Mechanisms of regulation of autophagy in Dictyostelium discoideum.

摘要

Dictyostelium is a social amoeba that lives a solitary life in the soil and feeds on bacteria. When food is scarce, Dictyostelium cells start development to form a fruiting body, which contains spores that are resistant to harsh conditions supported by inviable stalk cells.; Previously, our laboratory identified a mutant called chtA (now called fbxA) that cheated by preferentially forming viable spores, not the inviable stalk cells. The FbxA protein has F-box and WD domains, which are found in proteins that are involved in degradation of target proteins.; We asked how this gene is regulated and searched for proteins that interact with FbxA. We have recovered the promoter of fbxA and shown that its expression pattern is mostly prestalk-specific. We were able to select suppressor mutations of the fbxA mutant that allow the formation of fruiting bodies containing detergent-resistant spores. The suppressor mutations affect regA and dhkA, which are phosphodiesterase and histidine kinase genes respectively.; To cope with harsh conditions, eukaryotic cells developed a mechanism by which they recycle their cellular components, called 'autophagy'. Autophagy is essential for cells to survive during starvation. To provide energy and material for development, Dictyostelium cells induce autophagy.; We focused on Dictyostelium Atg1 (DdAtg1), which is a kinase that mediates initiation of autophagy. The atg1 - mutant had growth defects in minimal medium, unlike other autophagy mutants. We have eliminated the kinase activity of DdAtg1 and found that this activity is essential for autophagy and development. The C-terminal domain in DdAtg1, which has homologues in C. elegans and mammals, is also essential for the activity of DdAtg1 in autophagy and development.; To find novel proteins that are involved in autophagy, but not present in yeast, we screened thousands of colonies of Dictyostelium mutants. We screened for strains that are defective during development, can not survive well when starved, and do not change their density during starvation. One of the mutants, Hrm13 (now called atgN) behaved like an autophagy mutant. The disrupted gene was isolated and found to be a novel gene with homologues in worms and mammals, but not in budding yeast.