Recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth.

Autophagy is a bulk degradation process conserved from yeast to mammals. To examine the roles of autophagy in cellular metabolism, we generated autophagy-defective (atg) mutants in the X2180-1B strain background. We compared the growth of wild-type (WT) and atg cells in minimal (synthetic dextrose, SD) and rich (yeast extract/peptone/dextrose, YEPD) medium, and we found that mutations in the core autophagy machinery result in defects in the diauxic shift, the transition from fermentation to respiratory growth upon glucose depletion, specifically in SD. Furthermore, we confirmed that autophagy was induced prior to the diauxic shift, implying that it plays a role in this process. In YEPD, atg mutants grew normally, so we assumed that the insufficiency of certain nutrients in SD was responsible for the defects. We ultimately identified iron, which is a necessary cofactor for respiratory activity, as the nutrient required for the diauxic shift in atg mutants. Indeed, atg mutants exhibited defects in respiration, which was rescued by supplementation with iron. Based on these data, we hypothesized that autophagy is involved in iron recycling during the diauxic shift. smf3Δfet5Δ or smf3Δftr1Δ cells, which are unable to export iron from the vacuole, also exhibit defects in the diauxic shift, so iron released from the vacuole is important for the shift in SD medium. Finally, we observed that smf3Δfet5Δ cells accumulated nearly twice as much vacuolar iron as smf3Δfet5Δatg2Δ cells, suggesting that autophagy is involved in iron recycling by the vacuolar transport and degradation of iron-containing cargos.

Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression.

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. We have established an in vitro system that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. We find that Ago1-RISC (RNA-induced silencing complex) represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. Our finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis.