Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment

Wentao Gao, Jeong Han Kang, Yong Liao, Wen Xing Ding, Andrea A. Gambotto, Simon C. Watkins, Yong Jian Liu, Donna B. Stolz, Xiao-Ming Yin

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Autophagosomes and their precursors are best defined by electron microscopy but may also be traced in living cells based on the distribution of specific autophagy molecules. LC3, the most commonly examined autophagy marker in mammalian cells, labels structures that are frequently manifested as dots or rings using light microscopy; however, the nature of these structures is not entirely clear. We reported here a novel approach to examine the LC3-positive compartment in cell-free lysates, which revealed that they were actually tubulovesicular structures with considerable heterogeneity. Using affinity purification, we isolated these membranes for electron microscopy, which indicated that they possessed ultrastructural features consistent with autophagosomal membranes at various maturation stages. Further biochemical and proteomics analyses demonstrated the presence of multiple autophagy-related and other functional molecules. The different distribution patterns of Atg5, Atg16, Atg9, and p62/SQSTM1 on the LC3-positive compartment provided new clues on how these molecules might be involved in the dynamics of the autophagosomal membranes. Finally, several morphologically unique groups of LC3-positive membranes were categorized. Their topological configurations suggested that double-membrane vesicles could be derived from single membrane compartments via different means, including tubule-to-vesicle conversion, whose presence was supported by live cell imaging. These findings thus provide new information on the dynamics of the autophagosomal compartment.

Original languageEnglish (US)
Pages (from-to)1371-1383
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number2
DOIs
StatePublished - 2010
Externally publishedYes

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Membranes
Autophagy
Electron microscopy
Molecules
Electron Microscopy
Cells
Proteomics
Optical microscopy
Purification
Labels
Microscopy
Imaging techniques
Light

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment. / Gao, Wentao; Kang, Jeong Han; Liao, Yong; Ding, Wen Xing; Gambotto, Andrea A.; Watkins, Simon C.; Liu, Yong Jian; Stolz, Donna B.; Yin, Xiao-Ming.

In: Journal of Biological Chemistry, Vol. 285, No. 2, 2010, p. 1371-1383.

Research output: Contribution to journalArticle

Gao, W, Kang, JH, Liao, Y, Ding, WX, Gambotto, AA, Watkins, SC, Liu, YJ, Stolz, DB & Yin, X-M 2010, 'Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment', Journal of Biological Chemistry, vol. 285, no. 2, pp. 1371-1383. https://doi.org/10.1074/jbc.M109.054197
Gao, Wentao ; Kang, Jeong Han ; Liao, Yong ; Ding, Wen Xing ; Gambotto, Andrea A. ; Watkins, Simon C. ; Liu, Yong Jian ; Stolz, Donna B. ; Yin, Xiao-Ming. / Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 2. pp. 1371-1383.
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