Isolation and Characterization of Rat Liver Amphisomes. Evidence for Fusion of Autophagosomes With Both Early and Late Endosomes

J Biol Chem. 1998 Aug 21;273(34):21883-92. doi: 10.1074/jbc.273.34.21883.

Abstract

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonia / pharmacology
  • Animals
  • Asparagine / pharmacology
  • Endocytosis
  • Endosomes / physiology*
  • Gold / pharmacokinetics
  • Leupeptins / pharmacology
  • Liver / cytology*
  • Liver / metabolism
  • Male
  • Microscopy, Electron
  • Phagosomes / physiology*
  • Propylamines / pharmacology
  • Rats
  • Rats, Wistar

Substances

  • Leupeptins
  • Propylamines
  • Asparagine
  • Gold
  • Ammonia
  • leupeptin