Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

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.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 c...

Dictyostelium Hip1r contributes to spore shape and requires epsin for phosphorylation and localization

“…This regulated secretion is a prerequisite for both the synthesis and the organization of cellulose in the spore coat (Srinivasan et al, 2000b;Metcalf et al, 2003). After the spore coat proteins are secreted to the extracellular side, the enzyme responsible for cellulose synthesis, cellulose synthase, is positioned in the plasma membrane and synthesizes cellulose directly into the outer layer of the spore coat (Metcalf et al, 2007;West, 2003;Metcalf et al, 2003;Srinivasan et al, 2000b).…”

“…Hip1r mutant spores stain with SP70, a glycoprotein housed within the pre-spore vesicle, so Hip1r is not required for the overall secretion of PSV contents. The spore coat contains a minimum of ten different glycoproteins that are secreted through at least two different pathways (Metcalf et al, 2007;West, 2003). As a scaffolding protein with binding sites for actin, the plasma membrane and a coiledcoil domain, it is conceivable that Hip1r could be required for the proper positioning of any of the many factors involved in the organization of the spore coat.…”

Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium

“…A function for these two genes in GPS biosynthesis is documented here, and this has in turn revealed that the GPS plays an important role in cellulose deposition, protein incorporation, and the resistance of spores to stress. The effect on spore stress resistance is profound: while less severe than disruption of the dcsA cellulose synthase gene itself (47), it is more severe than coat structural protein mutants examined up to now (23,30,37).…”

“…This physicochemical barrier protects the enclosed amoeba from external stress and probably actively regulates terminal sporulation and spore germination. The spore coat is formed de novo from four known sources: (i) an early-formed pool of proteins and (ii) a galactose-rich polysaccharide (GPS) stored together in prespore vesicles (PSVs) of the slug, (iii) a late-formed pool of protein(s), including SP65 (23), and (iv) cellulose formed de novo at the cell surface. These components are separately deposited at the cell surface, where they organize into an asymmetrical trilaminar "sandwich" with proteins on either side enclosing the polysaccharides in the interior (see Fig.…”

“…More than 10 coat structural proteins are known and mutational studies have revealed essential roles for outer layer proteins in coat permeability (23,37). An inner layer protein, SP85, forms direct contacts with cellulose and the coat protein SP65 and is important for morphogenesis of both cellulose and the outer layer (22).…”