Current Projects
OVERVIEW
Phospholipids are the building blocks of biological membranes. Membranes leverage the amphipathic chemistry of lipids to form bilayers that encapsulate a cell and its multitude of organelles. Such compartmentalization has enabled cells to separate biochemical pathways, establish specialized functions that can respond when appropriate, and adapt to constantly fluctuating metabolic conditions. The Claypool laboratory’s research focus is on the underappreciated contribution of the mitochondrion to cellular phospholipid metabolism. In addition to being the sole producer of the canonical mitochondrial lipid, cardiolipin (CL), the mitochondrion hosts one of the two major pathways in a cell for the production of phosphatidylethanolamine (PE). PS decarboxylase 1 (Psd1) is an integral inner mitochondrial membrane protein that produces the vast majority of PE in the mitochondrion and can provide the entire cellular complement of PE in yeast. Ablation of the mitochondrial capacity to synthesize either CL or PE is embryonically lethal in mice. The mitochondrial pathway of PE production therefore provides a pool of this lipid that cannot be replaced by the other three PE biosynthetic pathways. Thus, both CL and PE are crucial for mammalian development and have distinct and yet overlapping properties that are essential not only for mitochondrial function, but life itself.
The lab currently has two major ongoing projects centered on different aspects of mitochondrial phospholipid metabolism in health and disease.
PROJECT ONE
CL and the ADP/ATP carrier — Mitochondrial ADP/ATP carriers (Aac) mediate the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix, an activity that is required for oxidative phosphorylation. Previously, we made the exciting discovery that the major yeast ADP/ATP carrier, Aac2, associates with the respiratory supercomplex (RSC; higher order assemblies of individual respiratory complexes) but only in the context of mitochondrial membranes that contain the unique phospholipid cardiolipin. Subsequently, we established that there is substantial overlap between the interactomes of yeast Aac2 and two human Aac isoforms. When combined, our results demonstrate that cardiolipin is of general importance to the extended and clinically relevant Aac family which participate in numerous evolutionarily conserved and cardiolipin-dependent protein-protein interactions that are therefore presumed to be functionally important. These collective findings strongly support our central hypothesis that the cardiolipin-dependent Aac interactome represents the mitochondrion’s “Achilles’ heel” in the multiple disease states that result from altered cardiolipin metabolism. In our ongoing efforts to drill into the cardiolipin-dependency of Aac2 we determined that cardiolipin promotes both the tertiary and quaternary assembly of Aac2, and excitingly, it does so via distinct mechanisms. We hypothesize that these two separable structural roles of cardiolipin with respect to Aac2 assembly reflect specific Aac2-cardiolipin interactions occurring within the folded carrier or on its periphery. From within, we speculate that three conserved cardiolipin-binding sites support the carriers folded structure and potentially enable its transport-related conformational dynamics. On the periphery, we hypothesize that the defining role of cardiolipin for the association of Aac2 with respiratory supercomplexes, composed in yeast of a complex III dimer and 1-2 copies of complex IV, involves individually weak interactions between Aac2-cardiolipin, Aac2-cardiolipin-RSC, and Aac2-RSC that when combined stabilize these multi-protein complexes. Results from testing these two hypotheses will significantly impact our understanding of the consequences of alterations in the Aac interactome that may occur due to mutations in Aac and/or perturbations in cardiolipin metabolism.
Funding: NIH – National Heart, Lung, and Blood Institute
PROJECT TWO
Mitochondrial PE metabolism — 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. Current projects seek to define the ER-associated mechanisms that 1) support Psd1 biogenesis; and 2) execute the swift elimination of mutant forms of Psd1.
Funding: NIH – National Institute of General Medical Sciences