Over the past decade, roles for the endoplasmic reticulum (ER) in the biogenesis of select nuclear-encoded mitochondrial precursors and the degradation of mutant, mis-localized, or non-productively imported proteins from the mitochondrial outer membrane (OM) have begun to emerge. Our interest in this unanticipated, novel ER-associated mitochondrial biology was serendipitous. In our ongoing efforts to characterize the lipid substrate trafficking requirements for phosphatidylserine (PS) decarboxylase 1 (Psd1 in yeast, PISD in humans), an evolutionarily conserved, integral inner mitochondrial membrane protein that produces phosphatidylethanolamine (PE), it became a priority for us to independently ascertain if a small fraction of wild type (WT) Psd1 is glycosylated and thus targeted to the endomembrane system, as recently claimed. Our generated results support the unavoidable conclusion that in yeast, the vast majority, if not all, of functional Psd1 is mitochondrially localized. However, we did uncover an intimate relationship between Psd1 and the ER: unlike the WT protein, non-functional forms of Psd1 are dually localized to the ER, where they are glycosylated, ubiquitinated, and rapidly degraded. Given the role of the ER as a staging ground for the efficient removal of non-functional Psd1, we then asked two questions ― 1) Is Psd1 biogenesis supported by a recently discovered Djp1-mediated Endoplasmic Reticulum Surface Retrieval pathway (ER-SURF)? and 2) Does the accumulation of ER-associated non-functional Psd1 depend on Msp1, an outer mitochondrial membrane resident AAA-ATPase known to remove mis-targeted proteins and non-productively imported mitochondrial precursors from the outer membrane? The resulting answers establish the premise for our proposed aims and identify Psd1 as an ideal model substrate to define novel mechanisms of ER-SURF and protein degradation that will undoubtedly apply to other mitochondrial proteins. The goal of Aim 1 is to define the early, pre- mitochondrial steps of Psd1 biogenesis that are mediated by specific interactions that occur at the ER. Other than a critical role for the Hsp40 cochaperone, Djp1, virtually nothing is known about ER-SURF. Results from Aim 1 will provide mechanistic insight about mitochondrial targeting through this novel pathway and whether it can be coerced in specific contexts to support non-mitochondrial biology. Over the past seven years, it has become increasingly appreciated that the accumulation of mitochondrial precursors outside of this organelle activates a range of cellular stress responses. In this context, our discovery that non-functional mutant Psd1 temporarily associates with ER membranes prior to being degraded is particularly exciting. The goal of Aim 2 is to determine the molecular mechanisms of non-functional Psd1 resolution. Results from Aim 2 will transform our understanding of how cells cope with mitochondrial precursors that fail to reach their correct destination, an emer...