The mannose-6-phosphate (Man6P) receptor is important for the sorting and transport of newly synthesized acid hydrolases (lysosomal enzymes) from the Golgi complex to lysosomes in most mammalian cells. The long-range goals of this research is to determine the structure of the Man6P receptor and to correlate this information with its known functional properties. the first set of specific aims of this research will be 1) to obtain amino acid sequence data on the receptor; 2) to use this sequence data to generate domain-specific antibody probes, and 3) to use these antibodies to further elucidate the structural and functional domains of the receptor. First, sufficient quantities of affinity-purified receptor protein will be isolated for amino-terminal sequencing by a gas-phase protein sequenator. Both the intact molecule (Mr=240,000) and smaller peptide fragments, generated by limited proteolysis or specific chemical cleavages, will be analyzed to reveal the 10-40 amino-terminal sequences of the intact receptor and of cleaved polypeptides. Second, molecular biological approaches will be receptor and of cleaved polypeptides. Second, molecular biological approaches will be used to obtain structural information on the Man6P receptor by screening several rat liver cDNA libraries in the phage Lambdagtll expression vector, for phages expressing Man6P receptors (or portions thereof) using existing polyclonal anti-receptor antibodies. Positive colonies will be cloned, and the inserted DNA will be isolated and sequenced. The amino acid sequence of the receptor, deduced from the nucleotide sequence, will be used to chemically synthesize homologous polypeptides which, in turn, will be used to generate peptide-specific antibodies. These anti-peptide antibodies will serve as probes to further elucidate receptor structure and function. These studies should aid in elucidating the structure of the Man6P receptor for lysosomal enzymes and provide numerous probes for identifying the functional domains of this important transport molecule. A second set of specific aims will be to: 1) expand upon previous studies on the localization of receptors in the Golgi complex by biochemically and immunocyto-chemically assaying Golgi fractions and subfractions for the receptor (a putative cis Golgi marker); and, 2) to localize lysosomal enzymes by immunoelectron microscopy in various cell types. These studies will add to our knowledge of the pathways taken by lysosmal enzymes.
