Lysosomes are primary components of the intracellular digestive system. Two processes that they participate in have been distinguished: sequestering of newly synthesized lysosomal enzymes, and processing of internalized membrane and its contents within an acidic environment. This project is concerned principally with developing a better understanding of lysosomal structure, biogenesis and function by studying two major integral lysosomal associated membrane glycoproteins, termed LAMP-1 and LAMP-2, which we have identified by use of monoclonal antibodies. The study will focus on the biochemical and cytological characterization of LAMP-1 and LAMP-2 as lysosome specific markers. Biochemical studies will involve protein purification, partial peptide sequencing, and characterization of the type and number of glycosylation sites. Structural studies are concerned principally with the topography of LAMP-1 and LAMP-2 in lysosomal vesicles of defined polarity. Genetic studies will include isolation and sequence analysis of full length cDNA. Biogenesis of these proteins will be analyzed by pulse-labeling and immunoprecipitation concomitant with analysis from specific subcellular fractions. An extension of this approach will be to prepare phenotypic mutants altered pharmacologically that will provide information regarding the importance of carbohydrate and peptide domains in proper cytological and biochemical expression. We expect that a comprehensive study of these major lysosomal components will enhance the understanding of the properties and dynamics of the lysosomal membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035405-02
Application #
3288095
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1986-04-01
Project End
1989-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
D'Souza, M P; Ambudkar, S V; August, J T et al. (1987) Reconstitution of the lysosomal proton pump. Proc Natl Acad Sci U S A 84:6980-4