Autophagy of cytoplasmic bulk cargo does not require LC3
Nikolai Engedal1, Per O Seglen1
1a Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo , Oslo , Norway.
Autophagy
|August 4, 2015
Summary
LC3 protein processing and bulk cargo flux in autophagy appear independent. LC3 is not a reliable general indicator for macroautophagy, despite GABARAP family proteins being crucial.
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- Autophagy is a cellular process for degrading damaged components.
- LC3 (microtubule-associated protein 1A/1B-light chain 3) is a common marker for autophagy.
- The relationship between LC3 processing and bulk cargo flux in macroautophagy remains unclear.
Purpose of the Study:
- To investigate the role of LC3 in bulk autophagy.
- To compare autophagic-lysosomal LC3 processing with bulk cargo flux.
- To determine if LC3 is a suitable general indicator for macroautophagy.
Main Methods:
- Quantitative immunoblotting for LC3 processing.
- Lactate dehydrogenase (LDH) sequestration assay for bulk cargo flux.
- Amino acid-starved rat hepatocytes treated with cycloheximide.
- Block-release experiments with 3-methyladenine (3MA) and thapsigargin (TG).
- siRNA-mediated knockdown of LC3 and GABARAP family proteins in LNCaP cells.
Main Results:
- 3MA suppressed LC3 lipidation, while both 3MA and TG inhibited cargo sequestration.
- LC3 flux resumed without bulk cargo flux after release from TG blockade.
- Bulk cargo flux continued despite LC3 flux halting after cycloheximide treatment.
- Cytoplasmic bulk cargo autophagy was LC3-independent, but GABARAP family proteins were essential.
Conclusions:
- Autophagic-lysosomal LC3 processing and bulk cargo autophagy (macroautophagy) can be mutually independent.
- LC3 is unsuitable as a general indicator for macroautophagy.
- GABARAP family proteins play a critical role in bulk cargo autophagy.
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