The project focuses on studies of the biosynthesis and function of D-LAMP-2, a Dictyostelium protein which appears to be a homologue of the human lysosomal associated membrane protein 2 (H- LAMP-2). Antibodies to H-LAMP-2 and oligonucleotide probes will be used to clone and sequence the cDNA coding for D-LAMP-2. Radiolabel pulse-chase, microscopy, and subcellular fractionation approaches will be used to examine the transport and processing of D-LAMP-2 in growing cells, and in mutant Dictyostelium cells altered in secretion and transport of lysosomal hydrolases. The biosynthesis and intracellular location of D-LAMP-2 will also be examined as a function of the developmental cycle of Dictyostelium. Also, antisense RNA and gene disruption techniques will be used to generate strains unable to produce D-LAMP-2. The function of the lysosomal system will be assessed by: determining if "normal" lysosomes are formed; examining the transport, sorting, and secretion of lysosomal enzymes; measuring the rate of endocytosis and phagocytosis; and determining if cells undergo normal differentiation. Together, these approaches will provide relevant new information on the role of LAMPs in the biosynthesis and function of lysosomes in growing and differentiating cells. Until now, most studies on lysosomal biogenesis (in both animal and slime mold systems) have focused on how the lysosomal enzymes enter the lysosomal compartment. Recently, a few lysosomal membrane marker proteins have been identified in mammalian cells; while these have been well characterized with respect to protein structure, there is as yet no clue as to their possible function. The discovery of a possible homologue of one of these lysosomal membrane proteins in the slime mold Dictyostelium comes at a fortuitous time, since it is only recently that Dictyostelium has become readily amenable to genetic and molecular-genetic manipulations. It is now possible to create mutant strains of the cells with specific genetic alterations. By creating mutants with specific alterations in the LAMP homologue, and assessing the resultant phenotypes with respect to lysosomal function, it should be possible to address the question of function of these proteins.
