Structure-function Analysis of the SP85/PsB Spore Coat Protein in Dictyostelium

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Cell walls are essential for the life of many microbial eukaryotes, plants and animals. Polysaccharides including cellulose are a major component of many walls. Studies of protein-rich walls in animals, Chlamydomonas and Volvox have shown that cell-matrix and matrix-matrix interactions are mediated by highly specific contacts involving discrete protein domains. The goal of this project is to study the spore coat of Dictyostelium in order to understand, in the long term, the critical roles played by proteins in the assembly of cellulose-rich walls. Like many cell walls, the spore coat consists of a central cellulose-rich region surrounded on either side by protein-rich layers. One of the major coat proteins, SP85 or PsB, is a prime suspect for coordinating cellulose- protein interactions in the coat. SP85 is located in the inner layer of the coat near the plasma membrane, and can simultaneously bind cellulose and another coat protein, SP65, in vitro . Based on the phenotypes of SP85 knockout strains and strains in which individual domains have been overexpressed, SP85 is thought to contribute to both the timing of cellulose synthesis (early function) and the organization of the outer layer including incorporation of proteins and confinement of cellulose (late function). One construct, a fusion of the N-terminal and cysteine-rich C1-domains, blocks a checkpoint required for cellulose synthesis in 85% of the cells, and disrupts the outer layer in the 15% that break through the block. The C1-domain uniquely binds both cellulose and SP65.To determine the role of binding of SP85 to cellulose and SP65 for function in vivo, the following experiments are planned: 1), the gene for full-length SP85 expressed in growing cells, from which it is secreted away from other coat proteins, will be subjected to random mutagenesis in its C1-domain to specifically block either cellulose-or SP65-binding; 2), the disulfide-bonding and glycosylation patterns of the C1-domain and nearby mucin-like domains will be mapped and used to monitor the structures of normal and mutant SP85s; and 3), the inactivating mutations defined in aim 1 will be introduced into SP85 and NC1 expressed in prespore cells and incorporated into the coat. Phenotype analyses will assess how the early and later SP85-dependent functions are affected. The results are expected to provide information about how SP65 regulates SP85 action in checkpoint inhibition, the hypothesized adaptor role for SP65 in bridging inner-and outer-layer proteins, and whether cellulose-binding is important for localizing SP85 and/or in tethering and organizing cellulose microfibrils. An additional set of experiments, aimed at testing the specificity of mutations that block SP65-binding, will also be carried out if time permits. These approaches will test the in vivo relevance of specific domain activities defined by in vitro studies, therefore avoiding potential artifacts intrinsic to the sole use of partial-length expression constructs. The cellulose-SP85-SP65 trimer is predicted to be a coat assembly core module which can be extended in future studies to understand how discrete protein domains elsewhere in SP85 and in other coat proteins function to organize wall assembly.Broader impacts: The structure-function relationships to be defined for the cysteine-rich domains of SP85 are expected to be generalizable to related domains of other cellulose-rich walls found in plants and eukaryotic microbes of economic and health significance. Ultimately, these studies are likely to establish useful precedents for how cellulose synthase is regulated, how cellulose is crystallized, how cellulose fibrils are constrained within the outer protein layer, and how cell wall layers are formed. The project will continue to engage the participation of graduate,undergraduate and high school students, including women and underrepresented minorities,as research trainees in execution of the aims consistent with the educational commitment of the PI in his academic setting. There will be significant interaction with core laboratories and other research laboratories in both the USA and internationally. These activities will contribute to the overall critical mass of effort in proteomics, glycobiology and cell wall biology, and is expected to constitute a continuing resource for the overall university community. All findings will be shared with the scientific community in timely fashion via meetings and publications.