Abstract 1811 Unraveling the Temporal and Spatial Regulation of Proteasome Biogenesis in Living Cells

Mitochondrial DNA (mtDNA) is essential for eukaryotic cells because it encodes subunits of the electron transport chain, necessary for the ATP production.The expression of mtDNA is regulated by proteins associated with it.Such functional nucleoprotein complexes, namely nucleoids, undergo dynamic rearrangements in response to the metabolic status of the cell.When ATP is in abundance, nucleoids are saturated with histone-like protein, TFAM, which suppresses mtDNA expression and promotes its stability.Conversely, when ATP is needed, mtDNA is accessible to replication and transcription factors.The capacity to efficiently transition nucleoids between these states is critical for meeting energetic needs of a cell.However regulatory mechanisms governing this process remain poorly understood.One such regulatory mechanism entails nucleoid-associated protease, Lon, which has been demonstrated to eliminate excess of the TFAM protein.We have recently demonstrated that mtDNA replication can be upregulated by mitochondrial chaperones that localize to the nucleoid.In this work I sought to assess whether mtDNA replication can be down regulated by elimination of these chaperones by Lon protease.To that end, I performed a series of proteolysis assays in vitro using recombinant proteins in question.We found that indeed Lon protease can efficiently degrade molecular chaperones.Interestingly, however, this activity appears to be suppressed in the presence of DNA.Currently I seek to evaluate our in vitro results, by testing whether inhibition of Lon protease in HeLa cells (by supplementation with CDDO) has an effect on cellular levels of chaperones under study, and levels of mtDNA.