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Related Experiment Videos

Polypeptide import and degradation by isolated lysosomes

S R Terlecky1, J F Dice

  • 1Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.

The Journal of Biological Chemistry
|November 5, 1993
PubMed
Summary

Lysosomes import and degrade proteins via a pathway selective for KFERQ-like motifs, enhanced by serum deprivation and heat shock cognate protein 73 (hsc73). This pathway involves specific substrate binding and is saturable.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Lysosomes are key organelles for protein degradation.
  • Previous work showed lysosomes import and degrade polypeptides, stimulated by ATP and hsc73.
  • The precise mechanisms and regulation of this lysosomal proteolytic pathway were not fully understood.

Purpose of the Study:

  • To further characterize the in vitro proteolytic pathway of lysosomes.
  • To identify substrate selectivity and binding characteristics.
  • To investigate the role of cellular conditions and hsc73 in regulating lysosomal protein degradation.

Main Methods:

  • In vitro assays using lysosomes from human diploid fibroblasts.
  • Testing of various polypeptides for import and degradation.
  • Analysis of substrate binding to lysosomal membranes.
  • Comparison of lysosomal activity under different serum conditions.
  • Investigation of hsc73 localization and regulation.

Main Results:

  • The pathway selectively degrades polypeptides containing KFERQ-like peptide motifs.
  • Substrate proteins bind to a specific protein site on lysosomal membranes.
  • Lysosomes from serum-deprived cells exhibit doubled proteolytic activity.
  • Intracellular hsc73 associates with lysosomes, increasing upon serum withdrawal.
  • The import process is saturable (Km = 5 microM for RNase S-peptide) and activated by reducing agents.

Conclusions:

  • Lysosomal protein import and degradation is a selective, saturable process regulated by substrate motifs and cellular conditions.
  • Serum deprivation and associated hsc73 increase lysosomal proteolytic capacity.
  • This pathway represents a significant mechanism for cellular protein turnover.

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