The flies were prepared for scanning electron microscopy through a series of increasing concentrations of acetone. Dehydrated flies were then incubated in 1:1 acetone and HMDS for 24 hrs followed by incubation in 100% HMDS. The flies were allowed to air dry in HMDS in the hood. Dehydrated flies were mounted on Electron microscopy stubs. Flies were coated with gold using a Denton vacuum sputter coater and analyzed using a Hitachi S-4800 High Resolution Scanning Electron Microscope. We thank Mary Konsolaki and the Bloomington Stock Center for the Drosophila strains; and Kyung Ok Cho and the Developmental Studies Hybridoma Bank for the antibodies; and members of the Singh and Kango-Singh lab for critical comments on the manuscript. The ubiquitin proteasome system is well known to be involved in diverse cellular processes, including development, proliferation, transcription, signal transduction, apoptosis, and DNA repair[1,2,3,4]. Ubiquitin E3 ligases play central regulatory roles of UPS in that they provide substrate specificity and catalyze the ligation of ubiquitin to the substrate.

Our understanding of E3 ligases has been improved dramatically with the discovery of the RING domain as a module for E3 ligases. The RING domain comprises eight cysteine and histidine residues together that bind two atoms of zinc to form one unique cross-braced minidomain, yielding a rigid, globular platform for protein-protein interactions. RING domain proteins are comprising.95% of all predicted human E3 ligases[5], implying a very broad involvement of RING-dependent ubiquitination in vivo[6,7]. Ubiquitination plays the important regulatory role mainly targeting substrates for degradation by the 26S proteasome. However, the proteasome is limited in its capacity for degrading individual proteins. Removal of aggregated proteins, larger macromolecular complexes and whole organelles is mediated by autophagy, a catabolic process in which cytosolic cellular components are delivered to the lysosome for degradation. Ubiquitination has been proposed as a signal for selective autophagy[8], and the autophagy receptor proteins, such as p62 and NBR1, interact with both ubiquitin and autophagosomespecific Atg8-family proteins LC3/GABARAP, to promote autophagy[9]. The role of autophagy in the control of mitochondrial degradation is now generally recognized[10,11,12,13]. The autophagic uptake of mitochondria and their subsequent degradation in lysosome accentuates the importance of mitochondrial degradation by autophagy for cellular homeostasis. However, how mitochondria are selected for degradation by autophagy remains largely unknown.

The removal of mitochondria can be specific, and the signals that specify mitochondria as targets of the autophagical process have recently begun to be elucidated both in yeast and mammalian cells. In mammalian cells, the activation of mitochondrial permeability transition and loss of mitochondrial membrane Dabrafenib Abmole PAX5 interacts with RIP2 to promote NF-��B activation and drug-resistance in B-lymphoproliferative disorders potential appear to be common features of mitochondrial autophagy[18]. The reactive oxygen species of mitochondrial origin are also proposed as signaling molecules for mitochondrial autophagy regulation[19,20]. Bif-1 is involved in the regulation of mitochondria autophagy by stimulating Bax and interacting with Beclin 1 through UVRAG[ 21,22]. The fission/fussion machinery of mitochondria has also been associated with autophagy[11,23], although direct involvement has not been demonstrated. Despite the considerable progress in characterizing mitochondrial autophagy, relatively little is known about the genes that regulate selective autophagy of mitochondria through ubiquitination.