Introduction

Exocytosis is a fundamental membrane trafficking event in eukaryotic cells in which membrane proteins or lipids are incorporated into the plasma membrane and vesicle contents are secreted to the exterior of the cell. It is essential for cells to absorb nutrients, communicate with the environment and each other, generate and maintain functional shapes1. The precise temporal and spatial regulation of polarised exocytosis is important for diverse biological events such as the establishment of a new bud in yeast2,3, epithelial cell polarisation4 and neuron development5. Polarised exocytosis takes place in several steps, which are evolutionarily conserved in eukaryotes. First, post-Golgi vesicles are transported by the type V myosin, Myo2, along polarised actin cables to sites of cell surface growth. Second, the vesicles are tethered at specific sites of the plasma membrane6,7. Finally, the vesicle membrane fuses with the plasma membrane in a process mediated by interactions between the SNARE family proteins located both on the vesicles and at the target membrane8.

The tethering of the secretory vesicles at the plasma membrane is mediated by the exocyst9,10. The exocyst is an evolutionarily conserved protein complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. The exocyst components are localised at the bud tip of small-budded cells or mother-daughter cell junction (bud neck) of large-budded cells, the sites of plasma membrane where active exocytosis and membrane expansion take place11,12,13,14. The exocyst has been shown to be involved in diverse cellular processes requiring polarised exocytosis such as yeast budding, epithelial polarity establishment, neurite outgrowth and ciliogenesis15,16,17,18. However, the mechanism of the exocyst regulating yeast budding has not been fully elucidated.

C. albicans is a multimorphic opportunistic human fungal pathogen19. In recent years, the role of exocyst components in the morphogenesis especially in the hyphal morphogenesis has been studied extensively in C. albicans20,21. Most recently, C. albicans Sec15 and Sec6 have been shown to play an important role in the delivery of cell wall components and hyphal branching22,23. In the budding yeast Saccharomyces cerevisiae, Sec15 has been shown to interact genetically as well as physically with activated Sec4, one of the founding members of the small rab GTPase family and a master regulator of post-Golgi vesicle trafficking14,24. Sec15 has also been shown to interact with the polarity establishment component Bem1 in yeast25. In Drosophila, Sec15 has been identified as a synthetic suppressor of oncogenic Ras by interacting with Eiger/TNF26. Here we show that C. albicans Sec15 interacts both physically and genetically with the Ras-family GTPase Rsr1, a master regulator of the bud site selection system, thus providing a direct link between the bud site selection system and exocytosis. We also show that Sec15 directly associates with the polarity determinant Bem1 and the unconventional myosin motor myosin V, Myo2. Therefore, Sec15 may play a central role in the polarised growth and exocytosis in C. albicans.

Results

Sec15 is required for normal cell growth and the maintenance of hyphal extension

Because SEC15 is essential for viability in S. cerevisiae, we generated a C. albicans SEC15 conditional mutant by deleting one copy of SEC15 and placing the other copy under the control of MAL2 promoter which can be shut off when grown in medium containing glucose. Strain construction was verified by colony PCR. The sec15 shut off cells grown under repressive conditions showed slow growth and significant morphological defects compared with the SEC15 on cells grown under permissive conditions. First, the sec15 shut off cells formed much smaller colonies than the SEC15 on cells after 3 days of growth on agar medium (Fig. 1A). Furthermore, the doubling time of the sec15 shut off cells was almost 3 times longer of the doubling time of the SEC15 on cells (Fig. 1B). These results indicate that the sec15 shut off cells grew much more slowly than the SEC15 on cells. Second, the sec15 shut off cells were much larger than the SEC15 on cells. Cell wall staining with Calcofluor White revealed that the sec15 shut off cells showed random budding pattern in contrast to the bipolar budding pattern of the SEC15 on cells (Fig. 1C). Furthermore, the sec15 shut off cells showed cell separation defect (Fig. 1C). Third, the sec15 shut off cells showed severe hyphal growth defects. More than 20 percent of the sec15 shut off cells were not able to generate germ tubes under hyphal inducing condition (100 cells were scored each for three repeats). While most of the sec15 shut off cells were able to generate a germ tube during the initial phase of hyphal development, they were not able to maintain the hyphal extension in prolonged growth. As a result, the sec15 shut off cells produced much shorter hyphae than the SEC15 on cells (Fig. 1D).

Figure 1
figure 1

C. albicans sec15 shut off mutant cells showed morphological defect.

(A) SEC15 shut off strain (strain AHU2) was first grown at 30 °C in maltose medium overnight, then diluted and equally inoculated onto maltose or glucose agar medium and incubated at 30 °C for 3 days. (B) The same overnight culture was diluted into liquid maltose or glucose medium and grown at 30 °C with vigorous shaking. Growth was monitored by measuring OD600 at intervals. Doubling times were calculated from the average OD values during the exponential phase of growth from three independent experiments. Data were means ± SD from three independent experiments. (C) SEC15 shut off strain (strain AHU2) was first grown at 30 °C in maltose and glucose medium overnight respectively. The overnight cultures were diluted into fresh liquid maltose or glucose medium and grown at 30 °C for further 3 hours. An aliquot of cells was then stained by Calcofluor White. Bars, 5 μm. (D) The same overnight cultures as above were diluted into liquid maltose or glucose medium plus 10% serum and grown at 37 °C for 3 hours. Bars, 5 μm.

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To get more information about the function of SEC15, we then examined the localisation of Sec15-GFP. The Sec15-GFP fusion protein was fully functional as reported previously27. Results by time-lapse microscopy showed that Sec15-GFP localisation could only be observed in two phases of the cell cycle: it localised to bud tips in the small-budded cells and at the bud neck in cells undergoing cytokinesis (Fig. 2). These results are consistent with the typical localisation pattern of exocyst proteins in S. cerevisiae1,7.

Figure 2
figure 2

Sec15-GFP shows cell cycle-dependent localisation in C. albicans.

Cells expressing Sec15-GFP (strain WYL41) were examined by time-lapse microscopy at intervals of 10 mins. Bars, 5 μm.

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Sec15 interacts with Rsr1-GDP in vitro and in vivo

An interaction between Rsr1-GDP (but not Rsr1-GTP) and Sec15 has been detected by a large scale two-hybrid study in S. cerevisiae28. To verify whether there is any physical interaction between Rsr1-GDP and Sec15 in C. albicans, in vitro binding assays were carried out. To this end, a recombinant Rsr1 GDP-locked form GST-Rsr1K16N was constructed and purified from E. coli by glutathione-Sepharose affinity chromatography. Indeed, GST-Rsr1K16N was found to bind with the N-terminal regions of Sec15, His-Sec151–300 (amino acids 1 to 300, expected size about 45 kDa) and His-Sec151–549 (amino acids 1 to 549, expected size about 74 kDa)(Fig. 3A). In contrast, the C-terminal region His-Sec15550–892 (amino acids 550 to 892) did not bind with GST-Rsr1K16N. These results indicate that Rsr1-GDP physically interacts with the N-terminal region of Sec15 in vitro.

Figure 3
figure 3

Sec15 binds to Rsr1-GDP in vitro and in vivo.

(A) Recombinant GST-Rsr1K16N purified from E. coli was pre-immobilized on glutathione beads and mixed with equal amounts of 20 μg of purified His6-Sec151–549, His6-Sec151–300 and His6-Sec15550–892. Bound GST fusion proteins were detected by Coomassie brilliant blue (CBB) staining and associated His6-tagged proteins were detected with anti-His antibody. (B) Cell lysates (CL) were prepared from C. albicans cells co-expressing Sec15-GFP and Rsr1K16N-Myc or Rsr1K16N-Myc alone, subjected to anti-GFP immunoprecipitation (IP) followed by anti-Myc western blot (WB). (C) Cell lysates were prepared from C. albicans cells co-expressing Sec15-GFP and Rsr1K16N-Myc or Sec15-GFP alone, subjected to anti-Myc immunoprecipitation followed by anti-GFP western blot.

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To further verify whether Rsr1-GDP interacts with Sec15 in vivo, a C. albicans strain co-expressing Sec15-GFP and Rsr1K16N-Myc and strains expressing Sec15-GFP or Rsr1K16N-Myc alone were constructed respectively. When Sec15-GFP was pulled down from the cell lysate using anti-GFP antibody, Rsr1K16N-Myc was detected in the precipitate of the cells co-expressing Sec15-GFP and Rsr1K16N-Myc but was not detected in the precipitate of the cells expressing Rsr1K16N-Myc alone (Fig. 3B). Reciprocally, when Rsr1K16N-Myc was pulled down from the cell lysate using anti-Myc antibody, Sec15-GFP was detected in the precipitate of the cells co-expressing Sec15-GFP and Rsr1K16N-Myc but was not detected in the precipitate of the cells expressing Sec15-GFP alone. These results demonstrate physical association of Rsr1-GDP with Sec15 in vivo in C. albicans.

SEC15 genetically interacts with RSR1

Taken together, the above results indicate that Sec15 interacts with Rsr1-GDP both in vitro and in vivo. To determine whether there is any genetic interaction between RSR1 and SEC15, Sec15-GFP localisation was examined in the rsr1Δ null mutant. In a wild type background, Sec15-GFP localises to the bud tip of small-budded cells and the bud neck of large-budded cells (Fig. 2). However, Sec15-GFP signal was not observed either at the bud tip or the bud neck in the rsr1Δ null mutant cells (Fig. 4, top). Instead, randomly localised Sec15-GFP spots were observed at the cell cortex in the small bud in a fraction of rsr1Δ null mutant cells. These results indicate that the polarised localisation of Sec15-GFP was completely abolished in the rsr1Δ mutant cells. These results indicate that the polarised localisation of Sec15-GFP depends on Rsr1. Taken together, both the functional and physical interactions between Sec15 and Rsr1 strongly support the idea that Rsr1 plays an important role in determining the localisation of the exocyst proteins to the site of bud growth.

Figure 4
figure 4

Exocyst subunits showed localisation defects in rsr1Δ cells.

Left panel, cells expressing exocyst-GFP in wild type background and rsr1Δ mutant background were grown to early log phase in GMM at 30 °C. Cells were harvested and examined by fluorescence microscopy. Bars, 5 μm. Right panel, the percentage of cells with polarised GFP signal in small-budded and medium/large-budded cells was quantified. Error bars indicate SD for three separate experiments. For each experiment, 200–300 cells were scored for each strain.

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To determine whether other exocyst components also mislocalise in the rsr1Δ mutant cells, the localisation of all the other exocyst components except Sec8 (whose GFP-tagging was not successful) was examined. All the GFP fusion proteins were functional as shown previously21. In a wild type background, all these exocyst components showed similar localisation pattern to Sec15, i.e. they localise to the bud tips of small-budded cells and the bud-neck of large-budded cells undergoing cytokinesis (Fig. 4). However, all these exocyst components showed localisation defects in the rsr1Δ mutant cells, although to a lesser extent compared with Sec15. Similar to Sec15, the bud tip localisation in the small-budded cells was almost completely abolished for all the exocyst components except Sec3, which still showed bud tip localisation in about 30% of the rsr1Δ mutant cells. Contrary to Sec15, the bud neck localisation in the large-budded cells could be observed in more than 20% of the rsr1Δ null mutant cells for all the other exocyst components except Exo84, which showed no bud neck localisation in the rsr1Δ mutant cells. However, compared with wild type background cells, the bud neck localisation for all the exocyst components showed significant defects in the rsr1Δ mutant cells in the following two aspects: first, the number of the rsr1Δ mutant cells showing bud neck localisation was much less than that of wild type background cells; second, the GFP signal at the bud neck in the rsr1Δ mutant cells was much weaker than that in the wild type background cells.

Next we examined the exocyst localisation under hyphal growth condition. In a wild type background, Sec15-GFP localised to the hyphal tip at 1 h of hyphal induction (Fig. 5A). At 3 h of hyphal induction, both hyphal tip and septum localisation of Sec15-GFP could be observed. In contrast, hyphal tip localisation of Sec15-GFP was completely abolished in rsr1Δ mutant cells at both 1 h and 3 h of hyphal induction, although septum localisation of Sec15-GFP could be observed at 3 h of hyphal induction. Similar localisation pattern was observed for all the other exocyst subunits except Sec3 (Fig. 5B and supplemental Fig. S1–5). Contrary to Sec15-GFP, hyphal tip localisation of Sec3-GFP could clearly be observed in rsr1Δ mutant cells at both 1 h and 3 h of hyphal induction.

Figure 5
figure 5

Sec15-GFP was mislocalised in rsr1Δ mutant hyphae.

(A) cells expressing Sec15-GFP in wild type and rsr1Δ mutant were grown overnight in GMM at 30 °C. Cells were then diluted into fresh GMM medium plus 10% serum and incubated at 37 °C for the indicated time period. Bars, 5 μm. (B) cells expressing Sec3-GFP in wild type and rsr1Δ mutant were grown overnight in GMM at 30 °C. Cells were then diluted into fresh GMM medium plus 10% serum and incubated at 37 °C for the indicated time period. Bars, 5 μm.

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To summarize, all the exocyst components examined showed severe localisation defects in the rsr1Δ mutant cells. First, the bud tip and bud neck localisation of Sec15 and Exo84 was completely abolished in the rsr1Δ yeast cells. Second, the bud tip localisation of Sec3, Sec5, Sec6, Sec10 and Exo70 was abolished in most of the rsr1Δ yeast cells while the bud neck localisation of Sec3, Sec5, Sec6, Sec10 and Exo70 was abolished in more than 40% of the rsr1Δ yeast cells. Third, Sec3, Sec5, Sec6, Exo70 and Exo84 mislocalised to cytoplasm in most of the rsr1Δ yeast cells. Fourth, the hyphal tip localisation of all the exocyst subunits except Sec3 was abolished in the rsr1Δ cells under hyphal inducing condition. These results support the idea that Rsr1 plays a role in the positioning of the exocyst complex.

Sec15 interacts with the polarity determinant Bem1

A direct physical interaction between Sec15 and Bem1 has been demonstrated in S. cerevisiae25. To determine whether similar interaction exists in C. albicans, in vitro binding assays between the C-terminal 343 amino acids of Sec15 and Bem1 was performed. Indeed, GST-Sec15550–892 was found to bind with the N-terminal 300 amino acids of Bem1, His-Bem11–300 (expected size 43.5 kDa). In contrast, GST-Sec15550–892 did not bind with the C-terminal region of Bem1, His-Bem1301–632 (Fig. 6A). These results are consistent with the results in S. cerevisiae25. Therefore, C. albicans Sec15 associates with Bem1 in vitro.

Figure 6
figure 6

Sec15 binds to Bem1 in vitro and in vivo.

(A) Recombinant GST-Sec15 C-terminal (amino acid 550–892) purified from E. coli was pre-immobilized on glutathione beads and mixed with equal amounts of 20 μg of purified His6-Bem11–300 and His6-Bem1301–632. Bound GST fusion proteins were detected by CBB staining and associated His6-tagged proteins were detected with anti-His antibody. (B) Cell lysates (CL) were prepared from C. albicans cells co-expressing Sec15p-GFP and Bem1-HA or Bem1-HA alone, subjected to anti-GFP immunoprecipitation (IP) followed by anti-HA western blot (WB). (C) Cell lysates were prepared from C. albicans cells co-expressing Sec15-GFP and Bem1-HA or Sec15p-GFP alone, subjected to anti-HA immunoprecipitation followed by anti-GFP western blot. (D) Left panel, Bem1-GFP localisation was examined in SEC15 shut off strain (AHU19) after 3h of re-culture in maltose or glucose medium diluted from overnight culture. Bars, 5 μm. Right panel, the percentage of cells with polarised GFP signal in small-budded and medium/large-budded cells was quantified. Error bars indicate SD for three separate experiments. For each experiment, 200–300 cells were scored for each strain. Bars, 5 μm. (E) Bem1-GFP localisation was examined in SEC15 shut off strain under hyphal growth condition. Bars, 5 μm.

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To verify whether Sec15 interacts with Bem1 in vivo, a C. albicans strain co-expressing Sec15-GFP and Bem1-HA and strains expressing Sec15-GFP or Bem1-HA alone were constructed respectively. When Sec15-GFP was pulled down from the cell lysate using anti-GFP antibody, Bem1-HA was detected in the precipitate of the cells co-expressing Sec15-GFP and Bem1-HA but was not detected in the precipitate of the cells expressing Bem1-HA alone (Fig. 6B). Reciprocally, when Bem1-HA was pulled down from the cell lysate using anti-HA antibody, Sec15-GFP was detected in the precipitate of the cells co-expressing Sec15-GFP and Bem1-HA but was not detected in the precipitate of the cells expressing Sec15-GFP alone (Fig. 6C). These results are consistent with physical interaction of Sec15 with Bem1 in vivo in C. albicans.

To determine whether there is any genetic interaction between Sec15 and Bem1, Bem1-GFP localisation was examined in the SEC15 shut off strain. When SEC15 was on, Bem1-GFP localised to the bud tips of small-budded cells and the bud-neck of large-budded cells undergoing cytokinesis as in wild type cells (Fig. 6D). These results are consistent with Bem1 localisation in S. cerevisiae25. However, when SEC15 was shut off, the bud neck localisation of Bem1-GFP was lost in most of the large-budded cells although most of the small-budded cells still showed bud tip localisation of Bem1-GFP (Fig. 6D). Furthermore, Bem1-GFP mislocalised to the cytoplasm of the mother cells in most of the SEC15 shut off cells.

Under hyphal inducing conditions, Bem1-GFP localised to the hyphal tip when SEC15 was on (Fig. 6E, left panel). When SEC15 was shut off, hyphal tip localisation of Bem1-GFP could be observed at 1 h of hyphal induction. However, hyphal tip localisation of Bem1-GFP was completely abolished in SEC15 shut off cells at 3 h of hyphal induction (Fig. 6E, right panel). These results clearly show a severe localisation defect for Bem1-GFP in SEC15 shut off cells, indicating a strong genetic interaction between Sec15 and Bem1 in C. albicans.

Sec15 interacts with Myo2 both in vitro and in vivo

It has been reported that Sec15 interacts directly with Myo2 in S. cerevisiae29. To determine whether similar interaction exists in C. albicans, in vitro binding assay between the C-terminal 343 amino acids of Sec15 (His-Sec15550–892) and the cargo-binding domain (CBD) of Myo2 was performed. Indeed, recombinant GST-Myo2-CBD strongly pulled down recombinant His-Sec15550–892 (Fig. 7A). In contrast, recombinant GST only did not pull down His-Sec15550–892. These results indicate that the C-terminal of Sec15 interacts directly with the CBD of Myo2 in vitro. This result is consistent with the result reported in S. cerevisiae29.

Figure 7
figure 7

Sec15 binds to Myo2 in vitro and in vivo.

(A) Recombinant GST-Myo2-CBD or GST alone purified from E. coli was pre-immobilized on glutathione beads and mixed with equal amounts of 20 μg of purified His6-Sec15550–892. Bound GST fusion proteins were detected by CBB staining and associated His6-tagged proteins were detected with anti-His antibody. (B) Cell lysates (CL) were prepared from C. albicans cells co-expressing Sec15-GFP and Myo2-Myc or Sec15-GFP alone, subjected to anti-Myc IP followed by anti-GFP western blot. (C) Cell lysates were prepared from C. albicans cells co-expressing Sec15-GFP and Myo2-Myc or Myo2-myc alone, subjected to anti-GFP IP followed by anti-Myc western blot.

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To test whether Sec15 interacts with Myo2 in vivo, a C. albicans strain co-expressing Sec15-GFP and Myo2-Myc and strains expressing Sec15-GFP or Myo2-Myc alone were constructed respectively. When Sec15-GFP was pulled down from the cell lysate using anti-GFP antibody, Myo2-Myc was detected in the precipitate of the cells co-expressing Sec15-GFP and Myo2-Myc but was not detected in the precipitate of the cells expressing Myo2-Myc alone (Fig. 7B). Reciprocally, when Myo2-Myc was pulled down from the cell lysate using anti-Myc antibody, Sec15-GFP was detected in the precipitate of the cells co-expressing Sec15-GFP and Myo2-Myc but was not detected in the precipitate of the cells expressing Sec15-GFP alone (Fig. 7C). These results indicate that Sec15 physically interacted with Myo2 in vivo in C. albicans.

Discussion

The exocyst plays a crucial role in the targeting of secretory vesicles to the plasma membrane during exocytosis. It has been shown to be involved in diverse cellular processes including yeast budding2,3. However, the molecular mechanism of exocyst regulating yeast budding remains unknown. Here we present several lines of evidence supporting a direct interaction between the exocyst component Sec15 and bud site selection determinant Rsr1 in C. albicans. First, in vitro binding assays with recombinant Rsr1K16N and fragments of Sec15 indicate that the N-terminal region of Sec15 directly binds to Rsr1K16N in vitro. Second, co-immunoprecipitation experiments with C. albicans cells co-expressing Sec15-GFP and Myc-Rsr1K16N indicate that Sec15-GFP associates with Myc-Rsr1K16N in vivo. Third, a complete loss of polarised localisation of Sec15-GFP was observed in rsr1Δ null mutant cells under both yeast and hyphal growth conditions. In contrast, polarised localisation of polarity establishment protein Bem1 was observed in both germ tubes and mature hyphae of rsr1Δ null mutant cells30. These results indicate that the loss of polarised localisation in rsr1Δ null mutant is specific to the exocyst. Last, the SEC15 shut off cells showed a random budding pattern in contrast to the bipolar budding pattern of wild type cells. The fact that another exocyst component, the sec3Δ null mutant cells showed normal bipolar budding pattern27 suggests that the random budding pattern is specific to SEC15 shut off cells. Taken together, these results strongly support that Sec15 interacts with Rsr1 both physically and genetically. Rsr1 is a master regulator of the bud-site-selection system in S. cerevisiae31. Thus the direct interaction between Sec15 and Rsr1 indicates that Sec15 may provide an important functional link between the bud-site-selection system and the secretory machinery.

Furthermore, several lines of evidence have been obtained in this study indicating that Sec15 directly associates with the polarity determinant Bem1 in C. albicans. Similar results have been reported in the budding yeast S. cerevisiae25. Bem1 is a scaffold protein that is required to maintain the Cdc42 module at the incipient budding site and is therefore essential for the proper function of the Cdc42 signaling cascade which leads to the initiation and stabilization of cell polarity in S. cerevisiae32. The interaction between Sec15 and Bem1 may provide a tight functional link between the secretory machinery and the polarity establishment machinery. Another functional link between the secretory machinery and the polarity establishment machinery has been suggested by the detection of association between the exocyst subunit Sec3 and Cdc42 in S. cerevisiae33. Our previous study has shown that GFP-Cdc42 showed no significant localisation defect in sec3Δ null mutant cells under normal growth temperature of 30 °C in C. albicans27, while Bem1-GFP showed severe localisation defect in the SEC15 shut off cells under similar growth conditions (Fig. 6D). These results indicate that Sec15, rather than Sec3, might play a major role in linking the secretory machinery and the polarity establishment machinery in C. albicans.

A recent study of the role of Sec15 in C. albicans hyphal formation by Chavez-Dozal et al.23 shows that the Spitzenkörper marker Mlc1-GFP, polarisome Spa2-GFP and exocyst subunit Exo70-GFP are mislocalised in strain tetR-SEC15 under repressive conditions. Consistent with these results, polarity establishment protein Bem1 is mislocalised in the MAL2-SEC15 conditional mutant hyphae grown under repressive conditions (Fig. 6E). It is noteworthy that Chavez-Dozal and co-workers used the more strictly regulated TetR promoter, other than the leakier MAL2 promoter, for conditional expression of SEC15 and showed that SEC15 was absolutely required for viability (cell death occurred within 5 hours of SEC15 shut off). Interestingly, all of Mlc1-GFP, Spa2-GFP and Exo70-GFP show no localisation defect in strain tetR-SEC6 under repressive conditions23. These results further support that Sec15 might play a critical role in linking the secretory machinery and the polarity establishment machinery in C. albicans.

In yeast S. cerevisiae, the unconventional myosin motor myosin V, Myo2, transports secretory vesicles to sites of exocytosis34. The C-terminal cargo-binding domain of Myo2 plays a critical role in the attachment of Myo2 to cargoes. In this study we find that the C-terminal region of Sec15 binds directly to the CBD of Myo2 in C. albicans, consistent with a previous study in yeast29. Yeast Sec15 also binds to the rab GTPase Sec4, providing a physical link between activated Sec4 on the vesicle surface and the other subunits of the exocyst complex14. Therefore, Sec15 plays a crucial role both in the recruitment of exocyst complex to the surface of secretory vesicles through interaction with Sec4 and in the transport of exocyst/secretory vesicles to sites of exocytosis by direct interaction with Myo2.

Taken together, Sec15 provides multiple functional links via interaction with various proteins (Fig. 8). First, Sec15 provides a functional link between the bud-site-selection system and the secretory machinery by interacting with the Ras-family GTPase Rsr1. Second, Sec15 provides a functional link between the polarity establishment machinery and the secretory machinery by interacting with polarity determinant Bem1. Third, Sec15 provides a functional link between secretory vesicles and the exocyst complex by interacting with type V myosin Myo2 and Rab GTPase Sec4. These findings suggest that Sec15 play an important role in the polarised cell growth in C. albicans.

Figure 8
figure 8

Schematic representation of Sec15 interactions.

Interactions identified in both S. cerevisiae and C. albicans are indicated by solid lines. Interactions identified only in S. cerevisiae are indicated by dashed lines.

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Methods

Strains, media and growth conditions

C. albicans strains used in this study are listed in Table 1. The strains were grown in either YPD (1% [w/v] yeast extract, 2% [w/v] peptone, 2% [w/v] glucose) or GMM medium (2% [w/v] glucose, 0.67% [w/v] yeast nitrogen base without amino acids). For C. albicans yeast growth, cells were grown at 30 °C. For hyphal growth, the media were supplemented with 10% serum and incubated at 37 °C. G1 cells were prepared by centrifugal elutriation as previously described35.

Table 1 Candida albicans strains used in this study.
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Gene deletion

Gene deletion mutants were constructed by sequentially deleting the two copies of a gene from BWP17. For the deletion of RSR1 gene, 500 bp of the 5′ UTR (AB) and 500 bp of the 3′ UTR (CD) of RSR1 were sequentially cloned into the plasmid pGEM T-easy Vector (Promega, USA). Then the HIS1 and ARG4 gene sequence were inserted between AB and CD by a BamHI site respectively. These plasmids were used as templates for PCR amplification of RSR1 gene deletion construct. To construct SEC15 shut off strain controlled by the MAL2 promoter, a URA3 flipper cassette was constructed by flanking the 4.2 kb URA3 flipper36 with the AB and CD DNA fragments corresponding to the 5′ and 3′ UTR of the SEC15. The URA3 flipper cassette was used to disrupt one copy of SEC15 from BWP17 as described previously36. The second copy of SEC15 was placed under the control of MAL2 promoter as described previously37. All strain genotypes were verified by colony PCR.

Epitope tagging of proteins

To generate Exo84-GFP fusion protein under the control of its endogenous promoter, the C-terminal 600 bp of EXO84 (orf19.135) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned in frame at the KpnI/XhoI sites of the plasmid pGFPutr38. The resulting plasmid was linearized by EcoRV at nt 2213 for chromosomal integration. To construct GFP-Exo70 fusion protein under the control of MET3 promoter, the full length coding sequence of EXO70 (orf19.6512) together with 500 bp of its 3′ UTR sequence was amplified by PCR with ClaI and PstI sites added to 5′ and 3′ respectively, cleaved with ClaI and PstI and cloned in frame at the ClaI and PstI sites of the plasmid pMET3-GFP-GAL4UTR39. The resulting plasmid was linearized by HindIII at a unique HindIII site in the 3′ UTR for chromosomal integration. To construct Sec5-GFP fusion protein under the control of its endogenous promoter, the C-terminal 2300 bp of SEC5 (orf19.74) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned in frame at the KpnI/XhoI sites of the plasmid pGFPutr. The resulting plasmid was linearized by EcoRV at nt 742 for chromosomal integration. To construct Sec6-GFP fusion protein under the control of its endogenous promoter, the C-terminal 800 bp of SEC6 (orf19.5463) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned in frame at the KpnI/XhoI sites of the plasmid pGFPutr. The resulting plasmid was linearized by HindIII at nt 1909 for chromosomal integration. To construct Sec10-GFP fusion protein under the control of its endogenous promoter, the full length coding sequence of SEC10 (orf19.3086) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned in frame at the KpnI/XhoI sites of the plasmid pGFPutr. The resulting plasmid was linearized by NcoI at nt 453 for chromosomal integration. To construct Bem1-GFP fusion protein under the control of its endogenous promoter, the C-terminal 1500 bp of BEM1 (orf19.4645) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned in frame at the KpnI/XhoI sites of the plasmid pGFPutr. The resulting plasmid was linearized by HindIII at nt 755 for chromosomal integration.

To construct RSR1K16N- MYC-ARG4 fusion protein under the control of its endogenous promoter, the promoter sequence (1200 bp) together with the coding sequence of RSR1 was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned at the KpnI/XhoI sites of the plasmid pMET3-6Myc-GAL4UTR39 to replace the MET3 promoter, resulting plasmid pRSR1-6Myc. The plasmid pRSR1K16N-6Myc was generated using Quick Change Site-directed Mutagenesis Kit (Stratagene). Finally, plasmid pRSR1K16N-6Myc-ARG4 was generated by replacing the URA3 gene sequence with ARG4 gene sequence using NotI and MluI sites. This plasmid was linearized by EcoRV at nt -921 in the promoter region for chromosomal integration. To construct Myo2- MYC-ARG4 fusion protein under the control of its endogenous promoter, the C-terminal 686 bp of MYO2 coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned at the KpnI/XhoI sites of the plasmid pRSR1K16N -6Myc -ARG4 to replace RSR1 sequence. The resulting plasmid was linearized by BamHI at nt 4246 for chromosomal integration. To construct Bem1-HA fusion protein under the control of its endogenous promoter, the C-terminal 1500 bp of BEM1 (orf19.4645) coding sequence was amplified by PCR with KpnI and XhoI sites added to 5′ and 3′ respectively, cleaved with KpnI and XhoI and cloned at the KpnI/XhoI sites of the plasmid pMET3-6HA-GAL4UTR30 to replace the MET3 promoter. The resulting plasmid was linearized by HindIII at nt 755 for chromosomal integration.

In vitro binding assay

For the binding assay between Sec15 and Rsr1, the full length coding sequence of Rsr1 was amplified from a cDNA library and fused to GST (GST-Rsr1) using the pGEX vector system (Amersham Biosciences). The GDP-locked form of Rsr1, GST-Rsr1K16N was generated using Quick Change Site-directed Mutagenesis Kit (Stratagene). Three different fragments of Sec15 were amplified and subcloned into pET21-a (Novagen) to generate His-Sec151–300 (amino acids 1 to 300), His-Sec151–549 (amino acids 1 to 549) and His-Sec15550–892 (amino acids 550 to 892). The fusion proteins were purified from Escherichia coli according to the manufacturer’s protocol and used for in vitro binding assays. For a typical binding experiment, the GST-Rsr1K16N fusion protein immobilized to glutathione-Sepharose beads (20 μl of beads) was incubated with 20 μg of purified His-Sec151–300, His-Sec151–549 and His-Sec15550–892 respectively in binding buffer (1× PBS buffer containing 1 mg/ml ovalbumin, 10 mM β-mercaptoethanol and 0.1% IGPAL-30) for 1 hour at room temperature. The total volume of incubation mixture was 200 μl. After the resin was washed four times with the binding buffer excluding ovalbumin, bound GST fusion proteins were detected by Coomassie brilliant blue staining and associated His-tagged proteins were detected with anti-His antibody. Similar strategies were used for in vitro binding assays between Sec15 and Bem1 or Myo2.

Co-immunoprecipitation

A culture of 20 ml was grown overnight in GMM. The culture was diluted to 200 ml the next day and grown for another 2 h. Cells were harvested and lysed in 1.5 volumes of HEPES lysis buffer (50 mM Hepes, pH 7.5, 1 mM EDTA, 5% glycerol, 0.05% Tween 20, 105 mM KCl) with complete protease inhibitor mix (Roche). Cells were broken by five rounds of 45-second beating at 5000 rpm in Tomy Micro SmashTM MS-100 (Tomy Medico, Japan) with 1 minute incubation on ice between rounds. Cell lysate was clarified by spinning at 10,000 g twice. Antibody-conjugated beads (sc-8334 AC for GFP, sc-40 AC for Myc and sc-7392 AC for HA, Santa Cruz Biotechnology) were washed twice with 500 μl of lysis buffer and mixed with the clarified lysate and incubated at 4 °C for 2 h. After four times of wash with lysis buffer, the beads were boiled for 5 minutes with 1X loading buffer. Proteins were separated by SDS-PAGE and probed by Western Blot using appropriate antibodies (sc-8334 for GFP, sc-40 for Myc and sc-7392 for HA, Santa Cruz Biotechnology).

Cell wall staining and fluorescence microscopy

Cell wall staining and fluorescence microscopy were performed as described by Pringle and co-workers40. A Leica DMR fluorescence microscope with 100× objective and a Hamamatsu digital camera interfaced with METAMORPH software (Universal Imaging) were used for imaging.

Additional Information

How to cite this article: Guo, P. P. et al. Sec15 links bud site selection to polarised cell growth and exocytosis in Candida albicans. Sci. Rep. 6, 26464; doi: 10.1038/srep26464 (2016).