We discovered that Nε-lysine acetylation occurs in the lumen of the endoplasmic reticulum (ER) in 2007. From that initial finding, we went on to discover the entire ER acetylation machinery (one membrane transporter, AT-1/SLC33A1, and two acetyltranferases, ATase1 and ATase2) and uncover a novel piece of ER biology. Specifically, we discovered that the ER acetylation machinery regulates proteostasis within the ER and secretory pathway by maintaining the balance between quality control/engagement of the secretory pathway and reticulophagy. By using a combination of biochemistry and high-definition mass spectrometry, we discovered that SLC25A1 and SLC13A5 act as important “metabolic partners” of AT-1. Homozygous mutations in AT-1/SLC33A1, SLC25A1 or SLC13A5 are associated with developmental delay of the brain and early forms of encephalopathy while heterozygous mutations are associated with similar forms of hereditary sensory and autonomic neuropathies (HSANs), including specific forms of spastic paraplegias. Important, these mutations either introduce a premature STOP codon or cause loss-of-function of the transporters. Furthermore, gene duplication events of AT-1/SLC33A1, SLC25A1 or SLC13A5 are associated with autism spectrum disorder (ASD), intellectual disability, and progeria-like dysmorphism. To expand our studies, we generated neuron-specific (AT-1 nTg, SLC25A1 nTg, and SLC13A5 nTg) and systemic (AT-1 sTg, SLC25A1 sTg, and SLC13A5 sTg) overexpressing mice. These animals display important phenotypic similarities, supporting the conclusion that we have identified a unified metabolic pathway that is at the basis of closely related neurodegenerative and neurodevelopmental diseases across lifespan. To complement the above studies and dissect the specific role of the two ATases, down-stream of AT-1, we have also generated Atase1-/- and Atase2-/- mice. Their phenotype supports the idea that these two ER-based acetyltransferases have evolved to play partially divergent roles. The GENERAL HYPOTHESIS of this research is that SLC25A1, SLC13A5, and AT-1 act in concert to regulate engagement of the secretory pathway and induction of reticulophagy. Specific Aim 1 will test the hypothesis that the ER acetylation machinery is the downstream target of a dysfunctional cytosol-to-ER flux of acetyl-CoA caused by the duplication of AT-1, SLC25A1 or SLC13A5. Specific Aim 2 will use our newly generated Atase1-/- and Atase2-/- mice to test the hypothesis that ATase1 and ATase2 have partially different biological functions. Specific Aim 3 will test the hypothesis that specific structural features of newly identified AT-1 downstream targets allow fine tuning of reticulophagy. In conclusion, this proposal is the result of novel discoveries made in our laboratory; it will help us dissect the molecular mechanisms of severe neurodegenerative and neurodevelopmental diseases across lifespan and it will allow us dissect essential molecular and biochemical functions of the ...