The Smk1p MAP Kinase Negatively Regulates Gsc2p, a 1,3-ß-Glucan

The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-␤-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae

Linda S. Huang*†‡, Hugh K. Doherty†, and Ira Herskowitz*§

*Department of Biochemistry and Biophysics, University of California, San Francisco, 513 Parnassus Avenue, Box 0448, San Francisco, CA 94143-0448; and †Department of Biology, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved July 19, 2005 (received for review March 21, 2005) Spore formation in Saccharomyces cerevisiae involves the sequen- We identify Gsc2p, a 1,3-␤-glucan synthase subunit, as a protein tial deposition of multiple spore wall layers between the pros- that physically associates with Smk1p. We find that glucan synthase pore membranes that surround each meiotic product. The Smk1p (GS) activity is elevated in smk1 mutants, but not in gsc2 smk1 mitogen-activated protein (MAP) kinase plays a critical role in double mutants, suggesting that SMK1 negatively regulates GSC2. spore formation, but the proteins that interact with Smk1p to We further examine spore wall deposition in smk1 mutants and find regulate spore morphogenesis have not been described. Using that, although deposition of the inner two spore wall layers is mass spectrometry, we identify Gsc2p as a Smk1p-associated independent of SMK1, deposition of the third chitosan layer is protein. Gsc2p is a 1,3-␤-glucan synthase subunit involved in highly aberrant in smk1 mutants. Elimination of Gsc2p suppresses synthesizing an inner spore wall layer. We ﬁnd that 1,3-␤-glucan the chitosan layer deposition defects of smk1 mutants. These results synthase activity is elevated in smk1 mutants, suggesting that suggest that Smk1p promotes proper deposition of the spore wall SMK1 negatively regulates GSC2. Although deposition of the two through negative regulation of 1,3-␤-glucan production via Gsc2p. inner spore wall layers is normal in smk1 mutants, deposition of the We propose that Smk1p regulates a transition between early and outer layers is aberrant. However, eliminating GSC2 activity re- late phases in spore wall deposition by regulating an enzyme stores normal deposition of the third spore wall layer in smk1 important for early spore wall biosynthesis. mutants, indicating that negative regulation of GSC2 by SMK1 is Materials and Methods important for spore wall deposition. Our ﬁndings suggest a model for the coordination of spore wall layer deposition in which Smk1p Yeast Strains and Growth Conditions. S. cerevisiae strains used are facilitates the transition between early and late phases of spore listed in the supporting information, which is published on the wall deposition by inhibiting a spore wall-synthesizing enzyme PNAS web site. All strains are in the SK1 genetic background and important for early phases of spore wall deposition. isogenic to LH177, which is essentially identical to KBY259 (9). All strains are homozygous diploid; only relevant genotypes are shown.

cell wall ͉ sporulation ͉ yeast ͉ chitosan Standard media and growth conditions were used (10). Synchro- CELL BIOLOGY nous sporulation was performed in 1% KOAc and 0.02% raffinose using a standard regimen (11), and the kinetics of sporulation were he coordination of sequential events is a regulatory challenge monitored by DAPI staining of cells withdrawn during sporulation Tin biological processes from cell-cycle control to cell differen- (12). Cultures that did not sporulate with normal kinetics or to tiation. Proper ordering of events can involve coupling the com- Ն85% efficiency were discarded. Yeast cell transformations were pletion of one event to the initiation of the next. Spore formation performed as described (13, 14). Gene disruptions and epitope in Saccharomyces cerevisiae involves the sequential deposition of tagging were carried out by using PCR-mediated techniques (15– four spore wall layers (1, 2), but the mechanisms that coordinate the 17); details are provided in supporting information. All gene fusions deposition of the different spore wall layers are unknown. were tested for function by analyzing their ability to undergo ͞␣ Sporulation in S. cerevisiae occurs in an a diploid cell and successful sporulation with kinetics and efficiencies similar to wild involves meiosis followed by the packaging of the meiotic products type and for their ability to support normal germination. during spore formation (reviewed in refs. 3 and 4). During spore formation, the four layers of the spore wall are sequentially Protein Immunoblotting, Kinase Assays, and Immunoprecipitation. deposited within the lumen of a double membrane structure (the Denaturing protein lysates for immunoblotting were prepared by prospore membrane) surrounding each meiotic product (1, 2), TCA precipitation as described (18). Native lysates for immuno- providing a system in which to examine the sequential deposition of precipitation were prepared from 50 ml of cells lysed by using a a structure. The spore wall enables the spore to withstand harsh MiniBeadBeater8 (Biospec) and glass beads into IP buffer (300 environmental conditions and is comprised of two inner spore wall mM NaCl͞5 mM EGTA͞50 mM Tris, pH 7.4͞0.5% Nonidet P-40) layers, which resemble the vegetative cell wall, and two outer layers, and a mix of protease inhibitors and phosphatase inhibitors as which contain spore-specific materials (Fig. 1A). The inner layers described (9). Lysates were clarified by centrifugation in a micro- are comprised of a mannan layer and a glucan polysaccharide centrifuge or a Beckman TLX centrifuge using a TLA100.4 rotor at layer, whereas the outer two layers consist of a chitosan layer and 23,000 ϫ g. Smk1-HA was immunoprecipitated by using HA-11 a dityrosine layer. beads (Covance). Smk1-zz was immunoprecipitated by using IgG Proteins involved in the synthesis of the spore wall layers have Sepharose (GE Healthcare). Trichloroacetic acid (TCA)- been identified and include Gsc2p, a component of 1–3-␤-glucan precipitated proteins, native lysates, and immune complexes were synthase (5, 6). Several proteins have been proposed to act as regulators of spore morphogenesis including Smk1p, a mitogen- activated protein (MAP) kinase essential for spore morphogenesis This paper was submitted directly (Track II) to the PNAS ofﬁce. (7). Although Smk1p is not required for progression through Abbreviations: MAP, mitogen-activated protein; GS, glucan synthase. meiosis, electron microscopic studies on smk1 mutant spores re- ‡To whom correspondence should be addressed. E-mail: [email protected]. vealed perturbations to spore wall structure indicating that the §Deceased April 28, 2003. nuclei are not properly packaged (7, 8). © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502324102 PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12431–12436 Downloaded by guest on September 26, 2021 polyacrylamide gel, and visualized by using Kodak X-Omat film or a PhosphorImager using IMAGEQUANT software (Molecular Dynamics).

Protein Purification and Mass Spectrometry. Pilot purifications using 250 ml of synchronously sporulating cells per strain (Ϸ1 ϫ 1010 cells) were done by using the SMK1::zz strain LH414 and the untagged control strain LH177. Purifications were carried out as below, except cells were lysed in a MiniBeadBeater8 (Biospec) and clarified by using a TLA100.4 rotor. Proteins were separated by using a 4–15% gradient gel and visualized by silver staining. For large-scale purification, a total of 13 liters each of LH414 and LH177 were grown under conditions of synchronous sporulation. Given the high aeration requirements of sporulating cells, cells were grown as 250-ml cultures in Fernbach flasks. Cells were harvested 8 h into sporulation and stored at Ϫ80°C until enough cells were collected for purification of SMK1-zz. Yeast cells were lysed in IP buffer with protease and phosphatase inhibitors (see above) by using glass beads in a BeadBeater (Biospec) and checked micro- scopically for at least 80% cell breakage. Lysates were clarified with a Beckman ultracentrifuge using a 70Ti rotor at 23,000 ϫ g at 4°C for 45 min. IgG Sepharose (GE Healthcare) was added to clarified lysates and incubated at 4°C for 2 h. Lysate and beads were applied to a column and washed with Ն10 bed volumes of IP buffer followed by 250 mM MgCl2 in IP buffer and 1M MgCl2 in IP buffer. Protein was eluted by using 0.5 M acetic acid, pH 3.4, collected into 1 M Tris (pH 7.4), concentrated by using a Centricon 30 (Millipore), and precipitated (19). Samples were electrophoresed in SDS͞ PAGE sample buffer on a 1.5-mm-thick 9% polyacrylamide gel. Bands were visualized by using Sypro Ruby Red (Bio-Rad) and excised. Sequence analysis was performed at the Harvard Micro- chemistry Facility by microcapillary reverse-phase HPLC nano- electrospray tandem mass spectrometry (␮LC͞MS͞MS) on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer. The MS͞MS spectra were correlated with known sequences by using previously developed algorithms (20, 21). Fig. 1. Smk1p is active during spore morphogenesis. (A) Diagram showing the spatial and temporal order of spore deposition. (B) In vitro kinase assay on GS Assay. GS was partially purified from sporulating yeast by sporulating cells. Smk1p was immunoprecipitated from untagged (LH177), membrane isolation essentially as described (22), with the addition SMK1::zz (LH414), and smk1(K69R)::zz (LH484) strains. (C) Kinase assay per- of the protease and phosphatase inhibitor mixture described above. formed on strains carrying Smk1p untagged (LH177), tagged with HA (LH178), Membrane sedimentation was performed in a Beckman Opti- and HA-tagged smk1 mutations containing the following amino acid changes: MAX centrifuge using a TLA100.2 rotor at 125,000 ϫ g at 4°C for K69R (LH415), T207A (LH416), Y209F (LH418), and T207A Y209F (LH417). 40 min. GS activity was measured by using [14C]UDP-glucose (MP Upper gel shows in vitro kinase assay. Lower gel is an immunoblot probed with anti-HA containing one-ﬁfth of the immunoprecipitation reaction used for Biomedicals). Measurements were normalized to the number of the kinase assay in the upper gel. (D–F) Sporulation time course examining cells collected because we found that the OD600, cell pellet weight Smk1p level and activity. Numbers at top of each panel indicate number of per ml of culture, and amount of membrane protein purified per ml hours yeast were in sporulation media. All assays in D–F use cells from the same culture fluctuated over time during sporulation. Within a particular sporulating culture. (D) Anti-HA immunoblot. Blot contains extracts from time point, experiments normalized to either cell weight or ␮gof SMK1::HA (LH178) strain undergoing sporulation. (E) Kinase assay using im- membrane purified yielded similar results. munoprecipitated Smk1-HA. (F) DAPI staining to monitor progression through sporulation. A cell with a single DAPI staining body has not undergone Microscopy and Spore Wall Assays. Visualization of mannan, glucan, meiosis, a cell with two DAPI staining bodies has completed meiosis I, and a cell with four DAPI staining bodies has completed meiosis II. Numbers listed are in and chitosan layers was performed as described (1), except that the percentages; a total of 200 cells for each time point were counted. described class 5 (mannan and chitosan) and class 6 (mannan and faint staining for chitosan) were grouped into a single class, as we found the classification of ‘‘not faint’’ versus ‘‘faint’’ staining sub- resuspended in SDS͞PAGE sample buffer and separated by SDS͞ jective. At least 200 cells were counted per time point; each strain PAGE. Gels were blotted onto nitrocelluose and probed with rabbit was grown in at least two independent cultures on different days. preimmune antiserum to detect the zz tag (gift from K. Benjamin), Only cells with four DAPI staining bodies were counted. Cells were with ␣-HA (12CA5; Covance) or with ␣-myc (9E10; Covance). scored by using a ϫ100, numerical aperture 1.45 objective on a Kinase assays were performed by using Smk1p immunoprecipi- Zeiss Axioskop Mot2 with an Orca-ER cooled charge-coupled tated from Ϸ1 ϫ 109 yeast cells. Immunoprecipitated Smk1p was device camera (Hammamatsu) and OPENLAB (Improvision) soft- added to a 20-␮l reaction containing 20 mM Tris (pH 7.4), 10 mM ware. Dityrosine fluorescence was performed as described (23). MgCl2, 10 mM MnCl2,1mMDTT,10␮MATP,5␮Ci of Don1-GFP and differential interference contrast (DIC) images [␥-32P]ATP plus 1 ␮g of Elk-1, presented as a recombinant protein were acquired from living cells by using the system described above. of amino acids 307–428 of Elk-1 fused to GST (Cell Signaling All other DIC microscopy was performed by using an Olympus Technologies). Kinase reaction was carried out at 30°C for 45 min, BX-60 microscope with an attached SPOT camera and software stopped by addition of SDS͞PAGE sample buffer, run on a 12.5% (Diagnostic Instruments).

12432 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502324102 Huang et al. Downloaded by guest on September 26, 2021 Fig. 2. Smk1p and Gsc2p interact and SMK1 modulates GS activity. (A) Silver stained gel from pilot puriﬁcation compar- ing an untagged control strain (LH177) with the SMK1::zz strain (LH414). Location of protein size standards denoted at left. Arrow with asterisk points to an Ϸ250-kDa protein that appears to bind the Smk1-zz protein speciﬁcally. (B) Immuno- precipitation experiment from strains containing untagged Smk1p (LH177), Smk1-zz (LH414), Gsc2-myc (LH565), and Smk1-zz Gsc2-myc (LH566). Immunoblot at left contains 5% of the native lysates used for immunoprecipitations shown at the right. The two immunoblots on the right are from the same gel showing immune complexes isolated by immunoprecipitation of Smk1-zz. The top blot shows the top of the gel, where Gsc2-myc migrates, whereas the bottom blot shows the mid- dle of the gel, where Smk1-zz migrates. Blots were probed with a mouse monoclonal anti-myc IgG. Smk1-zz can be visualized by using this antibody, as the zz-tag binds IgG. (C)GS activity assay performed on membrane fractions isolated from sporulating cells. The wild-type strain is LH177, smk1⌬ is LH185, and gsc2⌬ is LH625, and gsc2⌬smk1⌬ is LH701. Error bars show the standard error of the mean. (D) Kinetics of sporulation of the wild-type culture used for the GS assay in C, as monitored by DAPI staining. Numbers listed are in percentages; a total of 200 cells for each time point were counted. The other cultures used in C were chosen to have kinetics similar to the wild-type culture.

Results matrix. A protein with an apparent molecular mass of Ϸ250 kDa The Smk1p MAP Kinase Is Active During Spore Morphogenesis. We was enriched specifically in lysates from SMK1::zz cells, suggesting determined when Smk1p was catalytically active by establishing an that it physically associated with Smk1p (Fig. 2A). The 250-kDa in vitro assay for Smk1p kinase activity during sporulation. The protein was analyzed by tryptic digestion followed by mass spec- endogenous SMK1 gene was modified with a C-terminal addition trometry analysis and identified as Gsc2p. of an IgG-binding zz epitope-tag (from protein A) or a HA epitope Gsc2p is required for proper sporulation (6) and encodes a tag. Smk1p was isolated from cell extracts by immunoprecipitation subunit of 1,3-␤-glucan synthase (5, 6), an enzyme responsible for and incubated with [␥-32P]ATP and Elk-1, a substrate of mamma- synthesis of the 1,3-␤-glucan layer of the spore wall. We confirmed CELL BIOLOGY lian MAP kinases. Immunoprecipitates from both SMK1::zz-- the physical association of Smk1p and Gsc2p by coimmunoprecipi- containing and SMK1::HA-containing yeast phosphorylated Elk-1 tating the two proteins from sporulating cells (Fig. 2B). Endoge- (Fig. 1 B and C). This kinase activity was associated with Smk1p, as nous Gsc2p was tagged with the myc epitope. When Smk1-zz was no activity was detected in immunoprecipitates from cells that immunoprecipitated from sporulating cells, Gsc2-myc was detected contained only untagged Smk1p or tagged forms of mutant Smk1p in the immunoprecipitate (Fig. 2B), reflecting a specific interaction with the conserved catalytic lysine residue (K69) changed to between Smk1p and Gsc2p. Although high levels of Gsc2-myc were arginine (Fig. 1 B and C). Kinase activity was also greatly reduced detected in sporulating cells containing either untagged Smk1p or by mutations of sites in the conserved potential activating T loop of Smk1-zz (Fig. 2B), Gsc2-myc was detected in immunoprecipitates Smk1p, from phosphorylatable threonine (T207) and tyrosine only in the presence of Smk1-zz. These results confirm that Smk1p (Y209) to nonphosphorylatable amino acids (Fig. 1C). These physically associates with Gsc2p. kinase-dead and nonphosphorylatable smk1 mutants failed to support sporulation. GS Activity Is Increased in Cells Lacking Smk1p. The physical inter- The time course of Smk1p activity during sporulation was action between Smk1p and Gsc2p raised the possibility that Gsc2p examined. Cells were induced to undergo synchronous sporulation. may be required to activate Smk1p or that Smk1p may regulate At 4 h, 98.5% of cells had not yet begun the meiotic divisions, Gsc2p activity. These possibilities were explored by directly mon- whereas by 10 h, 93.5% of cells had completed meiosis (Fig. 1F). itoring the biochemical activities associated with each protein. Smk1p was noticeably induced after5hinsporulation medium, Robust Smk1p kinase activity was detected in the absence of Gsc2p reaching maximal levels between 6 and 8 h (Fig. 1D). Smk1p kinase (data not shown), indicating that Gsc2p was not required for the activity was examined by using cells from the same time course and activation of Smk1p. appeared strongly induced by6haftertheinitiation of sporulation, We examined Smk1p regulation of Gsc2p activity by using a GS with detectable activity persisting through 10 h of sporulation, when assay (Fig. 2C) (22). In wild-type cells, GS activity is low at6h(when mature spore walls appear (Fig. 1E). Thus, the Smk1p MAP kinase 26% of cells have completed the meiotic divisions) and peaks at 7 h becomes active after completion of meiosis and remains active (when 65% of cells have completed the meiotic divisions). Mem- throughout the time when spore wall deposition occurs, consistent brane preparations from gsc2 mutant cells did not show a significant with a role in spore morphogenesis. increase between 6 and 7 h and polymerized Ϸ30% as much glucan as wild-type cells at 7 h. These data are consistent with the Smk1p Associates with Gsc2p, a 1,3-␤-Glucan Synthase Subunit. To Gsc2p-dependent enzyme contributing the majority of the GS identify proteins that interact with Smk1p during the time when it activity in sporulating cells. Residual GS activity in gsc2 mutants is active, lysates were made from SMK1::zz cells and wild-type likely reflects the activity of Fks1p (5, 6, 24), a Gsc2p homolog used untagged control cells harvested after the meiotic divisions are predominantly in vegetatively growing cells. Such Gsc2p- completed, during the time of spore morphogenesis. Smk1-zz and independent activity is neither necessary nor sufficient to support associated proteins were isolated from extracts by using an affinity sporulation, as gsc2 mutants do not form mature spores (see below),

Huang et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12433 Downloaded by guest on September 26, 2021 surround the associated nucleus. To determine whether SMK1 or GSC2 were required at early stages in spore wall morphogenesis, we examined Don1-GFP (a marker for the leading edge of the devel- oping prospore membrane; ref. 25) localization in smk1 and gsc2 mutants. As in wild-type cells, Don1-GFP rings are found in smk1 and gsc2 mutants, suggesting that the leading edge complex is intact (Fig. 3B). Because prospore membrane growth precedes spore wall synthesis, these data suggested that SMK1 and GSC2 act at later stages in spore wall formation. Spore wall deposition in smk1 and gsc2 mutants was assessed by using fluorescent probes to visualize the mannan, glucan, and chitosan layers (1). The innermost mannan layer binds the lectin concanavalin-A (ConA) and can be visualized by using FITC coupled to ConA. The overlying 1,3-␤-glucan layer can be detected by using an anti-1,3-␤-glucan antibody. The chitosan layer binds to Calcofluor White. Consistent with previous observations (1), the mannan layer appeared first in wild-type yeast, followed by the glucan layer and then the chitosan layer (Table 1, see also Fig. 1A). Thus, as spores progress in maturity, they begin with only a mannan layer, then have both mannan and glucan, followed by mannan, glucan, and chitosan. As spores continue to mature, the ability to detect the glucan layer with antibody staining is lost (why this occurs is unknown), resulting in cells that show staining for only mannan and chitosan. It is important to note that Calcofluor White staining does not allow the distinction between the deposition of and the assembly of the chitosan layer. The outermost layer, the dityrosine layer, can be detected by the inherent ability of dityrosine to fluoresce under UV light on patches of wild-type yeast grown on membranes (ref. 23; Fig. 3D). These methods were used to examine spore wall formation in smk1 and gsc2 mutants. As detailed in Table 1 and Fig. 3C, smk1 mutant cells produced the inner mannan and glucan layers, but did not deposit the Fig. 3. SMK1 negatively regulates GSC2.(A) DIC images of sporulating yeast. (B) outermost layers properly. Unlike in wild-type cells, where a Don1-GFP localization. Rings of Don1-GFP are shown from the side, such that all four rings in an ascus can be visualized in a single focal plane. The wild-type stain chitosan layer surrounds all four meiotic products, only a subset of is LH790, smk1⌬ is LH788, and gsc2⌬ is LH789. (C) Immunoﬂuorescence pictures the meiotic products deposit chitosan in smk1 mutant cells. Al- showing mannan, the nuclei (DAPI), and chitosan. Note that, in smk1 mutants, the though deposition of chitosan occurred in smk1 mutant cells, this top two spores do not show detectable chitosan staining but do stain for mannan. deposition was aberrant and incomplete. Finally, the outermost For B–D, the wild type is LH177, smk1⌬ is LH185, gsc2⌬ is LH625, and gsc2⌬smk1⌬ dityrosine layer was not detected in smk1 mutant cells (Fig. 3D), is LH701. (D) Dityrosine ﬂuorescence. Cells were grown on a membrane and consistent with previous biochemical studies demonstrating a lack subjected to UV light to visualize the dityrosine layer. (Left) Patches of yeast on a of dityrosine in the spore wall of smk1 mutant cells (8). Thus, SMK1 membrane. (Right) The same patches under UV light. appears specifically required for production of the outer spore wall layers. gsc2 mutants were severely compromised in the production of a whereas fks1 mutants sporulate and germinate with wild-type different spore wall layer, the 1,3-␤-glucan layer (Fig. 3C). The kinetics and efficiencies (data not shown). reduction in this layer is consistent with our biochemical measure- Like wild-type cells, smk1 mutant cells showed an increase in GS ment indicating that Gsc2p is required to support the majority of GS activitybetween6and7hintosporulation. However, unlike activity during sporulation (Fig. 2C).Afewgsc2 spores had the wild-type cells, GS activity levels continue to increase after7hin Ͼ glucan layer (Table 1), likely because of the activity of Fks1p (see smk1 mutants. smk1 mutant cells show a 5-fold increase in GS above). All four spores in an ascus showed staining for glucan activity compared to wild-type cells at 8 and 9 h. This increase in whenever staining was detected, consistent with our detection of GS activity depends on GSC2,asgsc2 smk1 double mutants do not refractile tetrads in gsc2 mutants (Fig. 3A). Despite the low fre- show this dramatic increase in GS activity. These observations quency of glucan deposition in gsc2 mutants, deposition of the outer suggest Smk1p may negatively regulate Gsc2p activity. chitosan and dityrosine layers proceeded at frequencies compara- ble to wild-type cells (Table 1 and Fig. 3 C and D). Thus, normal Spore Wall Deposition in smk1 and gsc2 Cells. Our biochemical data deposition of the glucan layer was not required for deposition of the indicated that Smk1p negatively regulated GS activity and pre- outer spore wall layers. Taken together, these observations dem- dicted that the underlying defects in smk1 and gsc2 mutants would onstrated that smk1 and gsc2 mutants were defective in producing differ, prompting us to analyze the spore morphology phenotypes different layers of the spore wall. As summarized in Fig. 4A,the of both mutants in greater detail. Sporulation of wild-type cells outer two layers of the spore wall were not produced normally in results in the creation of a compact ascus containing four tetrahe- smk1mutants, whereas in gsc2 mutants, the inner glucan layer was drally arranged spores. The majority of gsc2 mutant cells formed selectively disrupted. spores that appeared immature and failed to compact, whereas smk1 mutants failed to form any structures resembling spores (Fig. SMK1 Inhibition of GSC2 Is Important for Spore Wall Deposition. Our 3A). The observation that smk1 and gsc2 mutants generate mor- biochemical data suggested that Smk1p negatively regulated Gsc2p phologically distinct sporulation products is consistent with the activity. To test whether the inhibition of Gsc2p was an important existence of distinct underlying defects in the two mutants. aspect of Smk1p function, spore wall production was examined in Before deposition of the spore wall, the prospore membrane gsc2 smk1 double mutants. We reasoned that if SMK1 inhibition of grows from the spindle pole body of each meiotic product to GSC2 were important for spore morphogenesis, the elimination of

12434 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502324102 Huang et al. Downloaded by guest on September 26, 2021 Table 1. Spore wall deposition analysis Mannan Genotype͞ Mannan ϩ glucan Mannan staining DAPI Mannan ϩ glucan Mannan ϩϽ4 spores ϩϽ4 spores pattern, h only Mannan ϩ glucan ϩ chitosan ϩ chitosan with chitosan* with chitosan*

Wild type 6 9.5 9 12.3 33.2 36.1 – – 7 0.5 2.4 8 35.3 53.5 – – 8 – – 4.6 22.8 72.6 – – 9 – – 2.4 7.6 90.1 – – smk1 6 36.8 51.5 11.7 – – – – 7 18.8 54.5 17.4 – – 8.5 0.9 8 – 32.3 33.6 – – 31 3.1 9 – 4 52.6 – – 38.5 4.9 gsc2 6 14.5 45.4 2.4 8.2 29.5 – – 7 1.4 38.3 1.4 11 47.8 – – 8 – 18.3 2.4 11.5 67.7 – – 9 – 7.7 2.9 6.2 83.3 – – smk1 gsc2 6 21.2 71.7 3.8 – 3.3 – – 7 4.4 45.7 4.4 2.9 42.7 – – 8 0.5 23.6 4.2 10.8 60.8 – – 9 – 18.4 5.7 4.2 68.4 – 3.3

Numbers are in percentages. Because of rounding, not all amounts for a particular experiment add up to exactly 100%. *One, two, or three spores within an ascus are surrounded by chitosan. An example of this is depicted in Fig. 3D.

GSC2, might allow proper deposition of outer spore wall layers to GSC2-independent fashion. Taken together, our biochemical and proceed without SMK1. As predicted, the gsc2 smk1 mutant cells cell biological data demonstrate that SMK1 is required for proper did not exhibit the chitosan deposition defect of the smk1 single deposition of the two outer layers of the spore wall, the chitosan and mutant. In most gsc2 smk1 double mutant cells, all four meiotic dityrosine layers, and suggest that inhibition of GSC2 is one products have detectable chitosan deposition (Table 1 and Fig. 3C). essential aspect of SMK1 function. As in the gsc2 single mutant, gsc2 smk1 double mutant cells were compromised in their ability to produce the glucan layer, presum- Discussion CELL BIOLOGY ably due to the lack of GSC2 (Table 1). As summarized in Fig. 4A, Cell division and differentiation involve the construction of struc- the elimination of GSC2 allowed proper deposition of chitosan even tures specialized for particular tasks, from the mitotic spindle found in the absence of SMK1, consistent with SMK1 promoting chitosan in all dividing cells to the stereocilia of vertebrate hair cells. The deposition through the inhibition of GSC2. spore wall that protects the meiotic products of S. cerevisiae is a Although inhibition of GSC2 is critical for proper chitosan complex structure comprised of multiple layers deposited in a deposition, SMK1 clearly has additional functions during spore spatial and temporal sequence. Our work provides insight into the morphogenesis. The other outer layer of the spore wall, the regulatory strategies controlling spore wall formation and demon- dityrosine layer, was not produced in the gsc2 smk1 double mutant strates the existence of a previously unknown role for the Smk1p (Fig. 3D), suggesting that SMK1 controls dityrosine deposition in a MAP kinase in regulating spore morphogenesis. In many multistep processes, the initiation of subsequent events depends on the successful completion of prior events. This is not the case for late steps in spore morphogenesis, as the outer spore wall layers are laid down normally in gsc2 mutants that are defective in inner wall synthesis. Nonetheless, deposition of inner and outer spore wall layers does appear coordinated. Our data indicate that appropriate deposition of the outer chitosan layer requires Smk1p- dependent inhibition of GS activity. The ability to suppress the chitosan deposition defect of smk1 by simply eliminating GSC2 points to Smk1p-dependent inhibition of Gsc2p as an important step for chitosan deposition. These observations suggest a model in which SMK1 increases the fidelity of chitosan deposition by assisting the transition from exclusive deposition of inner spore wall material (mannan and glucan layers) to deposition of outer spore wall layers (chitosan and dityrosine layers; Fig. 4B). In this model, Smk1p is not active during early phases of spore wall production, whereas Gsc2p is fully active, producing the glucan layer and potentially inhibiting chitosan deposition. Later during sporulation, Smk1p kinase is Fig. 4. Summary. (A) Summary of spore wall deposition phenotypes shown activated by an unknown mechanism. Active Smk1p inhibits quantitatively in Table 1. (B) Model for Smk1p regulation of Gsc2p activity Gsc2p, both decreasing glucan layer production and facilitating during spore morphogenesis. See text for details. chitosan deposition.

Huang et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12435 Downloaded by guest on September 26, 2021 This model suggests that GS activity should normally rise early next layer can be deposited. We speculate that there may exist a in spore wall production and then decrease in response to Smk1p Smk1p-dependent timing mechanism that is activated indepen- activity. Our data (Fig. 2C) show a sharp increase in GS activity in dently of the success of spore wall deposition and shuts down the wild-type cells between the 6- and 7-h time point, with no subse- synthesis of a layer, permitting the deposition of the next. How quent increases in GS activity at the 8- and 9-h time points. In synthesis of one layer may disrupt the synthesis of a subsequent interpreting these data, it is critical to note that even in the most layer is unknown, but this inhibition could be as simple as the need highly synchronously sporulating culture, there is substantial asyn- for one completed layer to act as a template for the deposition of chrony. Over the course of our assay, an increasing percentage of the next. In the current example, continued synthesis of glucan cells will have passed the point in sporulation when glucan depo- could preclude proper deposition of chitosan, whereas the complete sition is initiated, and even at 8 and 9 h, some fraction of wild-type absence of glucan may lead to the next layer being deposited onto cells have not yet begun to deposit chitosan (Table 1). Thus, if GS the mannan layer. activity remained constant once induced, total GS activity should Because we see heterogeneity of chitosan deposition within an continue to increase as the assay progresses. Instead, although the ascus in smk1 mutants, we further speculate that individual spores percentage of cells completing meiosis rises by Ϸ35% between 7 may at some rate override the timing mechanism, perhaps analo- and9hofsporulation (Fig. 2D), GS activity remains roughly flat. gous to the ability of cells to override the DNA damage checkpoint Thus, GS activity per sporulating cell does not rise and may well (28). A spore may have ways of overriding the need for GS decrease as sporulation progresses. In contrast, the level of GS inhibition, and whether this occurs is a decision made on a activity in smk1 mutants continues to increase over time, rising spore-by-spore basis. Alternatively, it is possible that GS activity Ͼ4-foldbetween7and9hofsporulation, consistent with a failure varies from spore to spore in smk1 mutants; those spores that have to inhibit GS activity in smk1 mutant cells. less GS activity can support chitosan deposition, resulting in a Several genes required for the proper production of the outer heterogenous phenotype within an ascus. spore wall layers have been identified, and include SSP2, CRR1, Our results raise important issues for future investigation. We do SPO77, OSW1, and MUM3 (2, 26, 27). These genes may be not understand the mechanisms that activate Smk1p at the appro- important in working with or in parallel to SMK1 to regulate the priate time. How glucan activity may interfere with chitosan transition between inner and outer spore wall deposition. Thus, it deposition is also unknown. Understanding the biochemical mechanism of Gsc2p regulation by Smk1p is of interest, especially given is of interest to examine the relationship between SMK1 and these the existence of SMK1 homologues in other fungal species and the genes. importance of 1,3-␤-glucan synthase as a target for antifungal The transition from inner to outer spore wall layer deposition agents (29–33). Nonetheless, the association of Smk1p with a spore could be regulated in several ways. First, deposition of outer layers wall biogenesis enzyme provides a clear opportunity for this MAP may depend on successful completion of inner layers, with the kinase to act directly on the machinery responsible for generating completion of one layer triggering the deposition of the next. structure within the cell. Because our data show that the chitosan layer can be deposited even without proper glucan layer deposition in gsc2 mutants, this is likely We thank B. Lane and the Harvard Microsequencing Facility for not a major mechanism. Alternatively, deposition of different layers performing the mass spectrometry analysis; E. Cabib for advice regard- could be completely independent, perhaps relying on a timing ing the GS assay; A. Neiman for sharing advice regarding spore wall mechanism that switches on the synthesis, deposition, and͞or assay; K. Benjamin (Molecular Sciences Institute, Berkeley, CA) and K. assembly of the next layer at the appropriate time, irrespective of Irie (University of Tsukuba, Tsukuba, Japan) for gifts of reagents; M. the state of the previous layer. Because we do not see proper Shiaris and K. Campbell for use of laboratory space, equipment, and chitosan deposition in smk1 mutants and because mutants exist that reagents; A. Neiman, P. Garrity, and the Herskowitz laboratory for block in sporulation after synthesis of the glucan layer (2), this is also helpful discussions; and K. Benjamin, P. Garrity, K. Jaglo, and J. Urano for comments on the manuscript. This work was supported National likely not the case. Institutes of Health Grant GM59256 (to I.H.) and from start-up funds Instead, our data are consistent with a model where the depo- and a Faculty Scholarship Support Award from UMass Boston (to sition of inner and outer spore wall layers is interdependent, but L.S.H.). L.S.H. was a Herbert W. Boyer Postdoctoral Fellow and a where the deposition of one layer needs to be inhibited before the Special Fellow of the Leukemia and Lymphoma Society.

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