FORMATION AND MAINTENANCE OF LYSOSOME STRUCTURE-FUNCTION

Project: Research project

Description

The long term objective of my research is to describe the precise molecular
structure and function of lysosomes. From biosynthesis to turnover, how do
lysosomes assemble, organize and activate their constituent enzymes, while
protecting the cell from autodigestion? Within this general framework, the
proposed experiments will specifically ask: 1) what protein sequence
determinants of cathepsin D, a major lysosomal protease, and pepsin, a
secreted protease, are responsible for directing these two highly
homologous digestive enzymes to different cellular compartments; ii) what
are the general cellular effects of a loss of cathepsin D activity in
lysosomes; iii) what is the physical disposition of cathepsin D in the
various cellular compartments including lysosomes, i.e. is it membrane
bound or soluble; and iv) what parameters define cathepsin D turnover or
degradation? To answer these questions it will be necessary to isolate functionally
expressible recombinant DNA molecules coding for cathepsin D and pepsin and
isolate cultured cells conditionally defective in cathepsin D activity.
The recombinant clones will be used to construct deletion and fusion
proteins which will, in turn, be assayed for complementation of the
cathepsin D deficient cells. This approach will allow rapid screening of
randomly constructed (i.e. shotgun) recombinant molecules and thus, a
complete search of cathepsin D and pepsin primary sequence for putative
sorting signals. To evaluate the effect of a loss of cathepsin D activity,
the conditionally defective cells will be characterized in terms of their
biochemical defect, changes in protein synthesis and turnover, morphology,
and growth characteristics. Pulse-chase cell fractionation procedures will
be used to study the turnover of cathepsin D as it relates to lysosome
structure and function in normal cultured cells. The studies of lysosome physiology, described here, are basic in nature but
will be valuable to our understanding of lysosome related pathologies at
the cellular and molecular level.
StatusFinished
Effective start/end date1/1/8612/31/90

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $105,810.00
  • National Institutes of Health

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Cathepsin D
Lysosomes
Maintenance
Pepsin A
Cultured Cells
Peptide Hydrolases
Cell Fractionation
Recombinant DNA
Firearms
Enzymes
Protein Sorting Signals
Proteins
Clone Cells
Pathology

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)