Chapter 13 Cytosolic LC3 Ratio as a Quantitative Index of Macroautophagy

Motoni Kadowaki, Md Razaul Karim

Research output: Contribution to journalReview articlepeer-review

101 Scopus citations

Abstract

Macroautophagy, an intracellular bulk degradation process and a typical form of autophagy in eukaryotes, is sensitive to physiological regulation, such as the supply and deprivation of nutrients. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays a critical role in macroautophagy formation and is considered a suitable marker for this process. In mammalian cells, there is a limitation for biochemical and morphological methods to monitor autophagy within a short period of time. During analysis of the subcellular distribution of LC3, we found that the cytosolic fraction contains not only a precursor form (LC3-I), but also an apparently active form, denoted as LC3-IIs. Both LC3-I and LC3-IIs in the cytosolic fraction, and thus the LC3-IIs/I ratio (designated the cytosolic LC3 ratio), were more responsive to amino acids than monitoring LC3-II or the LC3-II/I ratio in the total homogenate, and remarkably reflected the total proteolytic flux in fresh rat hepatocytes and the cultured H4-II-E cell line. Thus, in addition to representing a sensitive index of macroautophagy, examining the cytosolic LC3 ratio is an easy and quick quantitative method for monitoring the regulation of this process in hepatocytes and H4-II-E cells.

Original languageEnglish (US)
Pages (from-to)199-213
Number of pages15
JournalMethods in Enzymology
Volume451
Issue numberC
DOIs
StatePublished - 2009
Externally publishedYes

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