membrane. TM1 indicates predicted first transmembrane segment. Amino acid positions are given and bHLH denotes the N-terminal basic helix-loop-helix leucine zipper DNA binding domain. B. Protein sequence alignment of the predicted first transmembrane segments of C. neoformans and human SREBP-2. Putative helix-destabilizing residues are underlined. C. Western blot of phosphatase-treated whole cell extracts (40 µg) from wild-type and stp1Δ cells transformed with empty vector, or plasmids expressing the indicated Sre1 truncations. Lanes 1 and 7, 2 and 6, 3 and 5 are duplicates of stp1Δ cells expressing empty vector, Sre1 (aa1-535), and Sre1 (aa1-501), respectively. Cells were grown to exponential phase in YES medium at ambient oxygen concentrations. Asterisks indicate non-specific, cross-reacting proteins detected by anti-Sre1 antiserum.

A. Venn diagram comparing statistically-significant genes more highly expressed in wild-type versus sre1Δ and stp1Δ cells under low oxygen. B. Venn diagram comparing statistically-significant genes with reduced expression in wild-type versus sre1Δ and stp1Δ cells under low oxygen. C. Quantitative PCR analysis on genes more highly expressed in wild-type versus sre1Δ or stp1Δ cells. Wild-type, sre1Δ, and stp1Δ cells were grown under ambient (21%) or 3% oxygen for 2 hours, and RNA was harvested. RNA level for the indicated genes was quantified by real time RT-PCR and normalized to that in wild-type cells at 21% oxygen. Error bars represent standard deviation from three biological replicate experiments.

A. Wild-type, sre1Δ and stp1Δ cells were grown for 48 hours in liquid YES medium. Final cell density was determined using a spectrophotometer (1 OD₆₀₀ = 2×10⁷ cells) at the indicated concentrations of itraconazole (left panel) or 25-thialanosterol (right panel). Error bars represent standard deviation from three biological replicate experiments. B. Wild-type, sre1Δ and stp1Δ cells were grown for 48 hours in liquid YES medium. Equal numbers of cells were plated and grown on YES medium for 2 days. Colony-forming units were counted and percent viability calculated. C. Wild-type, sre1Δ and stp1Δ cells were grown for the indicated times in the presence of 2.5 nM itraconazole, 5 nM 25-thialanosterol or DMSO as a vehicle control. Equal numbers of cells were plated and grown on YES medium for 2 days. Error bars represent the standard deviation from three biological replicate experiments.

A. Protein alignment of human Site-2 protease (NCBI refseq ID: NP_056968) and C. neoformans Stp1 (BROAD ID: CNAG_05742.2) using SIM alignment tool (http://ca.expasy.org/tools/sim.html). Putative catalytic residues are underlined. B. C. neoformans serotype A wild-type and stp1Δ cells were shifted from 21% oxygen to 3% oxygen for indicated periods of time. Immunoblot analysis was performed on whole cell extracts (40 µg) using anti-Sre1 antiserum. P and N denote the precursor and nuclear forms, respectively. Asterisks indicate non-specific, cross-reacting proteins detected by anti-Sre1 antiserum. C. C. neoformans cells from the indicated strains were grown under ambient (21%) or 3% oxygen for 2 hours, and RNA was harvested. SRE1 transcript levels were quantified by real time RT-PCR and normalized to that in wild-type cells at 21% oxygen. Error bars represent standard deviation from three biological replicate experiments. D. Five-fold serial dilutions of C. neoformans cells from the indicated strains were spotted on rich medium and rich medium containing 0.3 mM CoCl₂ and incubated at 30°C for 3 days. E. C. neoformans stp1Δ cells transformed with either empty vector, STP1, STP1 (H202A), STP1 (H202A, E203A) and STP1 (D467A) were grown for 2 hours at 3% oxygen. Immunoblot analysis was performed on whole cell extracts (40 µg) using anti-Sre1 antiserum. P and N denote the precursor and nuclear forms, respectively. Asterisk indicates non-specific, cross-reacting proteins detected by anti-Sre1 antiserum. F. C. neoformans stp1Δ cells were transformed with the indicated plasmids and five-fold serial dilutions of cells were spotted on rich medium and rich medium containing 0.3 mM CoCl₂ and incubated at 30°C for 3 days. Six independent isolates were plated.

A. C. neoformans serotype A wild-type and stp1Δ cells were grown in YES medium at 37°C. Cell numbers were determined at one hour intervals for 12 hours. Error bars represent one standard deviation from three biological replicates. B. Female Balb/c mice (n=10/strain) were infected via tail vein injection with the indicated C. neoformans strains and mouse survival was monitored.

A. Outline of C. neoformans ergosterol biosynthetic pathway. Genes encoding Sre1-dependent (bold) and Stp1-dependent (underlined) enzymes are listed. B. Wild-type, stp1Δ and sre1Δ cells were grown for 2 hours at 21% or 3% O₂. Total sterols were extracted and analyzed by gas chromatography. Ergosterol and sterol intermediates were quantified and normalized to wild-type 21% oxygen ergosterol levels and plotted. Error bars represent the standard deviation from three biological replicate experiments.