Transformations in C. albicans and S. cerevisiae.

The strains used in this study are listed in Table ​Table2.2. Transformation of C. albicans and S. cerevisiae strains was done by the lithium acetate method (21). To create a mutant in which both copies of the CaMYO5 gene were deleted, the Ura⁻ strain CAI4 (15) and the Ura⁻ His⁻ strain RM1000 (39) were transformed with pU21 digested with KpnI and SacI. Genomic DNA was prepared from Ura⁺ transformants, digested with SpeI, and analyzed for the correct integration event by Southern blotting with a digoxigenin system (Boehringer Mannheim). Positive transformants derived from strain CAI4 were plated on medium containing 5-fluoroorotic acid (Diagnostics Chemicals Ltd., Charlottetown, Prince Edward Island, Canada), a drug which inhibits the growth of Ura⁺ strains. Ura⁻ strains thus obtained were analyzed similarly for the loop-out event that occurs by homologous recombination between the hisG direct repeats flanking the URA3 gene. The CaMYO5/Camyo5::hisG strain was transformed with pU21 digested with KpnI and SacI. Ura⁺ transformants were analyzed similarly for the correct integration event resulting in the disruption of the second CaMYO5 allele with the hisG::URA3::hisG blaster. A positive Camyo5::hisG/Camyo5::hisG::URA3::hisG transformant, called COU42, was analyzed for the correct loop-out event by plating on 5-fluoroorotic acid as described above. In parallel, positive transformants derived from the disruption of the first allele of CaMYO5 in RM1000 were transformed with 5 μg of a PCR-amplified Camyo5::CaHIS1 cassette by using UO6 and UO36. Transformants that were Ura⁺ and His⁺ were screened by Southern blot analysis for the disruption of the second allele with the CaHIS1 marker as described above. Nineteen positive transformants were thus obtained that displayed phenotypes similar to that of COU42.

TABLE 2.

Strains

Strain	Genotype	Reference or source
C. albicans
SC5314	CaMYO5/CaMYO5 CaURA3/CaURA3	15
CAI4	ura3::1 imm434/ura3::1 imm434	15
RM1000	CAI4 Cahis1::hisG/Cahis1::hisG	39
COU13	CAI4 CaMYO5/Camyo5::hiG-URA3-hisG	This study
COU42	CAI4 Camyo5::hisG/Camyo5::hisG-URA3-hisG	This study
COU46	CAI4 Camyo5::hisG/Camyo5::hisG	This study
COU73	CAI4 Camyo5::hisG/Camyo5 (CaMYO5)	This study
COU186	CAI4 Camyo5::hisG/Camyo5 (CaMYO5-GFP)	This study
COU190	CAI4 Camyo5::hisG/Camyo5 (Camyo5S366A)	This study
COU201	CAI4 Camyo5::hisG/Camyo5 (Camyo5S366D)	This study
COU232	CAI4 Camyo5::hisG/Camyo5 (Camyo5S366D-GFP)	This study
COU243	CAI4 Camyo5::hisG/Camyo5 (Camyo5S366A-GFP)	This study
CLJ4	CAI4 Cacla4::hisG/Cacla4::hisG	28
S. cerevisiae HA31-9c	MATacan1-100 ade2-1 his3-11 leu2-3, 112 ura3-1 trp1-1 myo3::HIS3 myo5::TRP1	18

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The CaMYO5, CaMYO5^S366A, CaMYO5^S366D, CaMYO5-GFP, CaMYO5^S366A-GFP, and CaMYO5^S366D-GFP alleles were integrated into the Camyo5/Camyo5 Ura⁻ mutant. First, pU67, pU77, pU78, pU93, pU97, and pU98 were linearized with BglII and transformed by the lithium acetate method into COU46 (COU42 Ura⁻). The integration of each of these CaMYO5 alleles at the CaMYO5::hisG locus was verified by Southern blot analysis. The overexpressed CaMYO5^S366A and CaMYO5^S366D alleles were integrated into the Cacla4/Cacla4 Ura⁻ mutant (CLJ4) (28). For this purpose, 10 μg each of pU95 and pU96 was linearized with XhoI and transformed by the lithium acetate method into CLJ4. The integration of these alleles at the PCK1 locus was also verified by Southern blot analysis.

To determine functional complementation of the CaMYO5 gene in the S. cerevisiae myo3 myo5 mutant strain, pRS316/426, pVL62 (MYO5 in pRS316; a kind gift from Cunle Wu), pU47, and pU48 were transformed into HA31-9c (18). Single colonies were streaked on 1% yeast extract-2% peptone-2% d-glucose (YPD) plates and grown at either 30 or 37°C for 3 days.

Phenotypic analyses.

Media used for the hypha induction experiments were as follows: YPD supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL), 2% agar supplemented with 10% FBS, Spider medium (1% nutrient broth, 1% mannitol, 0.2% potassium phosphate [pH 7.2]), SLAHD medium (16), and Lee's medium (30).

Strains were grown in YPD overnight, and 30 μl of a 10⁻⁵ dilution was spread on one-sixth of a plate (agar-FBS, Spider, SLAHD, or Lee's medium). Plates were incubated for 4 days at 37°C. Colonies were visualized by using an inverted Nikon TMS microscope with a ×2 objective and a ×10 projection lens. Strains from the overnight cultures were also diluted to 1:20 in YPD supplemented with 10% fetal calf serum and were incubated for different times at 37°C. Cells were visualized by Nomarski optics by using an upright Leitz Aristoplan microscope with a ×40 objective or a ×100 immersion oil objective and a ×10 projection lens. For testing the phenotypes of the derived cla4 deletion strains, these and wild-type strain SC5314 were grown overnight in YPD or YPCa (1% yeast extract-2% peptone-2% Casamino Acids). Cultures were diluted to 1:20 in YPD or YPCa, respectively, supplemented with 10% FBS and were incubated for 3 h at 37°C.

Doubling times for each strain were determined starting from three independent colonies grown overnight in YPD and diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 at the beginning of the time course. The OD₆₀₀ values were determined every hour for up to 7 h.

Fluorescence.

Overnight cultures in YPD of the various strains were diluted to 1:20 in either YPD or YPD with 10% FBS and grown for 3 h at 30 or 37°C, respectively. For GFP fluorescence, cells were washed once in phosphate-buffered saline (PBS) and mounted on slides for visualization with the appropriate filter by epifluorescence microscopy by using an upright Leitz Aristoplan microscope with a ×100 immersion oil objective and a ×10 projection lens. For 4′,6′-diamino-2-phenylindole (DAPI; Sigma) and Calcofluor White (Sigma) staining, cells were fixed in 70% ethanol for 20 min and washed once with PBS. Cells were then stained for 5 to 10 min with either a 1:1,000 dilution of DAPI at 1 mg/ml or a 1:1,000 dilution of Calcofluor White at 1 mg/ml. Cells were washed extensively with PBS before visualization with the appropriate filter. For rhodamine-phalloidin (Molecular Probes, Eugene, Oreg.) staining, cells were fixed in medium with 3.7% formaldehyde (final concentration) for 30 min. After the cells were resuspended in 50 mM potassium phosphate buffer (pH 6.6) (PK buffer), they were fixed with 3.7% formaldehyde for 1 h. After one wash in PK buffer, cells were incubated for 30 min in PK buffer containing 0.1% Triton X-100. Cells were washed twice with PBS and incubated with a 1:10 dilution of rhodamine-phalloidin (200 U/ml) in PBS overnight at 4°C. After the supernatant was removed, cells were resuspended in PBS and visualized directly with the appropriate filter. DAPI staining was done at the last step with no further washing.

Video microscopy.

Overnight cultures of COU73, COU186, COU201, COU232, and SC5314 were diluted to1:1,600 in YPD supplemented with 10% FBS. Live video microscopy was done at 37°C by using a Leica DM-IRB inverted microscope equipped with a Ludl motorized stage, a temperature-controlled Δt culture dish system (Bioptechs), and a Sensys charge-coupled device camera. Pictures were taken with a ×40 objective and a ×10 projection lens every 15 min and in five different locations. Lengths of hyphae at different time points for more than 25 hyphae per strain were measured by using Openlab 3.1 software. The hyphal growth rate distribution for each strain was then determined. t tests were performed to determine the difference of means for COU73 versus COU201 and for COU186 versus COU232. The means were found to be significantly different in each set (P < 10⁻⁶).

Myosin I is not essential for viability in C. albicans.

To study the role of myosin I in C. albicans, we created a strain in which both copies of CaMYO5 were deleted (Δ/Δ Camyo5). Figure ​Figure11 shows the sequential disruption of both alleles of CaMYO5 in C. albicans (for details, see Materials and Methods). A single Δ/Δ Camyo5 transformant was obtained starting from strain CAI4 (15), and 19 Δ/Δ Camyo5 transformants were obtained starting from strain RM1000 (39). The latter strain is auxotrophic for histidine as well as uracil, enabling the disruption of each allele with a different marker (CaHIS1 and CaURA3). These 20 Δ/Δ Camyo5 mutants displayed similar phenotypes, as described below. The ability to generate viable double-mutant strains indicates that the myosin I gene is not an essential gene in C. albicans.

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FIG. 1.

Sequential disruption of both alleles of CaMYO5. Genomic DNA from strains was digested with SpeI and analyzed by Southern blotting with a probe encompassing the 5′ noncoding region of CaMYO5 (thick line). Lanes: 1,CAI4 (wild type; Ura⁻); 2, CaMYO5/Camyo5::hisG-URA3-hisG; 3, CaMYO5/Camyo5::hisG; 4, Camyo5::hisG-URA3-hisG/Camyo5::hisG; 5, Camyo5::hisG/Camyo5::hisG; M1, bands of 23, 9.4, 6.6, 4.4, 2.3, and 2.0 kb; M2, bands of 21, 5.1, 5.0, 4.3, 3.5, 2.0, 1.9, 1.6, 1.4, 0.9, and 0.8 kb.

Myosin I is required for the polarized distribution of cortical actin patches.

We examined cells of the Δ/Δ Camyo5 mutant and the wild-type strain (SC5314) microscopically for phenotypes that would indicate the role played by myosin I in budding yeast cells. Cells of the Δ/Δ Camyo5 mutant viewed by Nomarski optics looked abnormally round, in some instances enlarged, and were clumped compared to wild-type cells (Fig. ​(Fig.2).2). To determine whether cortical actin patches were mislocalized in the Δ/Δ Camyo5 mutant, we treated cells grown exponentially with rhodamine-phalloidin to stain filamentous actin. Localization of cortical actin patches in budding cells of S. cerevisiae and C. albicans has been described extensively (4, 44). In summary, cortical actin patches localize to the growing bud tip, redistribute evenly in an isotropic growing bud, and finally localize at the mother bud neck after cytokinesis. In our experiments, cortical actin patches in 69% of wild-type cells were entirely localized to the buds during early bud emergence and growth (Fig. ​(Fig.22 and Table ​Table3).3). These patches remained in the buds of 18% of cells during and after nuclear division. These patches also were localized at the mother bud neck in 10% of cells. Strikingly, for the Δ/Δ Camyo5 mutant, only 3% of cells retained cortical actin patches exclusively in the buds. In this mutant, cortical actin patches were localized evenly in both the mother cell and the buds for 44% of cells before nuclei had divided and in 31% of cells during nuclear division. Finally, in 7% of cells, the patches were localized at the mother bud neck while also localizing in daughter and mother cells. Overall, cortical actin patches are dramatically mislocalized in the Δ/Δ Camyo5 mutant, suggesting that myosin I plays a role in the organization of the actin cytoskeleton.

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FIG. 2.

Cortical actin patch patterns in wild-type and mutant cells. Cells were fixed and stained with rhodamine-phalloidin. Nuclear DNA was stained with DAPI. Scale bar, 10 μm.

TABLE 3.

Cortical actin patch patterns in yeast cells^a

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^aDAPI, nuclear staining; RP, rhodamine phalloidin, F-actin staining. Polarized in bud, cortical actin patches localize exclusively in bud; loose, cortical actin patches localize preferentially in bud but also in mother cell; depolarized, cortical actin patches localize equally in bud and mother cell; nuclear division, nuclei dividing; bud neck, cortical actin patches localize exclusively at neck; bud neck, loose, cortical actin patches localize at bud neck preferentially but also in daughter and mother cells; cytokinesis, nuclei have completed division, and mother and daughter cells are about the same size; G₁, no apparent budding. Scale bar, 1 μm.

Myosin I plays a role in chitin deposition in the cell wall and in the budding pattern.

To determine whether myosin I also plays a role in cell wall biogenesis in C. albicans, cells of the mutant and wild-type strains were labeled for chitin with Calcofluor White. In the mutant, a small proportion of cells that were unusually enlarged and round showed aberrant chitin deposition in the cell wall (Fig. ​(Fig.3).3). These findings were not observed in the wild-type strain. At the same time, we found that nearly 20% of mutant cells exhibited a random budding pattern; the value for wild-type cells was 2% (Table ​(Table4).4). Mutations in actin or other cytoskeletal proteins lead to a high frequency of random budding patterns in diploid S. cerevisiae cells (11, 60). The higher incidence of random budding in the Δ/Δ Camyo5 mutant than in the wild-type strain correlate with these data, suggesting that the actin-dependent mechanisms for bud site selection are similar in C. albicans and S. cerevisiae. In addition, C. albicans myosin I may be important for the regulation of cell wall biogenesis, based on the abnormal chitin deposition observed in the cell wall of the mutant.

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FIG. 3.

Budding patterns of wild-type and Camyo5/Camyo5 mutant strains. Cells were fixed and stained for chitin with Calcofluor White. (Panel a) Nomarski view. (Other panels) Calcofluor White staining. (a to c) SC5314. (d to f) reintegrant Δ/Δ (Camyo5) . Arrows indicate cells exhibiting random budding. In the large panel (Camyo5/Camyo5), the enlarged cell in the center shows abnormal chitin deposition in its cell wall. Scale bar, 4 μm.

TABLE 4.

Budding patterns^a

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^aCells with more than two buds and/or bud scars were counted. Scale bar, 1 μm.

Reintegration of wild-type myosin I in the null mutant leads to the recovery of wild-type characteristics.

To determine whether the phenotypes of the mutant strain are due to the deletion of both copies of myosin I, the wild-type CaMYO5 gene was reintegrated at one of the CaMYO5 loci in a Ura⁻ strain (see Materials and Methods). Six transformants or “reintegrant” strains were thus obtained, and all exhibited normal cell shape. One reintegrant (COU73) was selected for more detailed analysis. This reintegrant exhibited normal cell shape (Fig. ​(Fig.2),2), cortical actin patch distribution (Fig. ​(Fig.22 and Table ​Table3),3), and budding pattern (Fig. ​(Fig.33 and Table ​Table4).4). Thus, the deletion of myosin I alone is responsible for the phenotypes of the Δ/Δ Camyo5 strain.

Myosin I is required for the formation of hyphae on solid and in liquid media.

We examined the ability of the Δ/Δ Camyo5 mutant to form hyphae under different hypha-inducing conditions. The Δ/Δ Camyo5 and wild-type (SC5314) strains were plated for single colonies on agar containing 10% FBS, Spider medium, SLAHD medium, or Lee's medium and were grown for 4 to 5 days at 37°C. The wild-type strain could form extensive hyphae on all media (Fig. ​(Fig.4A4A and data not shown). In contrast, the Δ/Δ Camyo5 strain did not form hyphae on any medium (Fig. ​(Fig.4A4A and data not shown). Occasional extensions could be observed on parts of a Δ/Δ Camyo5 colony grown on agar containing 10% FBS, but microscopic examination showed that these were formed by pseudohyphal cells (data not shown). To confirm that the deletion of myosin I alone was the cause of this nonhyphal phenotype, we tested the ability of the reintegrant strain to form hyphae under these conditions. This strain could form hyphae to the same extent as wild-type strain SC5314 (Fig. ​(Fig.4A4A and data not shown). These results indicate that myosin I is required for hypha formation on solid hypha-inducing media.

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FIG. 4.

Hypha formation in wild-type and mutant strains. (A) Hypha formation on solid media was determined by plating single colonies of each strain on 10% FBS (a) or Spider medium (b) plates. Plates were incubated for 4 days at 37°C. Scale bar, 1 mm. (B) Hypha formation in liquid YPD supplemented with 10% FBS was determined at 37°C after 2.5 h of incubation. Scale bar, 10 μm.

To determine cellular morphology under hypha-inducing conditions, cells were also incubated in liquid YPD medium containing 10% FBS or in Lee's medium at 37°C. After 2 to 3 h of incubation, cells of the wild-type strain formed germ tubes that extended into hyphae (Fig. ​(Fig.4B4B and data not shown). In contrast, cells of the Δ/Δ Camyo5 strain occasionally formed germ tubes but were unable to form hyphae after more than 6 h of incubation in YPD medium containing serum or overnight incubation in Lee's medium, by which time the wild-type and reintegrant strains had formed extensive hyphae (data not shown). Pseudohyphal cells could be observed in the Δ/Δ Camyo5 mutant instead (Fig. ​(Fig.4B).4B). Because the Δ/Δ Camyo5 strain could form pseudohyphae but not hyphae, it is possible that myosin I is required for maintaining polarized growth but not for initiating it.

Myosin I localizes to the bud and hyphal tips and partially colocalizes with cortical actin patches.

To determine the localization of myosin I in C. albicans, we fused in frame the sequence encoding GFP (38) at the 3′ end of the CaMYO5 open reading frame. We then introduced CaMYO5-GFP into the Ura⁻ Δ/Δ Camyo5 strain, such that CaMyo5-GFP was the sole source of myosin I. Two independent transformants that were examined could form hyphae in the presence of serum. The CaMyo5-GFP protein in these transformants was detected by Western blot analysis as a band of 175 kDa (Fig. ​(Fig.5A).5A). These results suggest that CaMyo5-GFP functions properly in the Δ/Δ Camyo5 strain. Figure ​Figure5B5B shows the localization of CaMyo5-GFP in exponentially growing cells. CaMyo5-GFP clearly localized in patches at the tip of emerging buds. CaMyo5-GFP localization could also be observed at the mother bud neck after nuclear division was completed (Fig. ​(Fig.5B5B and data not shown). The localization of CaMyo5-GFP in hyphal cells was examined as well (Fig. ​(Fig.5C).5C). CaMyo5-GFP localized in patches at the hyphal tip. The patch-like distribution of CaMyo5-GFP in budding and hyphal cells was similar to that of actin in cortical patches. To determine whether CaMyo5-GFP colocalizes with actin, we stained yeast cells and hyphal cells with rhodamine-phalloidin (Fig. ​(Fig.6).6). In most of the cells examined (108 of 120), there was either very good colocalization or partial colocalization of CaMyo5-GFP and cortical actin patches (two or more spots coinciding) either at the bud or hyphal tips or at the mother bud neck or septae (Fig. ​(Fig.66 and data not shown).

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FIG. 5.

Localization of wild-type and mutant CaMyo5-GFP. (A) Protein extracts (50 μg) prepared from cells expressing CaMYO5-GFP (Wt), CaMYO5^S366D-GFP (S366D), or CaMYO5^S366A-GFP (S366A) or from SC5314 (−) grown in YPD to late exponential phase were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 6% acrylamide gel. GFP fusion proteins were analyzed by Western blotting. Molecular weight markers (MW; in thousands) are indicated on the right. (B and C) Cells growing exponentially (B) or induced with 10% FBS at 37°C for 3 h (C) were visualized for GFP fluorescence. One of the wild-type hyphal cells moved during exposure, leading to a double exposure. Scale bars, 10 μm.

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FIG. 6.

Overlay of rhodamine-phalloidin and GFP signals. Budding and hyphal cells expressing CaMyo5-GFP were fixed and stained with rhodamine-phalloidin (RP) and DAPI. Pictures were taken with the appropriate filters to visualize CaMyo5-GFP (GFP), actin (RP), and nuclei (a). Overlays of the images were created with Adobe Photoshop, version 6.0. Scale bar, 4 μm.

Serine 366 in myosin I is essential for normal growth and hypha formation.

In several organisms, the ATPase activity of myosin I is increased by phosphorylation of a conserved serine residue in the head domain (10, 31, 56). In S. cerevisiae, this phosphorylation is catalyzed by activated Ste20 and Cla4 kinases (57). Ste20 and Cla4 are members of the PAK family of protein kinases which may be activated by Cdc42p and function to regulate cell morphology (26, 27). To determine whether the phosphorylation of myosin I by C. albicans PAK kinases is important for hypha formation, we created mutant alleles of CaMYO5 that code for alanine or aspartate instead of serine in the unique PAK phosphorylation site (serine 366). These alleles were reintroduced into the Δ/Δ Camyo5 strain at the Camyo5::hisG loci (see Materials and Methods). Proper integration and expression of these alleles in several transformants were confirmed by Southern and Northern blot analyses, respectively (data not shown).

To determine whether Camyo5^S366D and Camyo5^S366A can rescue the phenotypes of the Δ/Δ Camyo5 strain, we measured the growth rates of strains carrying these alleles and characterized their budding patterns. We found that the wild-type strain and the strain expressing Camyo5^S366D (COU201) divided every 55 min at 37°C in 2× YPD (Table ​(Table5).5). In contrast, the Δ/Δ Camyo5 strain and the strain expressing Camyo5^S366A (COU190) divided every 69 min under the same growth conditions (Table ​(Table5).5). We also found that the S366A mutation increased random budding 10-fold, similar to the complete deletion of myosin I, while the S366D mutation increased random budding 5-fold (Table ​(Table4).4). Although there was no abnormal chitin deposition in cells expressing Camyo5^S366D, abnormal chitin staining could be observed in cells expressing Camyo5^S366A (data not shown). These results suggest that the phosphorylation of serine 366 is important for optimal growth and for cell wall biogenesis. Furthermore, phosphorylation of serine 366 may also be important, albeit to a lesser extent, for localization of the bud site. We also observed that the mutant expressing Camyo5^S366D did not form hyphal cells in YPD (Fig. ​(Fig.2),2), suggesting that other components of the actin cytoskeleton need to be activated to allow hypha formation under non-hypha-inducing conditions.

The mutant strains were plated for single colonies on agar containing 10% FBS and on Spider medium to determine their ability to form hyphae. All six transformants that expressed Camyo5^S366D formed fuzzy colonies similar to those of the wild-type and reintegrant strains (Fig. ​(Fig.4A).4A). Under liquid hypha-inducing conditions, these cells were able to form hyphae as well. However, the numbers and rates of growth of the hyphae formed were lower than those of the wild-type strain (Fig. ​(Fig.4B4B and Table ​Table6).6). The majority of seven transformants that expressed Camyo5^S366A formed fuzzy colonies with much shorter fringes than the wild-type and reintegrant strains on agar containing 10% FBS, but on the whole periphery of the colony, unlike the Δ/Δ Camyo5 strain (Fig. ​(Fig.4A).4A). On Spider medium, the colonies of the strain expressing Camyo5^S366A were consistently larger than those of the wild-type and Δ/Δ Camyo5 strains. Microscopic examination revealed that colonies of this strain consisted of pseudohyphal cells (data not shown). In liquid YPD supplemented with 10% FBS or in Lee's medium, cells of the strain expressing Camyo5^S366A were unable to form hyphae but formed pseudohyphae to a greater extent than cells of the Δ/Δ Camyo5 strain (Fig. ​(Fig.4B4B and data not shown). These results suggest that CaMyo5^S366A still retains some activity sufficient for more uniform pseudohyphal growth. Overall, the phosphorylation of serine 366 appears to be important for the proper function of CaMyo5 during hyphal growth.

To determine whether the TEDS-rule site serine 366 is important for the localization of myosin I, we tested the localization of CaMyo5^S366A-GFP and CaMyo5^S366D-GFP in normal cells and elongated pseudohyphal or hyphal cells. These proteins were also detected by Western blot analysis as 175-kDa bands, and their levels were similar to the CaMyo5-GFP protein level (Fig. ​(Fig.5A).5A). We found that the majority of CaMyo5^S366A-GFP did not localize in cortical actin patches but localized in the cytoplasm (Fig. 5B and C). The majority of CaMyo5^S366D-GFP did not localize to cortical actin patches either (Fig. 5B and C). Occasionally, some patches could be observed at the tips of buds, but these were not observed at hyphal tips. Mainly cytoplasmic staining and minor punctate staining at the periphery could be observed in these hyphae. These results suggest that serine 366 is important for the proper localization of myosin I and, surprisingly, that the proper localization of myosin I at the hyphal tip is not required for hyphal growth.

Regulation of C. albicans myosin I function.

We suggest that serine 366 of C. albicans myosin I is phosphorylated and that phosphorylation is required for the activity of myosin I, either by controlling the actin-dependent ATPase activity of the motor (56) or by controlling the interaction of myosin I with actin. That serine 366 is subject to phosphorylation is consistent with previous studies and is based on mutant phenotypes produced by mutations affecting this residue. Previous studies demonstrated that the phosphorylation of the TEDS-rule site in myosin I of different organisms is important for function. In one particular S. cerevisiae strain background (W303α), the phosphorylation of serine 357 in myosin I is essential (57). In A. nidulans, an S371E mutation introduces a residue that mimics a phosphorylated serine and leads to the accumulation of membranes in growing hyphae as a result of the hyperactivation of endocytosis. In contrast, an S371A mutation does not drastically affect this process (58). However, both of these mutations affect other aspects of cell growth and morphogenesis to the same extents, suggesting that the phosporylation of serine 371 per se does not regulate all functions of myosin I. In Dictyostelium, serine 332 in myoB is essential for function, although it is not clear if phosphorylation plays a critical role (42). In C. albicans, an S366A mutation produces a slow-growth phenotype similar to that of the Δ/Δ Camyo5 strain, while an S366D mutation does not impair growth in the yeast form. These data are analogous to the function of serine 361 in myosin I of fission yeast, where mutation to an alanine does not rescue the growth defect produced by a myosin I deficiency, but mutation to an aspartate does (53). Finally, a C. albicans myosin I mutant allele with the S366A mutation also does not allow the formation of hyphae, while the S366D mutation restores significant hyphal growth in response to hypha-inducing conditions. However, it appears that myosin I with aspartate 366 is less functional than wild-type myosin I. As discussed above, the S366D mutation results in the cytoplasmic localization of myosin I, as observed for myosin I fused to GFP. Moreover, the S366D mutation does not restore the polarized distribution of cortical actin patches in budding or hyphal cells. These results suggest that myosin I localization is required for the polarized distribution of cortical actin patches but is not a prerequisite for polarized growth.

To determine if the CaCla4 PAK kinase regulates the activity of myosin I by phosphorylating serine 366, we assessed whether the hyphal defect of the Δ/Δ Cacla4 mutant can be overcome by overexpressing the phosphorylation mimic allele of myosin I, CaMYO5^S366D. We detected no major morphological differences between the Δ/Δ Cacla4 mutants expressing wild-type levels of CaMYO5 or overexpressing CaMYO5^S366D under hypha-inducing conditions (data not shown). Based on observations with S. cerevisiae, where the expression of Myo3^S357D could not overcome the lethality caused by the deletion of both STE20 and CLA4 (57), our results suggest that other targets of CaCla4 must be activated to bypass the hyphal defect of the Δ/Δ Cacla4 mutant.

We thank Daniel Dignard for the sequence alignment, Bernard Massie for antibodies, Josée Ash and Cunle Wu for plasmids, Catherine Bachewich for oligonucleotides, Doreen Harcus for help with processing C. albicans, and Jürg Oberholzer for analysis of data. We also thank Stanford University for making the C. albicans myosin I sequence available from the database. We are especially grateful to Catherine Bachewich, Cunle Wu, and Bill Zerges for critical reading of the manuscript. Finally, we thank members of our laboratories for fruitful discussions.

U.O. was the recipient of Swiss National Foundation and NSERC visiting fellowships.