The importance of phosphatidylethanolamine (PE) in biology is multi-faceted. PE is typically the second most abundant phospholipid component in biological membranes and thus plays a fundamental role in cellular autonomy and subcellular compartmentalization. In addition, PE is a precursor for other major lipids and is critical for a diverse range of specific biological functions. In eukaryotes, PE synthesis can occur via four separate pathways one of which is performed by phosphatidylserine decarboxylase 1 which resides in the inner mitochondrial membrane. Intriguingly, even though there are four distinct pathways to make PE, deletion of phosphatidylserine decarboxylase 1 is embryonically lethal in mice. Very little is known about regulatory mechanisms that govern flux through the mitochondrial PE pathway. The overarching goal of this application is to begin filling in the numerous gaps in our knowledge about how this essential biosynthetic pathway is regulated. Phosphatidylserine decarboxylase 1 has been traditionally modeled to generate PE by acting on substrate present in the intermembrane space-facing leaflet of the inner membrane. However, recently, it has been suggested that phosphatidylserine decarboxylase 1 can produce PE by acting on substrate present in the outer membrane. An important ramification of this new and yet unsubstantiated in trans model is that it does not require the lipid substrate to traffic across the aqueous intermembrane space. Since lipid trafficking steps represent a means to control access to substrate, knowledge about whether substrate transport across the intermembrane space is required for phosphatidylserine decarboxylase 1 activity, or not, is necessary to establish a framework of putative mechanisms capable of regulating flux through this pathway. The goal of aim 1 is to systematically test the in trans model utilizing a novel topologically inverted chimera of phosphatidylserine decarboxylase 1 whose ability to make PE is absolutely dependent on the movement of substrate across the intermembrane space. Recently, a novel tumor suppressor, LACTB, was discovered that when overexpressed in certain cancer cell lines, reduces cell proliferation and increases cellular differentiation via a mechanism that is at least in part explained by a significant decrease in the levels and function of human phosphatidylserine decarboxylase 1. Importantly, the underlying mechanism responsible for the decrease in phosphatidylserine decarboxylase 1 abundance, which was determined to be post-transcriptionally mediated, was not ascertained. In aim 2, we will continue to exploit a temperature sensitive allele of phosphatidylserine decarboxylase 1 to identify the proteases and define the rules that govern its efficient removal at non- permissive temperature. Ultimately, this information will be used as a guide to unravel how this enzyme and pathway are post-transcriptionally regulated in humans. By obtaining a more comprehensive understan...