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Figures
- Fig. 1.
The ArfGAP2/3 Glo3 interacts with the sterol C5 desaturase Erg3. (A) Schematic outline of the HBH purification. (B) Erg3 and Glo3 interact in a yeast two-hybrid assay. β-galactosidase activity expressed as Miller units are given. Arf1 served as a positive and Pub1 as a negative control. Standard deviation of experiments performed in triplicates are given. (C) Growth of Δglo3Δerg3 cells is not impaired on YPD. Drop test of indicated strains on YPD plates incubated at 30°C for 3 days. (D) GLO3 and ERG3 display synthetic genetic interaction on low tryptophan medium. Drop test of indicated strains on plates containing 5, 20 or 200 mg/l tryptophan; 200 mg/l is the standard tryptophan concentration in selective media.
- Fig. 2.
The localization of the tryptophan permease Tat2 is impaired in Δglo3Δerg3 cells. (A) Tat2 accumulates in intracellular foci in Δglo3Δerg3 cells. The localization of Tat2-GFP was assessed in early- to mid-log phase growing cells of different strains. (B) Quantification of A. The data of at least three independent experiments in which≥100 cells were counted per strain are displayed. Error bars represent standard deviation. The p-value corresponds to<0.01. (C) Tat2 does not accumulate in the Golgi. Double labeling of Tat2-GFP and the Golgi marker Anp1-mCherry. Arrows point to non-overlapping signals. (D) Tat2 accumulates in endocytic compartments. Double staining of Tat2-GFP and the lipophilic dye FM4-64, marking endocytic compartments. Arrows point to overlapping signals. (E) Overexpression of Tat2 rescues the growth defect of Δerg3 and Δglo3Δerg3 cells on low tryptophan plates. Drop assay of indicated yeast strains on selective media containing different concentration of tryptophan; 200 mg/l being the standard concentration. The scale bars in A, C and D represent 5 µm.
- Fig. 3.
The localization of the general amino acid permease Gap1 is impaired in Δglo3Δerg3 cells. (A) Gap1 accumulates in intracellular foci in Δglo3Δerg3 cells. The localization of Gap1-GFP was assessed in early- to mid-log phase growing cells of different strains in selective media, which only contained proline as nitrogen source. (B) Quantification of A. At least 100 cells in each of three independent experiments were counted. Error bars represent standard deviation. The p-value corresponds to<0.01. (C) Gap1 accumulates in endocytic compartments. Double staining of Gap1-GFP and the lipophilic dye FM4-64, marking endocytic compartments. Scale bar represents 5 µm.
- Fig. 4.
Not all cargo depends on Glo3 and ergosterol for proper plasma membrane localization. (A) The localization of glucose transporter Hxt2 is not impaired in Δglo3Δerg3 cells. Hxt2 was appended chromosomally with GFP. Early- to mid-log phase grown cells were analyzed by microscopy. (B) Chitin synthase III transport to the plasma membrane and recycling through the TGN is not perturbed in Δglo3Δerg3 cells. Early- to mid-log phase grown cells expressing Chs3-GFP were analyzed by microscopy. Chs3 is localized to the bud neck in small and in large budded cells. (C) Quantification of Chs3 bud neck localization in different strains. The data of at least three independent experiments in which≥100 cells per cell-cycle stage were counted are presented. Error bars represent standard deviation. Small and large budded cells are schematically represented. (D) Carboxypeptidase Y (CPY) transport is not aggravated in Δglo3Δerg3 cells. CPY-GFP transport to the vacuole was assessed in indicated strains. In all cases vacuolar localization was observed, albeit with a varying degree of efficiency. CPY accumulated in the ER in Δglo3 and Δglo3Δerg3 cells. (E) Quantification of the phenotype displayed in D. At least 100 cells in each of three independent experiments were counted. Error bars represent standard deviation. The p-value corresponds to<0.01. The scale bars in A, B and D represent 5 µm.
- Fig. 5.
In Δglo3Δerg3 cells, Pma1 accumulates in distinct areas in the ER. (A) Pma1-GFP is retained internally in Δglo3Δerg3 cells. Early- to mid-log phase grown cells were analyzed by microscopy. Scale bar represents 5 µm. (B) Quantification of the phenotype displayed in A. At least 100 cells in each of three independent experiments were counted. Error bars represent standard deviation. The p-value corresponds to<0.001. (C) Pma1 causes proliferation of ER subdomains. Electron microscopy analysis of strains expressing Pma1-GFP. The scale bar in the low magnification is 1 µm and for the enlargements 500 nm.
- Fig. 6.
Pma1-GFP does accumulate in a compartment enriched for an ER marker. (A) Pma1-GFP is retained in ER subcompartments in Δglo3Δerg3 cells. Cells co-expressing Pma1-GFP and the ER marker Sec63-RFP were analyzed. (B) The Pma1 accumulations in Δglo3Δerg3 cells are not positive for the Golgi marker Anp1. Early- to mid-log phase grown cells expressing Pma1-GFP and Anp1-mCherry were analyzed by microscopy. Scale bars represents 5 µm.
- Fig. 7.
Snc1 recycling to the plasma membrane requires Glo3 and ergosterol. (A) Snc1 recycling is impaired in Δglo3Δerg3 cells. Snc1-GFP or the endocytosis deficient Snc1PEM-GFP were analyzed in logarithmically growing cells. (B) Snc1-GFP accumulates in endosomes. Co-labeling of Snc1-GFP and FM4-64, which marks endocytic compartments. (C) Quantification of Snc1-GFP plasma membrane localization. (D) The BoCCS region and the GAP domain are both required for proper Snc1 plasma membrane localization. Δglo3Δerg3 Snc1-GFP cells were transformed with an empty plasmid or plasmids expressing either wild-type Glo3 or mutant version of Glo3. Plasma membrane localization of Snc1-GFP was quantified. (C,D) At least 100 cells in each of three independent experiments were counted. Error bars represent standard deviation. The p-value corresponds to<0.01. The scale bars in A and B represent 5 µm.
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