Induction of Autophagy by Amino Acid Starvation in Fish Cells

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish.