A combined proteomic and genetic analysis identifies a role for the lipid desaturase Desat1 in starvation-induced autophagy in Drosophila

Abstract

Autophagy is a lysosomal-mediated degradation process that promotes cell survival during nutrient-limiting conditions. However, excessive autophagy results in cell death. In Drosophila, autophagy is regulated nutritionally, hormonally and developmentally in several tissues, including the fat body, a nutrient-storage organ. Here we use a proteomics approach to identify components of starvation-induced autophagic responses in the Drosophila fat body. Using cICAT TM labeling and mass spectrometry, differences in protein expression levels of normal compared to starved fat bodies were determined. Candidates were analyzed genetically for their involvement in autophagy in fat bodies deficient for the respective genes. One of these genes, Desat1, encodes a lipid desaturase. Desat1 mutant cells fail to induce autophagy upon starvation. The desat1 protein localizes to autophagic structures after nutrient depletion and is required for fly development. Lipid analyses revealed that Desat1 regulates the composition of lipids in Drosophila. We propose that Desat1 exerts its role in autophagy by controlling lipid biosynthesis and/ or signaling necessary for autophagic responses.

DrosophilaAcyl-CoA Synthetase Long-Chain Family Member 4 Regulates Axonal Transport of Synaptic Vesicles and Is Required for Synaptic Development and Transmission

“…A dAcsl-null allele, dAcsl KO , was generated by flippase-recognition target-mediated deletion between PBac{RB} Acsl e02676 and PBac{WH}Acsl f02764 , removing most of the exons according to a procedure described by Parks et al (2004) and Thibault et al (2004). Other stocks include UAS-mito-GFP from W. M. Saxton (University of California, Santa Cruz, CA) (Pilling et al, 2006), UAS-SyteGFP (III ) (Zhang et al, 2002), UAS-RFP-Atg8 from E. Hafen (Swiss Federal Institute of Technology, Zürich, Switzerland) (Köhler et al, 2009), UAS-LAMP1-GFP from H. Krämer (University of Texas Southwestern Medical Center, Dallas, TX) (Pulipparacharuvil et al, 2005), elav-GeneSwitch from H. Keshishian (Yale University, New Haven, CT) (Osterwalder et al, 2001), the motoneuron-specific OK6-Gal4 from M. B. O'Connor (University of Minnesota, St. Paul, MN), D42-Gal4 from W. M. Saxton, and the ubiquitous Tub-Gal4 from the Bloomington Stock Center (Indiana University, Bloomington, IN). For GeneSwitch rescue experiments, dAcsl 05847 /dAcsl KO ; elav-GeneSwitch/UAS-ACSL4-Myc mutant larvae at 2.5 d after egg laying (AEL) were transferred to food containing RU486 (mifepristone; Sigma) at a final concentration of 0.5 mM to induce neuronal expression of human ACSL4-Myc following the original protocol (Osterwalder et al, 2001) and then were dissected at 5 d AEL to examine axonal aggregates.…”

“…Autophagosomes can be generated locally within axons and then transported retrogradely for fusion with lysosomes (Hollenbeck, 1993;Yue, 2007). We expressed the autophagosomal marker red fluorescent protein (RFP)-Atg8 (Köhler et al, 2009) in motoneurons driven by OK6-Gal4 and found obvious Atg8-positive aggregates in dAcsl mutants, but no appreciable aggregates in wild types (Fig. 3B1,B1Ј).…”

Dietary rescue of altered metabolism gene reveals unexpected Drosophila mating cues

“…Similarly to what occurs in the null homozygous mutant alleles, desat1 -1573-C'1 ( 19 ) and 11A ( 20 ), we found that the double transgenic genotype combining a copy of the full desat1 regulatory region (6,908 bp) fused with Gal4 driving a copy of the UAS-desat1 -IR transgene (+/6908-Gal4; UAS-desat1 -RNAi/+; this genotype is hereafter designated as "IR") died during second instar stage (L2) when raised on standard lab food (corn/yeast/agar). Therefore, this IR genotype produced no viable adult progeny ( Fig.…”

“…If these transgenes also affect desat1 in tissues where it is not expressed, this should have no functional consequence. The 1573-C'1 and 11A lines were generated by the imprecise excision of the P element, either from the original mutant desat1 1573 allele [1573-C'1 ( 19 )] or from the desat1 EY07679 allele [11A ( 20 )] using ⌬ 2-3 genomic source of transposase. These lines are missing the desat1 gene coding region and are homozygous lethal.…”

Quantitative proteomics for the analysis of spatio-temporal protein dynamics during autophagy

“…84 Also in Drosophila, a combination of genetic and proteomic analyses determined that the lipid desaturase Desat1 localizes to autophagosomes under starvation conditions and is important for autophagy induction. 85 The relative abundance of proteins in mammalian cells during 36 h of amino acid starvation was measured by SILAC-based quantitative MS. Kinetic profiles of 1,486 proteins were recorded suggesting an ordered degradation of cellular components during starvation-induced autophagy.…”