The study of mechanisms by which lysosomal enzymes are sorted is teaching us about the relationship of protein structure to protein transport. In complex organisms, lysosomal enzymes serve a key role in embryonic re-sculpting. Their sorting is regulated during development. The long range goal of this project is to discover the molecular details of this regulation. Dictyostelium discoideum is a simpler organism whose development is well studied. A complete CDNA copy of the Dictyostelium MRNA which encodes beta- hexosaminidase (beta-hex), a lysosomal enzyme, has already been cloned and sequenced. This was used to transform the organism with a vector designed to express proteins accurately. It was shown that the transformants overproduce active enzyme, which is appropriately sorted. Also, a beta-hex-invertase fusion system was developed for evaluation of candidate signals. That study led to the discovery of a signal which sorts proteins to a vesicle whose contents are stored during growth and secreted during development. It also led to the hypothesis that lysosomal enzymes may be directed from organelle to organelle by more than one receptor, or by the combination of one receptor ligand interaction and other features of the enzyme structure. This will be pursued through the following specific aims. The first is to determine the exact chemical nature of the signal which is required to sort protein to the lysosomal pathway. This includes: a, determination of the minimal biologically functional sequence and the extent to which that sequence can be varied but still lead to proper sorting; b, confirmation that the sequence acts as a ligand for a receptor and purification of the receptor; and c, analysis of the specificity of the ligand-receptor interaction with synthetic peptides. The second is to test the idea that features of proteins, other than recognition markers, are required for proper sorting to lysosomes, specifically, to test theories that explain why a terminal beta-hex sequence is necessary. %%% A key feature of eukaryotes is that their cells are physically compartmentalized; that is, the cytoplasm is not a uniform mixture of cellular components, but rather, is highly organized in such a way that specific cellular functions, and the components that carry out those functions, are localized in discrete regions of the cell. Some of these functionally-distinct compartments are bounded by membranes, and can be considered to be "organelles." Since most of the cell's proteins are produced on cytoplasmic ribosomes, it follows that there must be mechanisms to ensure that specific proteins get to their specific subcellular locations. One well- studied example of this is the problem of how newly-synthesized lysosomal (degradative) enzymes get to the lysosomes (specialized membrane-bounded compartments responsible for enzymatic degradation of macromolecules). Nature seems to have provided multiple mechanisms for this to occur. Although the best-studied mechanism involves membrane-bound receptors which recognize mannosyl-6- phosphate residues at the terminal position of asparagine-linked oligosaccharides on lysosomal enzymes, it is not the only mechanism for proper targeting of lysosomal enzymes. In the slime mold Dictyostelium discoidum, mannosyl-6-phosphate receptors have been looked for but not found; therefore lysosomal enzyme targeting in these cells must be via one or more alternative mechanisms. This project holds the promise of elucidating details of at least one of these alternative mechanisms. Detailed understanding of the structural requirements for a protein to be targeted to a specific subcellular location is of basic importance to the biotechnology industry; it is expected that the information learned from this project will allow molecular engineers to design proteins more intelligently for production in bioreactors.
