Rim101 proteolysis and nuclear localization are dependent on pH.

<p>(A) GFP-Rim101 is proteolytically processed from 140 kDa to ~100 kDa in response to increasing pH. GFP-Rim101 was immunoprecipitated from wild-type cells after incubating for 5 hr at the indicated pH 8 (SC medium buffered with McIlvaine’s buffer). Protein processing was determined by western blotting using an α-GFP antibody. (B) GFP-Rim101 nuclear localization increases in response to increasing pH. Cells were cultured in the same way as in (A). GFP signal was assessed by epifluorescence microscopy. Nuclei were stained using Hoechst 33342 live nuclei stain. Scale bar = 5 μm. (C) GFP-Rim101 proteolysis is not induced by 1 M NaCl or 150 μM BPS. Cells were cultured in each indicated condition for 3 hr. GFP-Rim101 was analyzed by western blot. (D) GFP-Rim101 localization in response to pH 7 SC (McIlvaine’s) or pH 4 SC (McIlvaine’s) with 1 M NaCl or 150 μM BPS. Cell assessed epifluorescence microscopy after 30 min incubation in each condition. Scale bar = 5 μm (E) <i>rim101Δ</i> is not NaCl sensitive at pH 4. Strains spotted onto YPD, YPD 150mM HEPES pH 4 1.5 M NaCl, and YPD 1.5 M NaCl.</p

Role of Rim13 and Rim23 orthologs in Rim101-regulated phenotypes.

<p>(A) The <i>C</i>. <i>neoformans RIM13</i> and <i>RIM23</i> orthologs are required for pH 8 and NaCl tolerance. 10-fold serial dilutions of the indicated strains were spotted onto YPD, YPD 150mM HEPES pH 8, YPD 1.5M NaCl and incubated at 30^°C for 48 hr -72 hr (B) The <i>rim13Δ</i> and <i>rim23Δ</i> mutants have a <i>rim101Δ-</i>like capsule defect. Cells were incubated in CO₂-independent media for 48hr at 37^°C. Capsule was visualized by counterstaining with India ink. (C) Rim101 proteolysis and localization are disrupted in <i>rim13Δ</i>, <i>rim20Δ</i>, and <i>rim23Δ</i> mutant strains. GFP-Rim101 was immunoprecipitated from each strain after 5 hr incubation in pH 7.4 YPD buffered with 150 mM HEPES. (D) GFP-Rim101 localization was assessed in the indicated strains after culturing for 5 hr in SC medium buffered with McIlvaine’s buffered to pH 8. Nuclei were stained with Hoechst 33342 live nuclei stain. Scale bar = 5 μm.</p

The <i>rra1Δ</i> mutant is phenotypically identical to other Rim pathway mutants.

<p>(A) <i>rra1Δ</i> insertional mutant has pH 8 and 1.5 M NaCl growth defects that are rescued by <i>GFP-RIM101T</i> expression. 10-fold serial dilutions were spotted onto YPD, YPD with 150 mM HEPES pH 8, and YPD with 1.5 M NaCl. (B) Independent <i>rra1Δ</i> mutant has a growth defect pH 8 and 1.5 M NaCl. (C) The <i>rra1Δ</i> strain has a capsule defect. Cells were cultured for 48 hr in CO₂-independent media at 37^°C to induce capsule. Capsule was visualized by India ink staining. Scale bar = 5 μm. (D) The <i>rra1Δ</i> mutation disrupts GFP-Rim101 proteolysis. GFP-Rim101 was immunoprecipitated from the indicated mutant strains after 5 hr incubation in YPD with 150 mM HEPES at pH 7.4. (E) GFP-Rim101 nuclear localization is disrupted in the <i>rra1Δ</i> mutant. GFP-Rim101 was assessed after 5 hr incubation in pH 8 SC McIlvaine’s buffer.</p

Strain list.

<p>Strain list.</p

A model of the canonical Rim pathway elucidated in ascomycete fungi [21,24].

<p>A model of the canonical Rim pathway elucidated in ascomycete fungi [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005159#pgen.1005159.ref021" target="_blank">21</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005159#pgen.1005159.ref024" target="_blank">24</a>].</p

Rim23-GFP forms plasma membrane-associated puncta under neutral/alkaline pH conditions.

<p>(A) Rim23-GFP was visualized at pH 4 SC McIlvaine’s buffer and after 30 min after shift to pH 7 SC McIlvaine’s buffer. (B) The number of Rim23-GFP puncta increase with time after shifting from pH 4 to pH 7. Quantification Rim23-GFP puncta/cell at pH 4 (0 min) and after 10, 20, 30 min incubation at pH 7. (C) Rim23-GFP puncta are closely associated with the plasma membrane. Cells were stained with FM-464 after 1 hr incubation at pH 7. (D) 1 M NaCl and 150 μM BPS does not induce Rim23-GFP puncta formation. Cells images after 30 min incubation in indicated culture media. (E) Rim23-GFP puncta formation was disrupted by <i>rra1Δ</i>, <i>vps23Δ</i>, and <i>snf7Δ</i> mutants. Cells were imaged after 30 min incubation at pH 7. The number of puncta/cell was quantified for 62–100 cells/strain. All strains in this figure were cultured in SC McIlvaine’s buffer media. All scale bars = 5 μm.</p

Effects of Rim pathway mutants on virulence.

<p>(A) <i>rim13Δ</i>, <i>rim23Δ</i>, and <i>rra1Δ</i> mutants are hypervirulent in a murine model of cryptococcosis. 8–10 A/Jcr female mice were intranasally inoculated with 1X10⁵ cryptococcal cells and monitored daily for survival. (B) Histopathological analysis revealed increased inflammatory cell infiltration in Rim-pathway mutants. Infected A/Jcr mouse lungs were harvested on day 7 post inoculation and H & E stain. Black arrows mark <i>C</i>. <i>neoformans</i> cells.</p

ESCRT complex proteins, Vps23 and Snf7, are required for Rim101 activation.

<p>(A) <i>snf7Δ</i> and <i>vps23Δ</i> capsule defects are partially rescued by <i>GFP-RIM101T</i> expression. Strains cultured for 24 hr in tissue culture media. India ink used to visualize capsule. (B) <i>GFP-RIM101T</i> expression rescues the <i>vps23Δ</i> and <i>snf7Δ</i> growth defects on pH 8 but not 1.5 M NaCl. (C) GFP-Rim101 was immunoprecipitated from the indicated strains after 5 hr incubation in YPD with 150mM HEPES at pH 7.4. (D) GFP-Rim101 (full-length) nuclear localization is disrupted in the <i>vps23Δ</i> mutant. Localization was assessed after culturing for 5 hr in SC with McIlvaine’s buffer at pH 8. Nuclei were stained with Hoechst 33342 live nuclear stain. Scale bar = 5 μm.</p

Primers.

<p>Primers.</p

<i>ERG5</i>, <i>STT4</i>, and <i>SNT1</i> are genetic interactors of <i>HSP90</i>.

<p>The fitness defect of the <i>tetO-HSP90/hsp90Δ</i> and <i>stt4Δ/Δ</i>, <i>erg5Δ/Δ</i>, or <i>snt1Δ/Δ</i> double mutant strains is greater than the product of the fitness defect of each individual mutant. The dotted line indicates the expected double mutant fitness defect. In the <i>tetO-HSP90/hsp90Δ</i> strains, <i>HSP90</i> depletion is achieved by transcriptional repression with 0.05 μg/mL doxycycline (DOX).</p