Acinus: A nuclear regulator of autophagy and endocytic trafficking

Fusion with lysosomes is the common last step of endocytic trafficking and autophagy. Accordingly, several proteins are required in both pathways for cargoes to reach their destinations. Among these proteins, Drosophila Acinus stands out, as it exerts opposite effects on these two pathways, and thus establishes a new paradigm. Loss of Acinus function destabilizes early endosomes, thereby promoting the delivery of their cargo to lysosomes. By contrast, the maturation of autophagosomes to autolysosomes is inhibited in acn mutant cells. The increase in autophagy upon Acinus overexpression and its location to the nucleus are consistent with Acinus being a novel regulator of autophagy.Key words: fat body, endosomes, lysosomes, nuclear protein, Notch signaling, EGF ReceptorMuch of the core machinery that is required for the formation and maturation of autophagosomes and endosomes has been identified by genetic screens in yeast. But as both types of organelles are charged with more complex functions in multicellular organisms, it is not surprising to find additional layers of regulation imposed on them. One such regulatory element was revealed by a genetic screen we conducted in Drosophila.The screen''s original idea was to take advantage of the observation that many proteins acting in trafficking to lysosomes also function in the biogenesis of lysosome-related organelles. Among these, the pigment granules—responsible for the characteristic color of the fly eye—are easily scored for defects. Thus, we set up a primary screen for eye color mutants. Among the more than 500 original hits, a secondary screen identified those mutants that altered endocytic trafficking. Importantly, the genetic tool kit assembled by the fly community allowed us to screen homozygous mutant eyes in otherwise heterozygous flies. This schema made it possible to identify mutations that are homozygous lethal as one might expect for null alleles of genes required for lysosomal delivery.One of the unexpected genes identified by this screen was acinus (acn). The Acn protein lacks any domain signatures and is most similar to human Acinus, which had been implicated in the destruction of chromatin during apoptosis. It is not clear yet whether the Drosophila protein contributes to this function as well, but in acn null alleles chromatin condensation and fragmentation during apoptosis appear normal.There is, however, a profound effect on endocytic trafficking, as acn is required for stabilization of early endosomes. Staining for endocytosed ligands, such as Boss or Delta, is drastically reduced, concomitant with a reduction in early endosomes marked by Rab5 or the SNARE Avl. By contrast, late endosomes marked by Rab7 appear normal. These changes do not represent a block in the initial internalization of the ligands, as inhibition of lysosomal degradation reveals the same accumulation of internalized ligands in wild-type and acn mutant cells.Reduced stability of early endosomes also causes reduced signaling from EGF receptors and Notch, consistent with the emerging notion that signaling from these receptors may be linked to their uptake into early endosomes.Many mutants that disrupt endocytic trafficking also affect autophagy. We found that this theme extends to acn. The most accessible form of autophagy in Drosophila is found in fat bodies after a short period of starvation. Activation of the AKT1/TOR pathway triggers the formation of autophagosomes, which mature into autolysosomes by fusing with lysosomes. Loss of acn interferes with this maturation step, as shown by the reduction in LysoTracker staining and also by quantitative electron microscopy. Consistent with an effect on the maturation of autophagosomes, acn is required downstream of TOR signaling. For example, expression of dominant-negative TOR kinase is a powerful tool to induce autophagy in the fat body of wild-type, but not acn larvae.Interestingly, overexpression of Acn induces autophagy. This does not appear to be merely a side effect. Ubiquitous expression of Acn is lethal, but flies survive when autophagy is suppressed by knockdown of ATG5, a core element of the autophagy machinery. We find that this enhanced autophagy is also independent of the TOR pathway.Taken together, this analysis of the first null mutant of an acinus gene in any system reveals its function as a regulator of endosomal and autophagosomal dynamics, modulating developmental signaling and the cellular response to starvation. Our investigation of acn loss-of-function phenotypes reveals defects in membrane trafficking during endocytosis and autophagy. We were therefore surprised that Acn protein localized to the nucleus, and that we failed to detect any consistent localization to endocytic or autophagic structures. This unexpected finding was further tested with transgenes expressing Myc-tagged Acn in the context of a genomic rescue construct. This tagged protein, under control of its endogenous enhancer/promoter elements, rescued all aspects of Acn function, and, nevertheless, localized to the nucleus, rather than any endosomal compartment.These findings suggest that the mechanism by which Acinus proteins modify endocytosis and autophagy may be indirect. One model for such an indirect effect is suggested by the interaction of mammalian Acinus proteins with several RNA binding proteins. Modulation of the levels or structure of RNAs that encode specific elements of the endocytosis or autophagy pathways may constitute an exciting new element of their regulation. Testing this possibility and identifying potential targets regulated by this Acn-dependent mechanism are important challenges that we have just begun to address.