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Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase

2010; Springer Nature; Volume: 29; Issue: 17 Linguagem: Inglês

10.1038/emboj.2010.158

1460-2075

Amy Bishop, Rachel Lane, Richard Beniston, Bernardo Chapa‐y‐Lazo, Carl Smythe, Peter E. Sudbery,

Carbohydrate Chemistry and Synthesis

Article16 July 2010free access Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase Amy Bishop Amy Bishop Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Rachel Lane Rachel Lane Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Richard Beniston Richard Beniston Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Bernardo Chapa-y-Lazo Bernardo Chapa-y-Lazo Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Carl Smythe Carl Smythe Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Peter Sudbery Corresponding Author Peter Sudbery Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Amy Bishop Amy Bishop Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Rachel Lane Rachel Lane Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Richard Beniston Richard Beniston Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Bernardo Chapa-y-Lazo Bernardo Chapa-y-Lazo Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Carl Smythe Carl Smythe Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Peter Sudbery Corresponding Author Peter Sudbery Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK Search for more papers by this author Author Information Amy Bishop1,‡, Rachel Lane1,‡, Richard Beniston2, Bernardo Chapa-y-Lazo1, Carl Smythe2 and Peter Sudbery 1 1Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, UK 2Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield, UK ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. Tel: +44 114 222 6186; Fax: +44 114 222 2800; E-mail: [email protected] The EMBO Journal (2010)29:2930-2942https://doi.org/10.1038/emboj.2010.158 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkörper, which acts to focus delivery of the vesicles to the tip. Post-Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue-sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkörper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin-dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion. Introduction Polarized growth is a fundamental property of cell growth and development. In the development of specialized cells such as the outgrowth of axons and dendrites from the cell body of neurons, or the extension of plant pollen tubes, new membrane material is inserted at the growing tip by the process of polarized exocytosis, in which post-Golgi vesicles travel along cytoskeletal tracks to fuse with the plasma membrane. Fungal hyphae also show highly polarized tip growth. This polarized growth is driven by a subapical structure called a Spitzenkörper, which is rich in secretory vesicles that contain enzymes for the biosynthesis of cell walls, as well as providing the material for the expansion of the plasma membrane necessary for hyphal elongation. (Girbardt, 1957, 1969; Grove and Bracker, 1970; Grove et al, 1970; Bracker et al, 1997; Harris et al, 2005; Steinberg, 2007). According to the Vesicle Supply Centre model, secretory vesicles accumulate in the Spitzenkörper before onward transport to the hyphal surface, possibly transported along actin cables (Bartnicki-Garcia et al, 1989, 1995). The proximity of the Spitzenkörper to the tip ensures that secretory vesicles arrive in greater densities at the hyphal tip than subapical regions, resulting in polarized tip growth. The human fungal pathogen Candida albicans can grow in three developmentally distinct states: hyphae, pseudohyphae and yeast (Sudbery et al, 2004). The yeast form closely resembles the budding yeast Saccharomyces cerevisiae; the pseudohyphal form consists of chains of elongated cells with constrictions at the septal junctions; the hyphal form consists of chains of tube-like cells that have no constrictions at the septal junctions. In pseudohyphae, cell elongation can be so extreme that they superficially resemble hyphae; however, multiple lines of evidence suggest that there are fundamental differences between hyphae and pseudohyphae, including the organization of the cell cycle and modes of polarized growth (reviewed in Sudbery et al, 2004). Polarized growth at the tip of the pseudohyphal bud is restricted to the first part of the cell cycle (Crampin et al, 2005); whereas in hyphae, growth from the tip is continuous (Soll et al, 1985). We have shown that the myosin light chain, Mlc1 fused to YFP (Mlc1-YFP), is localized to a subapical spot at hyphal tips that resembles a Spitzenkörper and is continuously present throughout the cell cycle (Crampin et al, 2005). In contrast, Mlc1-YFP is localized to a surface crescent in pseudohyphae that is only present during the first part of the cell cycle. On the basis of this observation, we proposed that polarized growth in C. albicans hyphae, but not pseudohyphae, is driven by a vesicle-rich Spitzenkörper, which we have subsequently confirmed by observing that the vesicle-associated proteins Sec2 and Sec4 co-localize with Mlc1 in this apical spot, whereas they localize to a surface crescent in pseudohyphae (present work and L Jones, unpublished observations). Understanding the mechanism of the developmental switch from yeast to hyphae thus requires an understanding of why secretory vesicles accumulate in the Spitzenkörper in hyphae, but not in yeast or pseudohyphae. Polarized secretion has been extensively studied in the related budding yeast S. cerevisiae (for a review, see Park and Bi, 2007). Post-Golgi secretory vesicles travel along actin cables to sites of polarized growth in which they dock with a multiprotein complex called the exocyst before fusion with the plasma membrane, mediated by the interaction of v-Snares on the vesicles and t-SNAREs on the plasma membrane. The actin cables are generated at sites of polarized growth by a second multiprotein complex called the polarisome and the motive force for vesicle transport is provided by the class V myosin, Myo2, complexed to its regulatory light chain Mlc1. These late stages of the secretory pathway require the Rab GTPase, Sec4, in its active GTP-bound form, which mediates the docking of secretory vesicles with the exocyst through its interaction with the exocyst subunit Sec15 (Walworth et al, 1989; Guo et al, 1999). Sec4 is attached to the secretory vesicles and thus localizes to sites of polarized growth at the tip of the young buds and at the site of septum formation during cytokinesis (Goud et al, 1988; Novick et al, 1988; Novick and Brennwald, 1993; Walch-Solimena et al, 1997). Localization of Sec4 and its conversion to its active GTP-bound state is mediated by its GEF, Sec2, which is also found on secretory vesicles at sites of polarized growth (Walch-Solimena et al, 1997). In temperature-sensitive mutants of Sec2, Sec4 is only partially localized at the permissive temperature; at the non-permissive temperature, Sec4 is completely delocalized and electron microscopy shows that secretory vesicles are randomly distributed throughout the bud and mother cell (Walch-Solimena et al, 1997). Thus, activation of Sec4 by Sec2 is required for the transport of secretory vesicles and/or their retention at sites of polarized growth. S. cerevisiae Sec2 is composed of 759 amino acids (Nair et al, 1990). Its GEF activity is located in an N-terminal domain (1–161), which also contains a coil-coil region, which promotes its dimerization (Walch-Solimena et al, 1997; Dong et al, 2007). Sec2 is recruited to secretory vesicles by an upstream GTPase, encoded by a pair of redundant genes YPT31/32, which weakly binds Sec2 between residues 161 and 374 (Ortiz et al, 2002). Sec2 also physically interacts with Sec15, the effector of Sec4 (Medkova et al, 2006). The region in which Sec15 interacts with Sec2 overlaps the region in which Ypt31/32 interacts and the two proteins compete for Sec2 binding. Deletion mapping located a critical 58-residue window, 450–508, which is required both for localization of Sec2 to sites of polarized growth and for its attachment to secretory vesicles (Elkind et al, 2000). The temperature-sensitive sec2-59 allele is a nonsense mutation that encodes a truncated protein that lacks this domain (1–374) (Nair et al, 1990), whereas another temperature-sensitive mutation, sec2-78, results in a cysteine to tyrosine substitution at residue 478 within this localization domain (Elkind et al, 2000). Importantly, Sec2 is phosphorylated within the 450–508 region (Elkind et al, 2000). Although the phosphorylated residue or residues and the kinase responsible have not so far been identified, Sec2 localization has been shown to be directly or indirectly dependent on the cyclin-dependent kinase Cdc28 (McCusker et al, 2007). Mutant Sec2 proteins lacking the localization domain show increased affinity to Sec15 and are found complexed to Sec15 in the cytosol rather than on secretory vesicles (Medkova et al, 2006). The current model is that after docking with exocyst, the Sec2/Sec15 complex is released into the cytosol. To participate in a new cycle of secretory vesicle transport and docking, Sec15 must be exchanged for Ypt31/32. The localization domain acts to lower the affinity of Sec2 for Sec15 and thus facilitate the interaction with Ypt31/32 (Novick et al, 2007). Owing to the central function played by Sec2 in the localization of secretory vesicles to polarized growth, we have studied the function of Sec2 in the accumulation of secretory vesicles in the C. albicans Spitzenkörper. We show that Sec2 localizes to the Spitzenkörper in hyphae, to a surface crescent in pseudohyphae and to sites of septum formation in yeast. We reasoned that the differences of Sec2 localization in the different growth forms may reflect its differential phosphorylation. We, therefore, sort to identify the site or sites of phosphorylation, the kinase(s) responsible, and to determine the physiological effect of phosphorylation. We show that phosphorylation of S584 is required both for normal hyphal morphology and localization of Sec2 to the Spitzenkörper. We show that soon after induction, S584 is phosphorylated by Cdc28 partnered by the cyclins Hgc1 or Ccn1 and that this phosphorylation is required to localize Sec2 to the Spitzenkörper during hyphal growth. Moreover, the early phosphorylation of Sec2 is not dependent on the hyphal-specific-transcription programme initiated by the signal transduction pathways mediated by Cph1 and Efg1. Results Sec2 localizes to a Spitzenkörper in hyphae To localize Sec2, we generated a C-terminal fusion to YFP in a sec2Δ/SEC2 heterozygous parent, so that Sec2-YFP was the sole form of Sec2. This strain grew and formed hyphae normally, showing the Sec2-YFP fusion protein to be functional. The intracellular location of Sec2-YFP in cells growing in the different morphological forms was examined (Figure 1). In yeast, Sec2-YFP was observed to localize to punctate spots and to the cytokinetic ring at the mother-bud neck (Figure 1A). In pseudohyphae, Sec2 was located to a surface crescent at the tip of short elongated buds, it was absent at the tip of long buds and reappeared at the cytokinetic ring (Figure 1B). In hyphae, Sec2 localized to an apical spot in the majority of hyphae (87% n=65), which was continuously present (Figure 1C). A faint ring was also transiently visible at the site of septum formation (not shown). Thus, the pattern of localization is different in hyphae compared with yeast and pseudohyphae. The apical spot co-localized with FM4-64, a Spitzenkörper marker (Crampin et al, 2005) (Figure 1D). Exocyst and polarisome components localize to a surface crescent, which is spatially and dynamically distinct from the Spitzenkörper (Crampin et al, 2005, Jones and Sudbery, unpublished observations). Sec2-YFP also localized to a distinct location compared with Exo70, an exocyst component (Figure 1E). We have also found in FRAP experiments that Sec2-YFP has the dynamic properties of a Spitzenkörper component, clearly distinct from those of exocyst and polarisome components (Jones and Sudbery, in revision). Thus, in hyphae, the secretory vesicle component, Sec2, localizes to the Spitzenkörper, supporting the conclusion that a vesicle-rich Spitzenkörper forms at the tip of hyphae in this fungus. Figure 1.Sec2 localizes to the Spitzenkörper in hyphae. A sec2Δ/SEC2-YFP strain was grown as yeast, hyphae and pseudohyphae and the localization of Sec2-YFP visualized by fluorescence microscopy. (A) In yeast cells, Sec2-YFP forms punctuate patches and a ring at the mother-bud neck during cytokinesis (arrows). (B) In pseudohyphae, Sec2-YFP localizes to an apical crescent in short buds (arrow head, enlarged in inset); however, it becomes depolarized in long buds (barbed arrow). (C) In hyphae, Sec2-YFP localizes to a bright apical spot in hyphae corresponding to a Spitzenkörper (arrows). (D) Sec2-YFP co-localizes with the Spitzenkörper marker FM4-64. E: Sec2-GFP does not co-localize with the exocyst component Exo70-YFP. Cell outlines were visualized by counterstaining with Calcofluor white (A, B, D) or DIC microscopy (C). Note the blue fluorescence produced by Calcofluor white in this image may not be reproduced well by some printers. Scale bars: A, B, main panel; C, 5 μm; D, E, 1 μm; inset panel B, 0.5 μm. Download figure Download PowerPoint Sec2 requires phosphorylation on residue S584 to support hyphal growth We investigated whether different patterns of phosphorylation during yeast and hyphal growth could be detected in C. albicans Sec2-YFP by an electrophoretic band shift (Figure 2). Sec2-YFP extracted from stationary phase and growing yeast cells showed a reduced electrophoretic mobility compared with the sample treated with calf intestinal phosphatase (CIP) (annotated DP in Figure 2A). In most, but not all experiments, the band-shifted protein resolved into two separate bands (annotated Y in Figure 2A). When stationary phase yeast cells were induced to form hyphae, the pattern changed to a single, hyper-retarded band, suggesting it was hyper-phosphorylated (annotated H in Figure 2A). This change was evident within 20 min, well before hyphal germ tubes evaginated from the unbudded mother cell (30–35 min). Thus, in stationary phase and growing yeast cells, Sec2 is constitutively phosphorylated, probably on more than one residue, but the pattern of phosphorylation rapidly changes upon hyphal induction and precedes the appearance of hyphal germ tubes. Figure 2.Sec2 exists in multiple phosphorylated forms. (A) Sec2-YFP isolated from stationary phase cells (Stat, left panel) or cells growing in yeast-promoting conditions for 180 min (Y180, right panel) migrates as a doublet, which is retarded compared with Sec2-YFP dephosphorylated by CIP treatment. In this, and subsequent figures, annotation of the bands are as follows: H, hyphal-specific band; Y, double band visible in yeast and stationary phase cells (this band does not always resolve into a double band and so appears as a single band migrating between the dephosphorylated and hyphal forms); DP, Sec2 dephosphorylated by CIP treatment. (B) In hyphal-inducing conditions, Sec2-YFP still shows a hyper-phosphorylated band in cells where the hyphal-transcription programme is blocked by the efg1Δ/Δ cph1Δ/Δ mutations. Download figure Download PowerPoint Environmental cues that induce hyphal formation are sensed by a network of signal transduction pathways and result in a hyphal-specific programme of transcription (Biswas et al, 2007; Brown et al, 2007). The most important of these is the cAMP-dependent pathway, which targets the Efg1-transcription factor. A second pathway operates through Cph1, a MAP kinase that is homologous to the Fus3/Kis1 MAP kinase that mediates the pheromone response and pseudohyphal formation in S. cerevisiae. A C. albicans mutant lacking both Cph1 and Efg1 is unable to form hyphae and fails to initiate the hyphal-specific programme of transcription (Lo et al, 1997). We made use of this mutant to ask whether the hyphal-specific programme of transcription was required for the rapid appearance of the hyphal-specific pattern of Sec2 phosphorylation. In a cph1Δ/Δ efg1Δ/Δ strain growing as yeast, Sec2-YFP migrated with the reduced electrophoretic mobility characteristic wild-type yeast form compared with the CIP-treated protein from wild-type cells (Figure 2B). Interestingly, when this strain was induced to form hyphae, Sec2-YFP underwent a time-dependent band shift to a hyper-phosphorylated species, characteristic of the hyphal form, despite the complete inability of this strain to form hyphae. Thus, the rapid phosphorylation of Sec2 upon hyphal induction does not depend upon hyphal-specific gene transcription. To map the site or sites phosphorylated in hyphae, we constructed a series of C-terminal deletions in such a way that the resulting truncated protein was fused to YFP. C. albicans is an obligate diploid. We constructed the Sec2-truncated alleles using both a parent strain, which was a wild-type diploid, so that a normal copy of Sec2 was also expressed, and also in sec2Δ/SEC2 heterozygous parent strain, so that the truncated Sec2 protein was the sole form of Sec2 expressed. We determined whether the truncated protein was phosphorylated using band shifts in western blots and whether the Sec2-YFP was localized to the Spitzenkörper upon hyphal induction. In addition, this approach also allowed us to define the extent of the N-terminal region of Sec2 protein required for viability and the extent of the region required for normal hyphal growth. The results are summarized in Figure 3. In Figure 3A, black bars indicate deletions where we conclude the truncated Sec2-YFP strain is unable to support viability because the deletion could not be constructed in a heterozygous sec2Δ/SEC2 parent, but could be constructed in a wild-type parent; hatched bars indicate deletions where the truncated Sec2 could support viability, but which could not programme normal hyphal growth; open bars indicate deletions where the truncated Sec2 could support viability and normal hyphal growth. Figure 3.Deletion mapping to dissect Sec2 function. (A) C-terminal truncations fused to YFP were constructed in sec2Δ/SEC2 or SEC2/SEC2 parents and challenged to form hyphae. Phosphorylation, identified through a band shift, formation of morphologically normal hyphae and localization of the truncated Sec2-YFP to a Spitzenkörper were recorded. Black bars: the indicated truncations are presumed to remove Sec2 sequences required to support viability because they could not be constructed in a sec2Δ/SEC2 parent, but could be readily constructed in an SEC2/SEC2 parent. Hatched bars: the truncated Sec2 protein could support viability as they could be constructed in a sec2Δ/SEC2 parent, but could not form normal hyphae. White bars: the truncated Sec2 protein constructed in a sec2Δ/SEC2 parent could support viability and formed normal hyphae. The box marked Sc denotes the region equivalent to the 58 residue required for Sec2 localization and phosphorylation in S. cerevisiae. (B) Cells expressing only Sec21−583-YFP cannot form normal hyphae and the truncated Sec2-YFP is not polarized (left panel). Some cells appeared to form a germ tube, but failed to elongate (arrow). Cells expressing only Sec1−591-YFP formed normal hyphae and Sec2 is localized to a Spitzenkörper (right panel). Inset shows enlargement of tip indicated by arrow. Scale bars: main panels 5 μm, inset 1 μm. (C) Quantitation of Sec2-YFP (black bars), Sec21−583-YFP (white bars) and Sec1−591-YFP (grey bars) cells cultured in hyphal-inducing conditions for 90 min (n⩾70). PH=pseudohyphae. Failed GT=failed germ tube illustrated by arrow in the Sec21−583-YFP image in panel B. (D) Stationary phase cells (time 0) were induced to form hyphae for 60 min (60). Dephosphorylated CIP-treated extracts were used as a control. Autoradiographs of the truncations with the indicated end points and Sec21−607-YFP truncations with the indicated substitutions. Download figure Download PowerPoint Residues 492–550 in CaSec2 correspond to 450–508 in ScSec2, the 58-residue window required for localization and which contains a phospho-acceptor site or sites. In C. albicans, this region is apparently required for viability (Figure 3A). Three rounds of deletion mapping defined an 8-residue window (amino acids 583–591) that was required for the formation of normal hyphae, localization of Sec2-YFP to the Spitzenkörper and for its phosphorylation. Cells expressing only Sec21−550-YFP or Sec21−583-YFP were viable, but when induced to form hyphae, hyphal development was rudimentary or absent (Figure 3B and C) and truncated Sec2-YFP protein showed no polarization (Figure 3B). In contrast, cells expressing only Sec21−591-YFP formed morphologically normal hyphae in which Sec21−591-YFP localized to the Spitzenkörper (Figure 3B). In western blots (Figure 3D), we consistently observed that Sec21−550-YFP and Sec21−583-YFP did not show any difference in electrophoretic mobility compared with CIP-treated controls and so were probably not phosphorylated, whereas Sec21−591-YFP was band shifted, and so phosphorylated. We conclude that residues between 583 and 591 contain a residue(s) that is/are phosphorylated and that this phosphorylation event is required for normal hyphal growth. The sequence 583 NSPRQSVDG 591 contains two possible phosphorylation sites: S584 and S588. To investigate which of these is phosphorylated and to determine the physiological significance of the phosphorylation, we attempted to mutate these sites to the non-phosphorylatable alanine residue, in a sec2Δ/SEC2 heterozygote parent, so that only the mutated allele was expressed. We could readily make the S588A allele, which resulted in a strain, which produced morphologically normal hyphae, but were unable to create a strain in which the only copy of SEC2 carried the S584A allele. However, we could readily generate the phosphomimetic glutamate substitution at this site to form the S584E allele, which formed normal hyphae. Thus, it seems likely that S584 is the phospho-acceptor residue. If this is the case, then the Sec2 S584E protein should not show the hyphal pattern of phosphorylation, even though it can support normal hyphal growth. For unknown reasons, we were unable to C-terminally tag this allele, despite persistent attempts. However, we could construct an N-terminally tagged pMET3-green fluorescent protein (GFP)-SEC2 S584E allele where GFP-Sec2 S584E was expressed from the regulatable MET3 promoter (Care et al, 1999). We adjusted the culture conditions so that the level of Sec2 S584E expressed from the MET3 promoter was comparable with Sec2-YFP expressed from its native promoter, as judged by western blots. In these conditions, cells expressing only GFP-Sec2 S584E formed morphologically normal hyphae and GFP-Sec2 584E localized to the Spitzenkörper (Figure 4A). However, GFP-Sec2 S584E failed to show the large band shift upon phosphatase treatment that is characteristic of the wild-type protein, although a small band shift was evident (Figure 4B). Thus, we conclude that phosphorylation of S584 is necessary for hyphal growth. Figure 4.SEC2 S584E does not show the hyphal-specific pattern of phosphorylation, but forms hyphae normally. (A) Both pMET-YFP-SEC2-S584E/sec2Δ and pMET-YFP-SEC2/sec2Δ strains form hyphae normally and both YFP-Sec2 and YFP-Sec2 S584E are localized to a Spitzenkörper. (B) As with the C-terminal fusion, YFP-Sec2 shows a band shift in yeast, which shows a further shift during hyphal growth. YFP-Sec2 S584E shows a small band shift compared with the CIP-treated sample, but does not show a further shift in the hyphal sample. Scale bars: 5 μm. Download figure Download PowerPoint Although Sec21−591-YFP supported hyphal growth, the hyper-retarded band, characteristic of hyphal growth was not evident, suggesting that the hyper-phosphorylation of Sec2 observed in hyphae is not actually required for hyphal growth. We, therefore, sought to identify the additional phosphorylation event evident in hyphae and to investigate whether or not it is actually required for hyphal growth. To this end, we constructed a further series of C-terminal truncations to map the additional site. A Sec21−607 truncation showed the hyphal pattern of phosphorylation, thus locating the phosphorylated residue to the region between residues 597 and 607 (Figure 3D). This region contains three potential serine phospho-acceptor sites located at 598, 600 and 601. We introduced substitutions at each of these sites into the Sec21−607-YFP truncation. An alanine substitution at S598 abolished the hyper-phosphorylation; whereas substitutions at each of the other sites had no effect on the pattern of phosphorylation (Figure 3D). We tested the ability of Sec21−607 S598A-YFP to support hyphal growth. The ability to form hyphae in liquid culture was normal as was the ability to maintain hyphal growth in colonies on agar plates induced by Spider medium and serum (data not shown). Thus, as suggested by the ability of the Sec21−591-YFP to support hyphal growth, the additional phosphorylation event observed in hyphae is not actually necessary for Sec2 to support hyphal growth. The ability of the all the other serine substitutions in this window to form hyphae was also normal. It is interesting to note that residue S601 occurs in the same SPRQ context as S584. Nevertheless, substituting this residue had no effect on the pattern of phosphorylation or on its ability to form hyphae. We sought to identify the kinase responsible for the phosphorylation of S584. Sec2 has recently been reported to be targeted by Cbk1, a kinase that is essential for hyphal growth (McNemar and Fonzi, 2002; Kurischko et al, 2008). To investigate the function of Cbk1, we constructed strains containing Sec2-YFP, which contained a kinase-dead mutation in the Cbk1 active site or in the S570 site required for its activation by phosphorylation. Although neither of these strains could form hyphae, Sec2 was still phosphorylated normally (data not shown). Thus, Cbk1 is not responsible for targeting Sec2 S584. Tpk1 and Tpk2 are a pair of cAMP-dependent kinases activated in hyphal-inducing conditions and are required for hyphal development (Sonneborn et al, 2000; Bockmuhl et al, 2001). A cell lacking both kinases is not viable. We, therefore, used a tpk1Δ/Δ tpk2Δ/tpk2-1as strain in which the sole cAMP-dependent kinase catalytic unit could be inhibited by the ATP analogue 1NM-PP1, to investigate whether this kinase targets Sec2 (Bishop et al, 2000). Again, as in the case of Cbk1, Sec2 was phosphorylated normally after the addition of 1NM-PP1, despite the inability of this strain to form hyphae (data not shown). The context of S584 is an SP site which forms the core motif recognized by cyclin-dependent kinases (Cdks) (Endicott et al, 1999). In C. albicans, the cyclin-dependent kinase Cdc28 is known to be required for normal hyphal growth (Sinha et al, 2007). We tested the hypothesis that S584 phosphorylation is dependent on Cdc28 by determining whether Sec2 shows the hyphal pattern of phosphorylation in cells lacking Cdc28 activity. As Cdc28 is essential, we constructed a strain SEC2-YFP cdc28-1as strain in which Cdc28 activity can be inhibited by the ATP analogue 1NM-PP1 (Bishop et al, 2000). Western blots showed that Sec2-YFP phosphorylation was reduced (Figure 5A). Figure 5.Sec2 phosphorylation and localization is Cdc28 dependent. (A) When Cdc28-1as is inhibited, Sec2-YFP shows the yeast, but not the hyphal pattern of phosphorylation upon hyphal induction. Stationary phase cells were size fra