Mitochondrial amino acid export preserves mitochondrial volume at the egg-to-embryo transition

In animals, the transition from egg to embryo marks a rapid shift in cellular physiology, yet how mitochondria adapt to support this conversion remains poorly understood. We performed a targeted RNAi screen of conserved, nuclear-encoded mitochondrial proteins in Drosophila melanogaster to identify maternal effects. We identified distinct phenotypic classes affecting mitochondrial distribution or morphology in early embryos. We focused on Sideroflexin-1/3 (Sfxn1-3), a conserved mitochondrial inner membrane transporter, because it uniquely affects embryonic mitochondrial morphology. Maternal loss of Sfxn1-3 causes striking mitochondrial enlargement in embryos but not in oocytes, placing the trigger at the egg-to-embryo transition. Combining mitochondrial immunopurification with LC/MS-based metabolomic profiling, we show that amino acids are markedly enriched in Sfxn1-3-deficient mitochondria in both oocytes and embryos. This accumulation developmentally uncouples the metabolic and morphological phenotypes. By selectively disrupting discrete steps of egg activation, including translational activation, cell cycle re-entry, and fertilization, we demonstrate that mitochondrial swelling is independent of each, prompted instead by egg hydration. Beyond its reported role in proliferating cells as a serine importer, our data support a role for germline Sfxn1-3 in regulating inner mitochondrial membrane permeability to osmotically active amino acids. This activity is crucial for preserving organelle volume during the osmotic challenge of oocyte hydration. When this export fails, amino acid accumulation amplifies osmotic stress and drives rapid mitochondrial swelling. Together, these findings identify mitochondrial inner membrane permeability to amino acids as a key regulatory step that couples pre-established metabolite compartmentalization to osmotic adaptation at egg activation, and reveal how mitochondrial metabolic integrity and volume control are coordinated at this critical developmental transition.