The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans

Abstract

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Delta mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Delta mutant are similar to the effects of inhibiting beta-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.

Figure 1.

Sur7-GFP is present in static patches in the plasma membrane. C. albicans strain YJA15 carrying a SUR7-GFP fusion gene was analyzed by fluorescence microscopy. (A) Cells were grown in YPD medium to promote budding growth (left panel) and in GlcNAc medium to induce hyphal morphogenesis (middle and right panels). (B) Stability of the Sur7-GFP patches was examined by comparing pictures of the same cells taken 30 min apart. The merged images demonstrate the high degree of overlap of the patches. (C) Cells were treated with 5 or 25 μM myriocin for 90 min to block sphingolipid synthesis, and then Sur7-GFP fluorescence was recorded. Bars, 10 μm.

Figure 2.

sur7Δ is defective in endocytosis. A control C. albicans strain (DIC185), a sur7Δ strain (YJA11), and a complemented version of this strain in which one copy of SUR7 was reintroduced (YJA12) were assayed for ability to undergo endocytosis in two different assays. (A) Cells were treated with the lipophilic dye FM4-64 and then monitored after 15, 30, and 60 min by fluorescence microscopy for the accumulation of the fluorescence in the vacuolar membrane. (B) Ligand-induced endocytosis of the α-factor mating pheromone receptor was examined in wild-type (YAW54) or sur7Δ (YJA13) cells engineered to express STE2-GFP from the constitutive ADH promoter to avoid the need to convert the cells to the homozygous MATa/MATa genotype. Cells were incubated in the presence or absence of 5 × 10⁻⁷ M C. albicans α-factor for 60 min.

Figure 3.

Lsp1-GFP localizes to plasma membrane patches in sur7Δ cells. (A) The localization of Lsp1-GFP was analyzed in wild-type SUR7 cells (YJA16) and sur7Δ mutant cells (YJA17) by fluorescence microscopy. The middle of the cells was mainly in the focal plane, so the Lsp1-GFP appears primarily as patches around the periphery of the cell. (B) The stability of the Lsp1-GFP patches in the sur7Δ strain YJA17 was assessed by comparing images of the same cells recorded 30 min apart. The merge shows that the Lsp1-GFP patches remain essentially unchanged. The tops of the cells were in the focal plane to more readily observe the stability of the Lsp1-GFP patches.

Figure 4.

Actin polarization defect in sur7Δ cells. The control strain (DIC185; A) and the sur7Δ mutant (YJA11; B) were grown to log phase and then fixed and stained with rhodamine-phalloidin to detect actin localization (see Materials and Methods). Note that essentially all sur7Δ cells showed abnormal actin polarization, even those that appeared to have relatively normal morphology.

Figure 5.

Abnormal septin localization at sites distal to the bud neck in sur7Δ cells. Septin localization was monitored by analysis of a Cdc12-GFP fusion protein in a wild-type control strain (YAW44; A) and sur7Δ strain (YJA14; B). (C) A higher magnification image of Cdc12-GFP localization in the sur7Δ strain. Top panels, Cdc12-GFP; middle panels, Calcofluor White stain of cell wall chitin; bottom panels, DIC images. Bars, 5 μm.

Figure 6.

EM analysis of abnormal cell wall growth in sur7Δ cells. The wild-type strain DIC185 (A) and the sur7Δ strain YJA11 (B) were analyzed by TEM. Note in the sur7Δ mutant the abnormal appearance of torus-shaped structures of cell wall material and also the variations in cell wall thickness. Arrows indicate sites of invaginations of cell wall growth. (C) Higher magnification view of the septum of a sur7Δ mutant cell. Primary septum indicated by an arrowhead. (D) Representative sur7Δ cells exhibiting different amounts of abnormal intracellular wall growth. (E) Higher magnification images of abnormalities associated with the cell wall. Cells were analyzed by TEM as described in Materials and Methods. (A–D) black bars, 1 μm; (E) white bars, 100 nm.

Figure 7.

Characterization of intracellular cell wall growth in sur7Δ cells. The cell wall composition of wild-type strain DIC185 (A), sur7Δ strain YJA11(B), or the sur7Δ mutant strain YJA12 (C), in which one copy of SUR7 was reintroduced, was assessed by staining as indicated with Calcofluor, Aniline Blue, and concanavalin A-FITC. Cells were washed with methanol and acetone to permeabilize the plasma membrane and allow the intracellular cell wall structures to be stained.

Figure 8.

Altered sensitivity of sur7Δ mutant to antifungal drugs. Etest strips were used to test the sensitivity to the indicated antifungal drug in the wild-type strain DIC185, the sur7Δ strain YJA11, and the sur7Δ mutant strain in which one copy of SUR7 was reintroduced (YJA12). Cells were spread on a plate containing RPMI 1640 medium, the Etest strip was applied, and then the plates were incubated at 37° for 48 h. The Etest strips release a gradient of drug causing a zone of growth inhibition. Note the increased sensitivity of the sur7Δ mutant to fluconazole.

Figure 9.

Hyphal morphogenesis defects of sur7Δ mutant. (A) Calcofluor staining of hyphal cells induced with serum for 75 min. (B) Filipin staining of cells induced with 100 mM GlcNAc for 70 min to form hyphae. (C) Cells were spotted on solid medium agar plates containing 4% serum or 2.5 mM GlcNAc as indicated and grown for 7 d at 37°C, and then the plates were rinsed to remove surface growth and reveal invasive hyphal growth. The strains used were wild-type strain DIC185, sur7Δ strain YJA11, and the sur7Δ mutant strain in which one copy of SUR7 was reintroduced (YJA12).

Figure 10.

YLR414C encodes a new Sur7 paralog in S. cerevisiae. (A) Colocalization of Ylr414c-GFP and Sur7-RFP to punctate membrane patches in S. cerevisiae. The YLR414c-GFP was constructed as part of a genome-wide analysis of protein localization (Huh et al., 2003). (B) The function of a new SUR7-related gene, YLR414C, was tested by constructing a quadruple deletion mutant (sur7::kanR ylr414c::HIS3 fmp45::LEU2 ynl194c::URA3) in S. cerevisiae. The mutant cells were compared with the wild-type control (BY4741) for staining with Calcofluor (chitin), rhodamine-phalloidin (actin), and FM 4-64 (endocytosis). FM 4-64 was allowed to internalize for 30 min before recording the image. (C) Multiple sequence alignment of the region surrounding the Cys-containing motif in extracellular domain 1 of Sur7 and human claudin proteins. Complete multiple sequence alignment of a larger set of fungal Sur7-related proteins is shown in Supplementary Figure S1 and an evolutionary tree of their relatedness is shown in Supplementary Figure S2.