NOA1, a Novel ClpXP Substrate, Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.Max Planck Society for the Advancement of Scienc

Regulation of the Cytoplasmic Dynein Motor

The large size and complex organization of eukaryotic cells necessitates active, directional transport for efficient relocalization of cellular components and fidelity in their placement. This transport is carried out primarily by motor proteins that move along cytoskeletal filaments. In the past few decades, biochemical and biophysical studies have added greatly to our understanding of the mechansisms of motility and force generation of cytoskeletal motors. Much less is known about how these motors are coupled to their cellular cargos, and how their activity might be regulated appropriately for each function. This is a particularly intriguing question for the microtubule minus-end directed motor cytoplasmic dynein, and is the focus of this dissertation. Only a single cytoplasmic form of dynein has been identified in any organism, and yet this single type of motor performs all cytoplasmic microtubule minus end-directed transport. In vivo, dynein forms a large complex with several other proteins and protein complexes that are required for dynein-mediated transport; these factors may help to adapt and regulate dynein for its cellular functions. Chapter 1 provides a review of the current literature regarding the four major dynein adaptors, dynactin, Lis1-NudE/EL, Bicaudal-D, and RZZ-Spindly, and a discussion of how they might work together to facilitate dynein function in the cell. In the study presented in Chapter 2, we focused on the activity of dynactin, a ubiquitously required dynein adaptor. Previous indirect observations of dynactin had suggested that it could increase dynein processevity by tethering dynein to the microtubule through its own microtubule binding domains. Using recombinant dynactin and dynein from S. cerevisiae for direct observation in a single molecule assay, we found that dynactin increases the run length of single dynein motors, confirming that dynactin is a processivity factor for dynein. We find that this enhancement of dynein processivity does not require the microtubule binding domains of dynactin, but instead require a projecting coiled-coil stalk. The dynactin coiled-coil stalk, but not its microtubule binding domains, is also required for dynein function in living cells