7B). Taken together these results clearly demonstrate that the structures stained by FM4-64 are not fragmented vacuoles, and are plasma membrane-derived. Figure 7 Vacuolar

structure in the C. albicans sur7 Δ mutant. (A) Carboxy-DCFDA was used in addition to FM4-64 to differentiate between vacuolar and non-vacuolar structures stained by FM4-64 in C. albicans yeast cells. Upon active endocytosis, FM4-64 stains the vacuolar PI3K Inhibitor Library membrane whereas CDCFDA is passively diffused into the cell and into the vacuolar lumen. Images were taken following sufficient incubation that allowed each dye to reach the vacuole. (B) Thin-section electron micrographs of yeast cells of the wild-type and sur7Δ null mutant strains are shown with arrows indicating abnormal invagination of the plasma membrane and subcellular structures of plasma membrane origin. A size bar is shown to indicate 1 μm. Thus, from a structural perspective, the overall click here plasma membrane architecture of both yeast and hyphal cells, the C. albicans sur7Δ null mutant is markedly abnormal. The Candida albicans sur7Δ mutant is impaired in lipase secretion but overproduces Sap2p Secretion of degradative enzymes is important to pathogenesis, thus we characterized secretion of aspartyl proteases (Saps), lipase, and phospholipases in the sur7Δ null mutant strain. When inoculated on medium containing BSA as

the sole nitrogen source, wild-type C. albicans secretes aspartyl proteinases which result in a halo surrounding the colony due to extracellular proteolysis. Compared to prototrophic control strain DAY185 and the isogenic complemented strain, the C. albicans sur7Δ null mutant secreted increased extracellular proteolytic activity on BSA plates (Fig. 8A) and in liquid BSA (Fig. 8B). We next examined extracellular Sap2p secretion using Western blotting of culture supernatants using anti-Sap2p antibodies (from M. Monod). The C. albicans sur7Δ mutant produced substantially greater amounts of extracellular Sap2p, compared to DAY185 and the complemented strain (Fig. 8C). Thus, the proteolytic degradation of BSA (Fig. 8A and

8B) is likely due to increased secretion of Sap2p. In contrast, the C. albicans sur7Δ mutant secreted slightly reduced amounts Adenosine of extracellular Sap4-6p compared to the control and complemented strain when analyzed by Western blotting (data not shown). Figure 8 Protease secretion in the sur7 Δ null mutant strain. (A) Extracellular protease secretion was assayed using a BSA degradation plate assay. Overnight cultures were spotted onto BSA plates and incubated at 30°C for 24 and 48 h. The relative amount of extracellular protease activity is indicated by the halo surrounding the fungal colony. (B) BSA degradation and Sap2p levels in liquid cultures were also assessed. Overnight cultures were shifted to medium containing BSA as the sole nitrogen source and incubated at 30°C for 6 hours.