Cytosolic LC3 ratio as a quantitative index of macroautophagy.
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.
Kadowaki
Motoni
M
Department of Applied Biological Chemistry, Faculty of Agriculture, Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Karim
Md Razaul
MR
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D051381:Rats
D008869:Microtubule-Associated Proteins; C526109:Anxa3 protein, rat