Mitochondrial protein nmd regulates lipophagy and general autophagy during development

Lipophagy engulfs lipid droplets and delivers them to lysosomes for degradation. We found that lipophagy levels were low in most fly tissues, except for the prothoracic gland (PG) during larval development. Therefore, we performed a small-scale screening in the PG to identify regulators of lipophagy. We discovered that the loss of nmd, a gene encoding a mitochondrial AAA-ATPase, led to developmental failure and reduced lipophagy in the PG. Further studies indicated that nmd was not only required for lipophagy but also essential for general macroautophagy/autophagy in both PG and fat body tissues. Autophagy was induced but blocked at the autophagosome-lysosome fusion stage upon nmd reduction. Additionally, nmd interacted with mitochondrial protein import machinery, such as Tom20, Tom40, and the import cargo, such as Idh. Loss of nmd decreased protein import into mitochondria. Similar to the loss of nmd, reduction of Tom20 or Tom40 also resulted in reduced lipophagy in the PG. In adult flies, reducing nmd expression in the eyes caused lipid droplet accumulation and severe degeneration during aging. Overexpression of bmm, a triglyceride lipase, reduced lipid droplets in the eye but did not rescue the eye degeneration caused by the reduction of nmd.Abbreviation: ATAD1: ATPase family AAA domain containing 1; Atg8a: Autophagy-related 8a; Atg9: Autophagy-related 9; Atg14: Autophagy-related 14; Atg18a: Autophagy-related 18a; ATP: adenosine triphosphate; bmm: brummer; CtsL1: Cathepsin L1; Idh: isocitrate dehydrogenase (NADP+); Cis1: CItrinin Sensitive knockout; GFP: green fluorescent protein; LDs: lipid droplets; LIRs:LC3-interacting regions; Lsd-1: Lipid storage droplet-1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Marf: Mitochondrial assembly regulatory factor; Miga: Mitoguardin; Msp1: Mitochondrial Sorting of Proteins 1; nmd: no mitochondrial derivative; PG: prothoracic gland; phtm: phantom; PNPLA2/ATGL: patatin like domain 2, triacylglycerol lipase; RFP: red fluorescent protein; RNAi: RNA interference; Syx17: Syntaxin 17; TA: tail-anchored; TEM: transmission electron microscopy; TOMM: translocase of outermitochondrial membrane; Tom20: Translocase of outer membrane 20; Tom40: Translocase of outer membrane 40.