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Eukaryotic Cell, July 2007, p. 1137-1149, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00329-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat{triangledown}

Talibah Metcalf,1,{dagger} Hanke van der Wel,1 Ricardo Escalante,2 Leandro Sastre,2 and Christopher M. West1*

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104,1 Instituto de Investigaciones Biomédicas Alberto Sols, C.S.I.C./U.A.M., Arturo Duperier 4, 28029 Madrid, Spain2

Received 13 October 2006/ Accepted 28 March 2007


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ABSTRACT
 
Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.


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INTRODUCTION
 
Cell walls protect cells from osmotic, physical, and predatory stress (32). The compositions of walls, which are found surrounding many free-living cells, the female gametes of most animals, and the somatic cells of plants, range from almost completely proteinaceous to almost completely polysaccharide. An important subclass of cell walls has a core layer consisting of cellulose fibrils, as found around somatic cells of vascular plants; green algae; certain oomycetes and the water mold Achlya ambisexualis; and spore or cyst walls of the social soil amoeba Dictyostelium discoideum, the free-living amoebae of the genus Acanthamoeba, the soil amoeba Hartmanella glebae, and the amoebae-flagellates Naegleria grubei and Schizopyrenus russelli. Much remains to be discovered about the role of proteins in the assembly of cellulose-rich cell walls.

Dictyostelium is amoeboid during growth, and the absence of a cell wall allows a phagocytic mode of feeding by wild-type cells. Axenic mutants, frequently used as a laboratory model, rely on constitutive fluid-phase endocytosis for nutrition (7). In response to starvation, the amoebae aggregate and form migrating slugs that emerge from the soil to form fruiting bodies, which consist of a mass of spores supported aerially by a cellular stalk. Walls form de novo around each of the prespore cells as they collectively rise to the top of the fruiting body and around each of the stalk cells (32). The spore wall or coat is distinct from the stalk cell wall and consists of three morphological layers. The major and central layer is 200 nm thick and composed of interlaced cellulose fibrils and a Gal/GalNAc polysaccharide (GPS). It is bounded by an outer layer composed of proteins that form the major permeability barrier to macromolecules and at the plasma membrane by an inner layer containing SP85, a novel protein consisting of multiple Cys-rich and mucin-type domains.

The known coat protein precursors are synthesized during slug migration and stored with the GPS in prespore vesicles (PSVs). The process of coat formation is first evidenced by their exocytosis and is followed by the formation of cellulose fibrils elaborated from transmembrane cellulose synthase complexes. The organization of the secreted proteins into morphological layers depends on cellulose (37). Analysis of an SP85 disruption strain and the effects of expressing discrete domains of SP85 show that this protein influences the timing of coat formation as prespore cells rise up the stalk (35). SP85 also plays a critical role in outer layer assembly because overexpression of SP85 domains exerts dominant-negative effects on outer layer morphology (17, 37). Some of these effects depend on SP85's cellulose binding activity (17, 39) and possibly other protein interactions (16).

SP65, first identified in a two-dimensional (2-D) gel proteomic analysis of the coat (34), was later found to partially copurify with SP85 in urea extracts of spore coats (39). SP65 was not incorporated into coats of SP85 mutant spores and was also selectively coimmunoprecipitated from the interspore matrix with exogenous SP85 by a mechanism that specifically involved the C1 domain of SP85 (38). These findings reveal a physiologically significant association between SP85 and SP65 but do not show if the interaction is direct. This interaction may mediate some functions of SP85. This possibility has now been addressed by identifying the SP65 gene (named cotE), examining biochemical properties of the recombinant protein expressed separately from the complex milieu of the coat and interspore matrix, and disrupting its gene. This has revealed that SP65 is expressed after SP85 and other known coat proteins, suggesting that interaction with SP85 is under biosynthetic regulation. Disruption of the SP65 locus (cotE) yields a coat phenotype that partially overlaps with that of SP85-null spores, supporting the importance of a cellulose-SP85-SP65 complex for outer layer organization.


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MATERIALS AND METHODS
 
Cells and cell culture. D. discoideum strains used are listed in Table 1. Cells were grown axenically in HL-5 growth medium or in association with Klebsiella aerogenes on SM agar (24). Axenically grown cells were induced to develop by centrifugally washing cells in PDF buffer (24) and plating them on 0.45-µm Millipore filters or by washing them in KP (10 mM potassium phosphate, pH 6.5) and plating them on nonnutrient agar plates. Bacterially grown cells were centrifugally washed free of bacteria in KP and deposited on nonnutrient agar plates. Spores were harvested either by picking them with a loop into 0.2% NP-40 in KP or slapping the inverted agar plate onto a countertop and recovering spores by rinsing the lid with the same buffer. Spores were washed by centrifugation (10,000 g x 10 s). For pretreatments, spores were resuspended in the solution indicated and then washed in KP by centrifugation. Plating efficiency was determined by counting colonies formed by plating a serial dilution, prepared in a bacterial suspension, of a known number of spores, determined by counting in a hemacytometer, on SM agar plates. Spore coats were isolated from spores of axenically grown cells by density gradient centrifugation as described previously (38).


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  		TABLE 1. Strains and Abs used

Cloning the SP65 gene. The previously reported peptide sequence VRGNPTCLRNHDGI (38) was found to be encoded perfectly by a 309-nucleotide (nt) sequence present in a database of random sequences of shotgun-cloned Dictyostelium genomic DNA (gDNA) available at the time. Primers derived from this nucleotide sequence were used to clone additional gDNA (unpublished data) using inverse PCR and linker-mediated PCR (20, 27, 28). Segments of the new sequences matched other gDNA sequences in the databases. This process was reiterated until a full-length candidate open reading frame (ORF) for the protein was obtained and then extended until ORFs corresponding to the presumptive neighboring genes were encountered to ensure that all potential exons were identified. The predicted coding region was deposited in GenBank as cotE (AF279135). The coding region was subsequently confirmed by automated gene prediction when sufficient sequence data for the locus were obtained by the Dictyostelium Sequencing Consortium and is referenced as DDB0214991 at www.dictybase.org.

Expression of SP65 in vegetative cells. The sequence of processed full-length SP65, whose N terminus was inferred from Edman degradation analysis of cyanogen bromide (CNBr) fragments of SP65 (38), was amplified in a PCR using 65-CoS1 (5'-CGGGATCCAGTTATGATGCATGTTACAATGTAGT) and 65-CoAS1 (5'-CGGGATCCATTTGTCAAACCACCTATTGAATTGGCAG) as primers (see Fig. 1 and 2), CsCl-purified strain Ax3 gDNA as template, and a 9:1 ratio of Taq and Pfu polymerases. Bold letters indicate BamHI sites used for subsequent cloning. The PCR products were ligated into pCR4TOPO and cloned into POP10 Escherichia coli cells (Invitrogen). The BamHI fragment from a plasmid whose insert had the correct sequence (pTOPO-SP65) was subcloned into the unique BamHI site of the integrating plasmid pVS (38), yielding pVSmycSP65.


Figure 1
Figure 1
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  		FIG. 1. Sequence of SP65/cotE DNA. The sequence was derived as described in Results and corresponds to GenBank accession no. AF279135 and DictyBase DDB0214991 (www.dictybase.org). Bold amino acids represent sequences derived by Edman degradation after CNBr treatment of SP65. Underlined amino acids represent the cleaved N-terminal signal peptide. N* refers to potential sites of N glycosylation. Yellow highlighting indicates C4C motifs, bright green highlighting indicates C2C motifs, magenta highlighting indicates a CC motif, and dark and light gray highlighting indicate mucin-like motifs. The bold nucleotide sequence corresponds to the predicted polyadenylation signal. The underlined nucleotide sequence corresponds to primers used for PCR. Olive green-shaded nucleotides correspond to the transcript beginning and end suggested by ESTs CFK257 and CFJ136 (www.dictybase.org).


Figure 2
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  		FIG. 2. Schematic diagram of the SP65/cotE locus, expression construct, and genetic modifications. (A) Intronless cotE locus, which encodes SP65. Transcript borders are deduced from the terminal sequences of available EST clones (Fig. 1). (B) Protein features deduced from sequence analysis. The position of the bsr cassette insertion (D) is indicated by a vertical arrow. (C) Coding sequence for processed SP65 (without signal peptide) inserted in the pVS expression vector (strain HW210). (D) Location of the Blasticidin S resistance cassette (bsr) insertion, resulting from a double-crossover event (strain HW207). (E) Location of the GFP insertion, resulting from a single-crossover event (strain HW211).

pVSmycSP65 was electroporated into strain Ax3, and transformed cells were selected at 10 µg/ml and then 120 µg/ml G418 (38). A high-level expression clone (HW210) was obtained by screening with monoclonal antibody (MAb) 9E10, which recognizes the myc epitope tag. SP65 was purified by pumping 3.8 liters of HL-5 growth medium, from a cell culture that had achieved stationary phase, onto a 75-ml column containing SP-Sepharose High-Performance (Pharmacia) equilibrated in 50 mM HEPES-NaOH (pH 7.0), and eluting SP65 using a linear gradient of 0 to 1 M NaCl in the same buffer. SP65 eluted from the column as a broad peak at 100 to 200 mM NaCl based on dot blot and Western blot analysis using MAb 9E10. Fractions were pooled and further purified on a Superose 12 gel filtration column (Pharmacia) equilibrated with 50 mM sodium phosphate buffer, pH 7.0. The protein eluted unexpectedly late as a broad peak, suggesting weak adsorption to the column.

Expression of SP85 in vegetative cells. SP85 coding DNA was amplified from pVSBW9M (39) using PCR primers SP85-Sdif (5'-AATGGATCCTCTAGAGGTACCTGATCAATGAAAATTTTAAAAAATTG) and SP85-AS (5'-AAGGGATCCGGTTAAAAACCATTGAGATCGTTTACGTCG) and then cloned into pCR4TOPO. pVSBW9M contains a version of SP85 in which the native signal peptide is replaced by the celA signal peptide, and the myc tag was not included in the construct. The SP85 DNA was excised using BamHI (bold in the primers) and cloned into the BglII site of the extrachromosomal vector pJK1 (9). Sequencing showed that 13 of the 17 tetrapeptide repeats of mucin domain 1 were deleted during cloning; instability has been observed previously for SP85 coding DNA (39). A clonal strain of Ax3 which secreted SP85{Delta}13 (HW219) was generated by electroporation with pJK1sp85{Delta}13.

GFP tagging of the SP65/cotE locus. pVSmycSP65 was modified by the addition of a green fluorescent protein (GFP) (S65T variant) coding sequence at the unique SacI site present in the vector immediately past the 3' end of the SP65 coding region. The two successive stop codons of pVSmycSP65 were changed to Ser residues by site-directed mutagenesis (nt changes are underlined) using the following sense and antisense primers: 5'-GGTGGTTTGACAAATGGATCCTCATCATCAGAGCTC and 5'-GAGCTCTGATGATGAGGATCCATTTGTCAAACCACC, as previously described (17). Coding DNA (711 bp) of GFP was amplified from pTX-GFP (generous gift of T. Egelhoff [12]) using GFP-SCS (5'-GAGCTCTCAGGTTCAGGTAGTAAAGGAGAAGAACTTTTCACTGG AGTTG) and GFP-SCAS (5'-GAGCTCTTATTTGTATAGTTCATCCATGCCATGTG) as primers in a PCR as described above. GFP-SCAS includes a stop codon (underlined) resulting in a GFP C terminus of ELYK. The PCR product was ligated into pTOPO and verified for the correct sequence at the OMRF DNA Sequencing Core using an ABI3730 capillary sequencer. The GFP coding insert was excised with SacI (bold in sequences above) and ligated into the unique SacI site of pVSmycSP65 to yield pVS(mycSP65-GFP). This yielded, after the C terminus GLTN of SP65, GSSSSEL, followed by SGSG from the GFP expression construct, followed by SKGE from the GFP N terminus. The bsr cassette was excised as a ClaI fragment from pbsr519 (21) and inserted into the unique ClaI site within the G418 resistance cassette of pVS(mycSP65-GFP). pVSbsr(mycSP65-GFP) was electroporated into strain Ax3, and transfectants were selected using 10 µg/ml Blasticidin S. Clones containing the desired single-crossover insertion event were screened by Western blot analysis of developing plaque scrapes using anti-GFP rabbit Ab (Molecular Probes, Eugene, OR) for reactive bands at the expected Mr value and were retrieved at a frequency of 10%. Genetic modification was confirmed by PCR analysis (38) as described in Results. Primer GFP-AS1 (5'-AAATTTAAGGGTAAGTTTTCCGTATGTTGCATACC) hybridizes at the 5' end of the GFP coding region (see Fig. 2E).

Interruption of the cotE locus. pTOPO-SP65 was modified by insertion of the BamHI fragment of pbsr519 (21) into the BglII site at nt 1306 with reverse orientation. The disruption DNA was excised with BamHI, purified on an agarose gel, recovered using a freeze-squeeze method (25), ethyl alcohol precipitated, and electroporated into strain Ax3 (38). Cells that grew in the presence of 10 µg/ml Blasticidin S (ICN) were screened for modification of the cotE locus using PCR methods, yielding 16/22 clones with the desired replacement. Primer bsr-S1 (5'-GAAAATCAAATCAAAAAGATAAAGCTGACCCGAAAGC) was used in PCR studies to confirm gene replacement (see Results).

Northern blotting. cotE mRNA was detected by Northern blotting as described previously (1), using a probe generated from pTOPO-SP65 by PCR using 65-CoS1 and 65-CoAS1 as primers.

Anti-SP65 Abs. A preparation of purified SP65 was mixed with either RIBI adjuvant (Ribi ImmunoChem Research, Hamilton, MT) or Immuneasy adjuvant (QIAGEN) and injected into 6-week-old BALB/c female mice. Preimmune and immune sera were collected and tested by an enzyme-linked immunosorbent assay (ELISA) using purified SP65 adsorbed to microplate wells. Mouse 2, initially immunized three times using RIBI adjuvant, followed by two boosts using Immuneasy adjuvant, was the best characterized as a result of its high titer after the Immuneasy boosts. MAb clone 6H6 (4A11) was selected as a representative of >36 hybridoma clones which secreted ELISA- and immunofluorescence-positive Abs. MAb 4A11 is an immunoglobulin G (IgG) and was purified from the medium of cell cultures grown in a CL350 chamber (Integra), using a protein G column. Ascites were induced in other immunized mice after an intraperitoneal injection of pristane. Abs used are listed in Table 1.

SP65 binding assay. Anti-myc tag MAb 9E10 was expressed in cell culture, purified by protein G affinity chromatography, and coupled to CNBr-activated Sepharose 4B (Sigma). For immunoprecipitation (IP) studies, 32 µl of a 50% slurry of 9E10-Sepharose was diluted with 200 µl IP buffer, consisting of 50 mM Tris-acetate, pH 7.5, 0.3 M NaCl, 1 mM EDTA, 1 mg/ml bovine serum albumin, 0.5% (vol/vol) NP-40, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin), and centrifuged at 13,000 x g for 1 min. The beads were resuspended with (i) 50 µl of a preparation of SP85 that had been concentrated 10 times from the HL-5 culture medium of the SP85 expression strain described above using a Vivaspin 2 centrifugal concentrator with a polyethersulfone membrane (molecular weight cutoff, 30,000) and 50 µl of HL-5 medium prepared in the same manner, (ii) 50 µl of mycSP65 prepared in the same fashion and 50 µl of HL-5, or (iii) 50 µl of both SP85 and mycSP65. The mixtures, which were supplemented with protease inhibitors, were incubated for 3 h at 4°C, with maintenance of the beads in suspension on a Vortex Genie 2. The supernatant was removed by centrifugation, and the beads were washed twice with 1 ml IP wash buffer (IP buffer lacking bovine serum albumin). The beads and supernatants were analyzed by Western blotting.

Immunofluorescence. Culminants were scraped from nonnutrient agar plates, resuspended in 25 mM EDTA in 10 mM KP (pH 6.5), dissociated by pipetting at room temperature, deposited on coverslips freshly precoated with 1-mg/ml poly-L-lysine in H2O, and allowed to dry. After 5 to 10 min, unbound cells were washed off with KP buffer. The coverslip was dipped in –20°C methanol for 30 min, air dried, hydrated in 5% nonfat dried milk in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 30 min, incubated in primary Ab for 1 h at 22°C, washed five times over 10 min with TBS, incubated in secondary Ab for 1 h, and washed again as before. Coverslips were mounted onto slides in Vectashield (Vector Labs) with DAPI (4',6'-diamidino-2-phenylindole) and sealed with fingernail polish. Rabbit anti-GFP (1:1,000) was from Molecular Probes, and affinity-purified Texas Red-conjugated goat anti-rabbit Ab (1:100) and affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ab (1:100) were from Jackson Laboratories. Epifluorescence images were collected through a 60x-numerical-aperture 1.2 water-immersion Nikon lens mounted on a Nikon Eclipse TE2000 microscope via an ORCA charge-coupled device digital camera using Improvision OpenLab software and processed in Photoshop.

Lectin incubation of spores. Spores from axenically grown cells were processed and examined using 4-µg/ml FITC-conjugated Ricinus communis agglutinin I (120) essentially as described previously (30).

Nucleotide sequence accession number. The predicted coding region was deposited in GenBank as cotE (AF279135). The coding region was subsequently confirmed by automated gene prediction when sufficient sequence data for the locus were obtained by the Dictyostelium Sequencing Consortium and is referenced as DDB0214991 at www.dictybase.org.


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RESULTS
 
Cloning the SP65 gene. SP65 was originally identified as a Coomassie blue- and silver-stained protein in 1-D and 2-D sodium dodecyl sulfate (SDS)-polyacrylamide gels of highly purified spore coats (34). The protein was purified in denatured form from coats by column chromatography and SDS-polyacrylamide gel electrophoresis (PAGE) (39) and cleaved with CNBr. Two major fragments were recovered by SDS-PAGE, and N-terminal sequence data were obtained by Edman degradation (38). A full-length ORF that encoded these peptide sequences was obtained by a combination of PCR from gDNA and sequence data from the Dictyostelium genomic databases available at the time. The predicted coding region (Fig. 1) lacks introns and was deposited in GenBank as cotE (AF279135). As is typical for Dictyostelium coding DNA (27, 28), this ORF has a higher GC content than does flanking DNA and exhibits a favorable Kozak context for the predicted start codon and a candidate polyadenylation signal after the stop codon. The predicted protein contains a single internal Met residue, consistent with the generation of only two bands after treatment with CNBr. Their Edman degradation-derived sequences are highlighted in bold in Fig. 1.

The cotE/SP65 coding region was subsequently identified by automated gene prediction when sufficient sequence data were obtained by the Dictyostelium Sequencing Consortium and is assigned as DDB0214991 at www.dictybase.org. No expressed sequence tags (ESTs) are reported for this sequence in cDNA libraries prepared from slug cells (26; www.dictybase.org). This was initially surprising because known spore coat proteins accumulate in prespore cells of the slug, and their mRNAs are abundantly expressed (29) and highly represented in the cDNA libraries. However, two EST clones in a cDNA library from culminating cells matched this locus, suggesting that cotE may be expressed unusually late in development. The EST sequences suggest possible transcription start and stop sites as highlighted in brown in Fig. 1.

cotE lies on chromosome 2, the location of all but one of the known coat protein genes, and is predicted to encode a 464-amino-acid protein. A signal peptide-like sequence is removed after Ala17 based on the Edman degradation sequence data (Fig. 1), yielding a predicted protein with a calculated Mr of 45,363 that is substantially smaller than the apparent Mr of 65,000 based on SDS-PAGE. The occurrence of two N-glycosylation sequons (N* in Fig. 1) suggests that the apparent Mr difference may be explained in part by N-glycosylation of the native protein. The encoded protein has a predicted pI of 7.6 compared to the pI range of 7.0 to 7.2 observed by 2-D gel electrophoresis (34), and the difference might be due to an acidic posttranslational modification as suggested by the charge stutter seen in the 2-D gels.

The predicted protein sequence contains motifs (Fig. 2B) characteristic of known spore coat proteins (34). The N-terminal half is composed of five Cys-rich C4C motifs (highlighted in yellow in Fig. 1), each resembling the N-terminal subdomain of the EGF motif, followed by a Cys-rich C2C motif (green) and a Cys-rich CC motif (pink). The even number of Cys residues is consistent with each participating in an intramolecular disulfide bond. The C-terminal half, devoid of Cys residues, consists of 12 mucin-like motifs of 16 residues each (light gray), which are potential targets of extensive O-glycosylation (6, 30) that may, in addition to N-glycosylation, contribute to the difference between the predicted and apparent Mr values.

Expression of SP65 mRNA. Northern blot analysis was performed to determine the time course of expression of cotE in developing cells collected every 2 h until fruiting bodies formed at 24 h. A nearly full-length probe against cotE detected a major band migrating at 1.5 kb (Fig. 3), consistent with the predicted length of the cotE mRNA, 1,550 nt (Fig. 1), and a slowly migrating minor species whose significance is not known. These bands were first detected at 22 h, coincident with the latest known prespore marker, spiA (22), and consistent with the EST data (discussed above). In contrast, most coat proteins are expressed starting at around 12 h of development (5), coordinated with the plasma membrane protein PsA/SP29 (Fig. 3). The MADS box transcription factor SrfA is required for expression of genes at late developmental stages and for spore coat stability (1-3). Examination of cotE expression in an srfA-null strain shows that, as for pspA but unlike spiA, cotE expression does not depend on this MADS box transcription factor (1).


Figure 3
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  		FIG. 3. Time course of SP65 mRNA expression. Strains Ax3 (normal) and srfA– were grown axenically and developed on filters for the indicated number of hours. Cells were scraped, and equal numbers of cells were extracted and subjected to Northern blot analysis using a full-length SP65 probe. A parallel blot was probed for expression of spiA and pspA.

Recombinant expression of SP65 and Ab generation. SP65 is normally covalently associated with other proteins in the coat and is therefore difficult to recover in native pure form for biochemical studies and raising specific Abs. To obtain a pure preparation of SP65 without having to resort to denaturation, SP65 was recombinantly expressed as a secreted protein. The full-length coding region of processed SP65, beginning with Ser18, was cloned into pVS (Fig. 2C), an expression plasmid whose discoidin promoter is active in growing cells. The expressed protein was designed to contain an N-terminal cleavable signal peptide from the celA gene and a c-myc epitope tag which becomes exposed as the new N terminus after processing of the signal peptide.

Recombinant SP65 (mycSP65) was purified from the HL-5 growth medium of cells transformed with pVSmycSP65 on an SP cation-exchange column. SDS-PAGE analysis revealed a Coomassie blue-stained protein product with an apparent Mr of 66,000 (Fig. 4, lane a), and Western blot analysis showed that this band was reactive with MAb 9E10 as expected for the c-myc-tagged protein (lane d). SP65 from spores migrates slightly behind the 66,000-Mr standard (38), indicating that mycSP65 produced in the growing cells is not modified in exactly the same manner. Secreted mycSP65 was susceptible to fragmentation.


Figure 4
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  		FIG. 4. Recombinant expression of SP65 in growing cells. The HL-5 medium from strain HW210 cells expressing mycSP65 under control of the discoidin promoter (Fig. 2C) was purified over an SP cation-exchange column and analyzed by SDS-PAGE and Western blotting. Lane a, 100 µl, gel stained with Coomassie blue; lane b, 50 µl, Western blot probed with anti-SP65 (mouse 2 ascites); lane c, like lane b, 100 µl; lane d, 100 µl, Western blot probed with anti-myc (MAb 9E10). Mr standard values (two types) are given in 103.

mycSP65 was further purified by gel filtration and injected into five mice. Sera were collected, and either ascites were induced or spleens were harvested to create hybridomas for production of MAbs. An ELISA based on the immunogen preparation indicated that reactive Abs were strongly induced (data not shown). Mouse 2 ascites (M2asc) was reactive against mycSP65 and its putative fragmentation products (Fig. 4, lanes b and c), including a major band at 50,000 Mr that was not recognized by MAb 9E10 and presumably represents an internal or C-terminal fragment of SP65. None of the many dozens of hybridoma supernatants from fusions of spleens from three mice, immunized according to distinct protocols, were reproducibly reactive in Western blot assays (data not shown). As described below, a representative MAb, 4A11, was specific for SP65 based on immunofluorescence, suggesting that it recognizes a conformational epitope that is affected by denaturation and/or disulfide bond reduction (data not shown). Although the ascites and antisera are probably also directed primarily toward conformational epitopes, they were more useful in Western blot assays, presumably because the multiplicity of reactivities permitted recognition of occasional epitopes that might renature after Western blotting. Alternatively, the process of creating hybridomas may not have captured the full spectrum of specificities present in the pool of serum IgG.

Tagging the SP65/cotE locus with GFP. Since the Abs were either not reactive with or monospecific for SP65 in Western blot assays (see below), a GFP gene-tagging method was employed to detect the cotE gene product. A previously described single-crossover strategy (8) was employed to replace the 3' end of cotE, starting just upstream of the stop codon, with DNA encoding a hydrophilic spacer and GFP, followed by the actin 8 terminator and a Blasticidin S resistance (bsr) cassette (Fig. 2E). This insertion was expected to result in expression of SP65-GFP at the same level and developmental timing as those of SP65 in normal cells. Two (strains HW211 and HW212) of 21 Blasticidin-resistant clones expressed a protein reactive with anti-GFP at an Mr position expected for SP65-GFP (see below). This protein was not labeled with MAb 9E10 against the myc epitope, as would be expected if the crossover event occurred within the homologous coding region, downstream of the N-terminal myc coding DNA of the plasmid. These clones were examined by PCR for evidence of the desired genetic modification. A PCR using a forward primer from 5' SP65 DNA not present in the targeting vector (65-Pr1) and a reverse primer from the 5' end of the GFP coding region (GFP-AS1) (Fig. 1 and 2) yielded the expected 1.5-kb DNA product from HW211 and HW212 DNA (Fig. 5C) and no products from the parental Ax3 strain DNA. In addition, a PCR using a forward primer from the interior of the coding region (65-CoS2) and a second primer from the 3' untranslated region (65-NCAS1) yielded the expected 0.8-kb product from parental DNA but not from HW211, consistent with the replacement of this part of the gene by the GFP fragment (Fig. 5D). Another reaction showed the 5' end of the coding region to be intact (Fig. 5A).


Figure 5
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  		FIG. 5. Genetic modification of the cotE locus. DNA from strains Ax3, HW207 (cotE-bsr), and HW211 (cotE-GFP) was subjected to PCR in the presence of the primer pairs 65-PrS1/65-CoAS2 (A), 65-PrS1/bsr-S1 (B), 65-PrS1/GFP-AS1 (C), and 65-CoS2/65-NCAS1 (D) (Fig. 1 and 2 show primer locations). Reaction products were separated on a 1.2% agarose gel containing ethidium bromide. Expected band sizes and Mr standard sizes in kb are indicated. HW211 and HW212 DNA yielded similar results (not shown).

Interruption of the SP65/cotE locus by bsr. To study the function of SP65, the cotE locus was modified by insertion of the bsr cassette within the coding region using a double-crossover strategy based on homologous recombination (Fig. 2D). Modified strains were identified based on PCR amplification of an expected 1.0-kb band from the DNA of clonal strains using primers 65-Pr1, located upstream of the gene targeting DNA, and bsr-S1 (Fig. 1 and 2). An example comparing strains HW207 and Ax3 is shown (Fig. 5B). In addition, amplification products starting from the 5' end of the gene (Fig. 5A) or from the 3' end of the gene (Fig. 5D) were 1.4 kb larger in size as expected due to the insertion. Both SP65-GFP and cotE-bsr strains developed to form normal-appearing fruiting bodies with elongate spores (see Fig. 11 below). Since the bsr cassette of the targeting DNA was in reverse orientation relative to cotE sequences, its transcription terminator may also terminate a truncated SP65 mRNA encoding most of the Cys-rich N-terminal half of SP65 (Fig. 2), up to R235 (Fig. 1) followed by SKLVFGSALSF-tga(stop) from the bsr cassette. This protein would have an Mr of 25,672 and a pI of 7.6.


Figure 11
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  		FIG. 11. Lectin labeling of spores. Spores from axenically grown cells were treated with (B) or without (A) hot 6 M urea in KP buffer, washed, and incubated in 4-µg/ml FITC-conjugated Ricinus communis agglutinin I (120) (RCA-I) to label the GPS of the coat interior. Spores were washed and examined by phase-contrast (left panels) or epifluorescence (right panels) microscopy.

Expression of SP65 in spores. To confirm that SP65 resides in the spore coat, spores, spore coats, and interspore matrix (the viscous fluid that accumulates between spores in the sorus) were analyzed by Western blotting. Samples from the normal strain Ax3 and the mutant cotE-modified strains and from cells grown on bacteria or axenically were compared. The blots shown were probed with serum from mouse 2, bleed 5 (M2Bl5), but the results were confirmed with sera and ascites from other mice (data not shown). As shown in Fig. 6A, lane a, M2Bl5 bound to a band in whole spores developed from bacterially grown normal (Ax3) cells at an Mr of 65,000, corresponding to the expected position of SP65, and a second band at an Mr of 85,000. This band is SP85, as it was absent from the SP85-null strain HW70 (38) (Fig. 6C, lanes b and d). The reason for cross-reaction with SP85 may be related to C4C sequence motifs or posttranslational modifications such as O glycosylation shared between recombinant SP65 and SP85. SP65 and SP85 were only barely detected in the interspore matrix, as expected (38). Similar results were obtained using spores from axenically grown Ax3 cells, except that the SP65 band (SP65a) migrated heterodispersely with an apparent Mr centered at 55,000 (lane c). The difference may be due to proteolysis in axenically grown cells (see below).


Figure 6
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  		FIG. 6. Expression of SP65 in spores and interspore matrix. Bacterially or axenically grown normal (Ax3) or cotE mutant (HW207) cells were developed to form fruiting bodies. (A to C) Sori from 48-h cultures were separated into spores (Sp) and interspore matrix (ISM) by centrifugation. (D) Spore coats (SC) were isolated from lysed axenic spores by density gradient purification. Equivalent proportions of each fraction, corresponding to 5 x 105 spores, were analyzed by Western blotting. Blots were probed with Abs as indicated. Lanes within the same panel are from the same Western blot. Relative molecular weights are given in thousands.

Analysis of bacterially grown SP65-GFP spores (strain HW211) showed that the 65,000-Mr band was replaced by a band at an apparent Mr of 95,000 (lane e), as expected owing to the additional mass of GFP (26,700). The new 95,000-Mr band was uniquely labeled with anti-GFP Ab (Fig. 6B, lane e), confirming its identity as the fusion protein. Band a at Mr position 47,000 represents a nonspecific reaction of anti-GFP to an unknown spore protein. Like SP65, SP65-GFP was associated with the spore rather than the interspore matrix (compare lanes f and e). A similar finding was observed in axenically grown cells, except that the majority of SP65-GFP was apparently cleaved near the junction of SP65 and GFP, with SP65 being retained with the coat and GFP accumulating in the interspore matrix (compare lanes g and h in Fig. 6A and B). Other minor bands presumably represent other proteolytic events.

Analysis of the cotE-bsr strain (HW207) confirmed the absence of SP65 at the 65,000-Mr position (Fig. 6A, lane k). However, the mutant spores accumulated a new reactive band at an apparent Mr of 28,000 (SP65N1), corresponding to the protein expected if a cotE message truncated at the bsr insertion site were formed and translated (see above). This protein, concluded to represent the N-terminal half of SP65 (Fig. 2B), also appeared in spores from bacterially grown cells (data not shown) but is not evident in lane i because of underloading. The N-terminal half of SP65, comprised of C4C repeats, contains the information required for insertion into the coat, as it is not observed in the interspore matrix (lane l).

Examination of SP85 mutant cells (HW70 or 85–) shows that, in contrast to Ax3, SP65 accumulates in the interspore matrix rather than the spore (Fig. 6C). This distribution, observed previously using Coomassie blue staining (38), confirms that SP65 depends on SP85 for incorporation into the coat.

To determine if SP65 is present in the spore coat, equal numbers of spores and purified spore coats were compared by Western blotting. Fig. 6D (lanes a and b) shows similar levels of SP65 in the two fractions, indicating that the majority of spore SP65 is in the coat. Lanes c and d show a similar distribution for two known coat proteins, SP96 and SP75, and serve as a loading control.

Developmental regulation of expression of the SP65 protein. Western blot analysis was used to determine when SP65 accumulated during development. Probing of SP65-GFP in strain HW211, using anti-GFP, showed that the cotE gene product first accumulates at 20 h, which is the time at which the prespore mass rises up the forming stalk in this strain (Fig. 7E). This is similar to the time of accumulation of SpiA (Fig. 7D) and corresponds to the late expression of cotE and spiA mRNA (Fig. 3). In contrast, other known coat proteins including SP96, SP85, and SP75 begin to accumulate much earlier at 10 h (Fig. 7A to C), as described previously (5). Probing of SP65 expression in strain Ax3 using anti-SP65 suggests that SP65 does not accumulate until even later than SP65-GFP (Fig. 7F). The discrepancy may be due to a change in Ab recognition, which apparently depends on conformation, or instability of free SP65 in these axenically grown cells (see above). A later time of accumulation was also observed for truncated SP65N1 (Mr of 28,000) in the cotE-bsr disruption strain (Fig. 7G). These results show that SP65 is expressed at or just prior to spore coat formation, which occurs at 19 to 21 h under these conditions.


Figure 7
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  		FIG. 7. Time course of SP65 protein expression. Normal and mutant strains were grown axenically and developed on filters for the indicated number of hours. Equal numbers of cells plated were subjected to SDS-PAGE and Western blot analysis for developmentally regulated proteins using the indicated Abs. Note that the strains used in this analysis culminate at 19 to 20 h, in contrast to the strain analyzed by Northern blotting in Fig. 3, which culminate at 22 to 24 h.

Localization of SP65 in the spore coat. A microscopic analysis was conducted to confirm the biochemical evidence that SP65 resides in the spore coat. SP65-GFP was localized initially based on the intrinsic fluorescence of GFP. Cells in the process of forming coats were obtained by squashing 20-h HW211 culminants between a coverslip and a slide. Spores, evident by their ellipsoid shape and phase brightness in phase-contrast microscopy, were outlined by green fluorescence (Fig. 8A). Since normal Ax3 spores were not fluorescent (data not shown), this indicated that SP65-GFP is at the spore surface. In addition, some nonspore cells were also GFP positive (see asterisks), indicating that SP65 appears at the prespore surface prior to the final steps of sporulation. When the cellulose layer of the coat was revealed using the fluorescent brightener Calcofluor White ST, spores with cellulose (blue) also fluoresced green, confirming localization of SP65-GFP in the coat (Fig. 8B). Some cells had surface GFP fluorescence but no Calcofluor fluorescence (see asterisks), consistent with deposition of SP65 at the cell surface prior to cellulose. Examination of differentiating stalk cells showed only minimal association of SP65-GFP with stalk cell walls (Fig. 8C). Western blot analysis revealed little degradation of SP65-GFP at this early stage of sporulation (data not shown), and even if the fusion protein were cleaved, free GFP accumulates in the interspore matrix rather than the spore coat (Fig. 6). Similar, albeit more intense, spore surface fluorescence was observed in spores from bacterially grown cells (data not shown).


Figure 8
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  		FIG. 8. Localization of SP65-GFP in spores. Nineteen-hour culminants from axenically grown HW211 cells were squashed under a coverslip in the presence of Calcofluor White ST. Cells were photographed using phase contrast or for Calcofluor-induced fluorescence or GFP fluorescence as indicated. Left and right panels are successive images from the same field which allowed minor cell movements. (A) Asterisks mark GFP-positive, phase-dark cells that have not yet encapsulated. (B) Asterisks mark GFP-positive, Calcofluor-negative cells. (C) Nascent stalk cells from the basal disc region.

Localization of SP65 in differentiating prespore cells. SP65-GFP fluorescence was rarely visualized intracellularly. Since GFP fluorescence might be delayed following biosynthesis, GFP was localized immunocytochemically using an anti-GFP Ab. Labeling was first seen in occasional HW211 cells dissociated at 17.5 h (Fig. 9A, asterisk). Intracellular labeling appeared specific for SP65-GFP because it largely coincided with labeling by MAb 4A11, which probably recognizes a conformational epitope of SP65 (see above). Anti-GFP did not label normal Ax3 cells (not shown), indicating specific binding to SP65-GFP. MAb 4A11 did not label 15-h normal Ax3 and cotE-bsr (expressing SP65N1) cells but labeled 17.5-h cells in a pattern similar to that of HW211 cells (data not shown). In these examples, intracellular labeling was concentrated in a central region reminiscent of the Golgi apparatus (31). By 19 h, when spores first appear, anti-GFP and MAb 4A11 labeled a larger fraction of cells with a similar intracellular pattern (Fig. 9B). Strong labeling of spores with anti-GFP indicates that the GFP domain is exposed at the spore surface. In contrast, MAb 4A11 did not label spores, indicating that the SP65 epitope is either inaccessible, as occurs for other coat proteins including SP85 (see below), or lost due to a conformational change. Together, these data suggest that anti-GFP is a valid reporter of SP65 expression and that an intracellular pool of SP65-GFP briefly accumulates prior to export to the cell surface.


Figure 9
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  		FIG. 9. Localization of SP65-GFP in prespore cells. Axenically grown HW211 cells (SP65-GFP) were developed for 17.5 (A and C), 18.5 (D), or 19 (B and E) h; dissociated; deposited on poly-L-lysine-coated coverslips; and methanol fixed. Cells were probed for SP65-GFP using anti-GFP and FITC-conjugated anti-rabbit IgG and/or with MAb 4A11 and Texas Red-conjugated anti-mouse IgG for SP65 and for the coat protein SP85 using MAb 16.1 and Texas Red-conjugated anti-mouse IgG. Specimens were mounted in DAPI to localize nuclei. psp, prespore cell; sp, spore cell; *, single FITC-positive cell; numbers denote labeling patterns as described in Results. Panels A to C are the same magnification, indicated in panel A; panels D and E vary as indicated. Merged images are created from FITC and Texas Red images. Adjacent panels are images of the same field.

To compare localization of SP65 with SP85, HW211 cells were double probed with anti-GFP and either MAb 5F5 (data not shown) or MAb 16.1 to detect SP85 (17). At 17.5 h, there was frequent expression of SP85, which is expressed earlier and accumulates in PSVs coordinately with other known coat proteins, and rare expression of SP65 (Fig. 9C). By 18.5 h, SP65-GFP was expressed in many cells in vesicle-like structures that are almost completely distinct from SP85-positive vesicles (PSVs), so only the merged panel is shown (Fig. 9D). This image was particularly well flattened by the coverslip, allowing visualization of the typical annular profile of PSVs (38). SP65-GFP-containing vesicles tended to be smaller than PSVs, though some were annular and a few were superimposed upon PSVs. These structures are reminiscent of the non-PSV class of vesicles populated by an expressed C-terminal fragment of SP85 (38). In Fig. 9E, cells from sorogens at 19.5 h were classified according to their labeling patterns with the two Abs (numbers correspond to Fig. 9E [merge]): (i) cells with intracellular PSV labeling for SP85 but little anti-GFP labeling, consistent with onset of SP85 expression prior to that of SP65; (ii) cells labeling intracellularly for both SP85 and GFP in largely separate patterns, suggesting that SP65 initially accumulates in vesicles distinct from PSVs; (iii) cells that were labeled intracellularly for both but in an overlapping pattern, suggesting that SP65 vesicles and PSVs can fuse; (iv) spore-like cells labeled at their surfaces for both SP85 and SP65-GFP, often in crescents at the cell surface that represent apparent initiation sites; and (v) spore-like cells labeled at their surfaces for SP65-GFP but not for SP85, suggesting that SP85 quickly becomes masked as the coat matures. It is likely that following initial expression in the Golgi apparatus, SP65 is transported rapidly to the cell periphery in non-PSVs, which either fuse with or coordinately exocytose with PSVs to form initial cell surface crescents representing sites of nascent coat assembly.

SP65 binds directly to SP85. The previously observed interaction between SP65 and SP85 (38) was detected in the interspore matrix and coat extracts. To determine if the interaction occurs independently of other coat factors, mycSP65 (Fig. 4) and {Delta}SP85 (lacking 13 mucin repeats) were expressed recombinantly in the HL-5 growth medium of growing cells, which lacks other coat components including the polysaccharides. MAb 9E10-Sepharose preferentially pulled down an apparent 16,000-Mr N-terminal fragment of mycSP65 (mycSP65N2) from concentrated HL-5 containing mycSP65 (Fig. 10). mycSP65N2 is probably a degradation product that includes most or all of its five Cys-rich C4C domains (155 amino acids). Though in the absence of mycSP65 the beads nonspecifically pulled down a small amount of {Delta}SP85, the amount was greatly increased if mycSP65 was present. This suggests that the Cys-rich N terminus of SP65 interacts directly with SP85, previously shown to involve its own Cys-rich C1 domain.


Figure 10
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  		FIG. 10. Binding of SP85 to SP65. MAb 9E10 (anti-myc) beads were incubated with concentrated HL-5 growth medium from cells expressing mycSP65 or {Delta}SP85, or a mixture of the two. After 3 h, the beads were centrifuged and washed. One equivalent of the first supernatant fraction (nonbound) and a half-equivalent of the washed bead fraction (bound) were analyzed by SDS-PAGE and Western blotting for the presence of SP65 using MAb 9E10 or {Delta}SP85 using MAb 5F5, whose positions are marked in the margin. mycSP65N2 (Mr, ~16,000) was the predominant isoform of mycSP65 present in the conditioned medium (Fig. 4) that bound to the MAb 9E10 beads.

SP65 mutant coats are hyperpermeable. Genetic deletion of most coat proteins allows increased labeling of spores by the fluorescent lectin FITC-Ricinus communis agglutinin I (RCA-I) (32), suggesting a breakdown of the outer layer barrier that normally masks the lectin's target, the GPS residing in the inner and middle layers. Here, spores from the cotE-bsr strain HW207 are labeled with FITC-RCA-I more intensely than normal spores (Fig. 11). Preextraction of the spores with urea resulted in similar levels of fluorescence between spore types, suggesting that the difference was not due to altered levels of GPS itself. To test if the permeability difference affected spore resistance to stress, 2-day-old spores from bacterially or axenically grown strains Ax3, HW207 (SP65N1), and HW211 (SP65-GFP) were incubated for 45 min in 6 M urea in KP at 22°C or 0.5% SDS in KP at 22°C, 42°C, 62°C, or 100°C. All spores showed normal and similar viability except at the elevated temperatures (data not shown), which were similarly lethal for all three strains (1% plating efficiency at 42°C and <0.1% at higher temperatures). This indicated that the mutant spore coats still provided protection to the enclosed cell.


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DISCUSSION
 
SP65 is confirmed here to be selectively if not exclusively associated with the spore coat, making SP65 one of 10 validated proteins characteristic of this cell wall type. However, SP65 is not synthesized and stored in PSVs with the other nine (32) but rather is formed and routed to the cell surface just prior to cellulose synthesis, the final known component to be delivered to the cell surface during coat formation. The novel timing and routing of SP65 suggest that the interaction of SP65 with cellulose, via the intermediate PSV protein SP85, may critically regulate coat assembly.

The conclusion that SP65 is a coat protein is based in part on use of Abs generated against recombinant full-length mycSP65. Polyclonal Abs showed that SP65 is enriched in coats of spores based on Western blotting (Fig. 6), but their polyspecificity precluded their use in microscopy for confirmation. The MAbs, which recognize conformational epitopes, did not bind spore coats (Fig. 9B), probably because the epitopes were either masked or altered in coats. For similar reasons, DP87/SP75 and EB4/SP35 gene products were originally identified as interspore matrix proteins before other approaches identified them as coat proteins (32). Therefore, tagging the SP65/cotE locus with GFP to yield an SP65-GFP fusion protein was particularly informative. Intrinsic GFP fluorescence and anti-GFP Ab labeling are observed only at the surface of spores and not in slugs or in stalk cells (Fig. 8 and 9C to E). Functional evidence for coat localization emerged from the finding that truncation of SP65 rendered the coat permeable to an exogenous macromolecular tracer (Fig. 11).

Western blotting showed that SP65-GFP is not expressed until within an hour of coat formation (Fig. 7), and this was corroborated by immunofluorescence using anti-GFP and MAb 4A11 (Fig. 9A and B). Analysis of native SP65 confirmed the late timing, but the strong bias of the Abs toward conformational epitopes, masking of the epitopes in the coat, and apparent ongoing processing including degradation thwarted precise characterization of SP65 expression in the absence of the GFP tag. Protein expression was closely linked to message accumulation as determined by Northern blotting (Fig. 3), indicating that SP65 expression is under transcriptional control. The timing is essentially identical to that of the previously described prespore-cell-specific, glycosylphosphatidylinositol-anchored protein SpiA, associated with the plasma membrane that is tightly bound to the coat. The transport pathway and function of SpiA are unknown (22). Recently, additional late genes have been found to be expressed under the control of the GATA-type transcriptional factor StkA (14) or the MADS-box-type transcriptional factor SrfA (1, 2). SigD, regulated by SrfA, has amino acid sequence motifs found in other coat proteins and is a candidate for another coat protein with similar regulation. Unlike SigD and SpiA, however, SP65 does not depend on SrfA (Fig. 3).

The intracellular localization of the SP65 precursor is distinctive from that of known coat proteins. SP65-GFP was first detected in a central location (Fig. 9A and C) suggestive of the Golgi apparatus (31) and confirmed using MAb 4A11 for native SP65. Subsequently, anti-GFP labeling was distributed in a dispersed punctate manner consistent with vesicles. But double-labeling experiments showed that these are not equivalent to PSVs (Fig. 9D and E, number 2), where other coat protein precursors and the GPS accumulate (32). However, overlap of SP65 and SP85 was occasionally observed (Fig. 9E, number 3), but it is not clear whether this reflects rare vesicle fusion or a temporal progression in which SP65 and SP85 vesicles fuse transiently close to the time of exocytosis consistent with evidence that PSVs undergo continual maturation (23). GFP fluorescence could not be used to dynamically track nascent SP65-GFP because vesicular GFP was rarely fluorescent. Fluorescence acquisition by GFP depends on time and O2 (36), whose level in vesicles may be limiting. Initial formation of the coat was evidenced by a fluorescent crescent at the cell surface. This arc was usually labeled for both SP65-GFP and SP85, which is most consistent with coordinate rather than sequential secretion of the two proteins. Distinct secretory trajectories for discrete cell wall precursors have been observed in other cell systems, though this is most often associated with sequentially deposited wall layers (11, 15, 18). When cellulose is imaged using Calcofluor, the presence of cells fluorescent for GFP but not Calcofluor (Fig. 8B) suggests that SP65 is secreted to the cell surface before cellulose is deposited.

The dynamic properties of SP65, revealed by changes in Ab binding and Mr differences between axenically and bacterially grown cells, are unusual and may reflect a combination of natural protein maturation and local microenvironmental differences between axenically and bacterially derived sorogens. The transient appearance of the MAb 4A11 epitope might be related to global disulfide rearrangements (34) or other posttranslational modifications such as proteolytic processing (32, 39) thought to occur during coat assembly. SP65 may be associated with the outer coat surface because the GFP of SP65-GFP is accessible at the spore surface based on Ab labeling and is likely exposed to proteases present within the spore coat and the interspore matrix (10, 19, 32). Indeed, SP65 is susceptible to proteolytic degradation when expressed in the growth medium (Fig. 4). SP65-GFP was less stable in culminants from axenically grown cells than in culminants from bacterially grown cells, which correlates with the earlier time of encapsulation in the rising sorogen from axenically grown cells noted when nascent spores were collected (data not shown). A longer period of turbulent exposure to proteases experienced by the earlier-forming spores might explain the higher degree of breakdown of SP65 and SP65-GFP.

Previous studies suggested that SP85 simultaneously contacts cellulose and SP65 to form a structural unit of the coat. Domain mapping studies showed this to be mediated by its 110-amino-acid C1 domain composed of four Cys-rich C4C motifs related to the N-terminal subdomain of EGF repeats (17, 35, 38). A direct SP65-SP85 interaction is confirmed here using recombinant proteins, which rules out a requirement for a coat-specific intermediary factor such as a coat polysaccharide. Interestingly, the in vitro interaction was detected using a spontaneously generated N-terminal fragment of SP65 (mycSP65N2) that was captured by the MAb 9E10 beads used to immunoprecipitate SP65. This region encompasses the C4C motifs of SP65. Full-length SP65 normally may fold in such a way that the C-terminal domain masks accessibility of the N terminus (where the myc epitope is located), but an effect of a distinct posttranslational modification of the recombinant protein cannot be excluded. Together these results imply a general function for C4C motifs in mediating protein-protein and protein-carbohydrate interactions. This is consistent with the observation that truncated SP65N1 (generated by the insertion of bsr) accumulates in the coat rather than the interspore matrix (Fig. 6). The accumulation of SP65 in the interspore matrix of SP85/pspB-bsr spores confirms previous findings that SP85 is required to retain SP65 in the coat in vivo (38).

The permeability and structural defects of SP65 and SP85 mutant coats and the dependence of certain SP85 functions on the cellulose binding activity of SP85 (17) suggest that the proposed cellulose-SP85-SP65 triad is essential for outer layer formation. Though the distinct properties of SP85 mutant spores, which tend to be spherical and to not rise to the top of the stalk (35), and SP65 mutant spores, which are more permeable without urea pretreatment (compare Fig. 11 and reference 38), suggest that the two proteins contribute separate functions, the comparison is complicated because the SP85 mutation is a null whereas the SP65 mutant produces a potentially dominant-negative N-terminal fragment (SP65N1). Nevertheless, the new data suggest that some activities of SP85 may be regulated by the availability of SP65, which is synthesized much later and transported to the cell surface via a largely distinct vesicle population.


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ACKNOWLEDGMENTS
 
We are grateful to Scherwin Henry at the University of Florida ICBR Hybridoma Laboratory for preparation of the anti-SP65 Abs, the OMRF DNA Sequencing Facility for DNA sequencing, T. Egelhoff for pTX-GFP, and D. Fuller and W. F. Loomis for anti-SpiA Ab. Scott Plafker and Margaret Clarke are thanked for access to fluorescence microscopes. Mandrin Shima is acknowledged for his help in the PCR studies.

This work was supported in part by National Science Foundation grant no. 0350516, the SURE Program sponsored by the Presbyterian Health Foundation, and grant BMC2002-01501 from the Dirección General de Investigación.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, 940 Stanton L. Young Blvd., BMS 853, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104. Phone: (405) 271-2227, ext. 1247. Fax: (405) 271-3139. E-mail: Cwest2{at}ouhsc.edu Back

{triangledown} Published ahead of print on 6 April 2007. Back

{dagger} Present address: Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, Baltimore, MD 21205. Back


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Eukaryotic Cell, July 2007, p. 1137-1149, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00329-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • West, C. M., Nguyen, P., van der Wel, H., Metcalf, T., Sweeney, K. R., Blader, I. J., Erdos, G. W. (2009). Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium. Eukaryot Cell 8: 27-36 [Abstract] [Full Text]  
  • Repass, S. L., Brady, R. J., O'Halloran, T. J. (2007). Dictyostelium Hip1r contributes to spore shape and requires epsin for phosphorylation and localization. J. Cell Sci. 120: 3977-3988 [Abstract] [Full Text]  

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