The long range goal of this research is to discover the biochemical mechanism of the regulation of lysosomal enzyme sorting. One of the most important steps toward this goal is to determine the underlying sorting mechanism in the organism. This laboratory has recently cloned and sequenced a complete cDNA copy of the Dictyostelium mRNA which encodes beta-hex, a lysosomal enzyme. The organism has been transformed with a vector designed to express the beta-hex and other coding sequences, and transformants overproduce active enzyme. Mutational analysis has begun and a beta-hex-invertase fusion has been prepared for use in testing candidate signals. These clones and vectors will be used to determine the sorting signal which directs the protein to lysosomes. The most likely signal is either a modified N-linked oligosaccharide, which is the signal for sorting of lysosomal enzymes in mammalian cells, or an amino-terminal polypeptide sequence which is the sorting signal for yeast vacuolar proteins. The immediate aims of this proposal are: 1. To evaluate the role of N-glycosides in the sorting of beta-hex. Potential glycosylation sites in the coding sequence will be altered, the mutated insert will be expressed in Dictyostelium, and the sorting of the altered protein will be determined. 2. To evaluate the role of amino-terminal domains in the sorting of beta-hex. Two approaches will be taken. In one, selected protions of the amino-terminal coding sequence will be deleted and the deleted insert will be subjected to the analysis described above. In another, a signal-reporter fusion system will be developed to search for or to confirm potential signals. The amino-terminal region may also contain information which directs intercalation of the protein into membrranes. Removal of this information may have indirect effects on sorting. Therefore, the planned analysis will include an evaluation of the effect of deletions and fusions on the membrane intercalation of the protein. The study of mechanisms by which lysosomal enzymes are sorted is a topic of intense scientific interest because of what it can teach us about the relationship of protein structure to protein transport into specific cellular organelles. Also, in complex organisms, lysosomal enzyme sorting is closely regulated during development because these enzymes serve a key role in molding of embryonic shape. Dictyostelium discoideum is the simplest organism in which such regulation is known to occur. Study of this organism thus offers a particularly interesting system in which to study the molecular mechanisms of lysosomal enzyme sorting in cells.
