Adam S. Frost
Structural and Functional Characterization of the Ribosome Quality Control or RQC Complex
Age-associated diseases like neurodegeneration and heart failure are among the most prevalent causes of human morbidity. These diseases are also exceptionally difficult to study mechanistically. At the most fundamental level, we do not how long-lived cells such as neurons or cardiac myocytes counteract the accumulation of damaged or aggregated proteins over a lifetime. My lab seeks to understand the protein quality control and recycling mechanisms that protect such cells from age-associated decline. This effort led us and our collaborators to co-discover a new quality control pathway that utilizes a ribosome-bound assembly named the Ribosome Quality Control complex (RQC). The RQC complex comprises an E3 ubiquitin ligase (Ltn1), two uncharacterized proteins (Tae2 and Rqc1), and the AAA+ ATPase Cdc48/p97. These factors are conserved throughout eukaryotic evolution, permitting us to perform functional and structural studies in yeast in order to dissect molecular mechanisms that pertain to humans. We demonstrated that the RQC recognizes stalled ribosomes due to defective or inhibited translation and triggers the degradation of flawed or stuck translation products. Failures or deficiencies of this pathway have already been implicated in age-associated diseases. Mice harboring a hypomorphic allele of LTN1/Listerin suffer from a profound neurodegeneration syndrome. Mutation of Cdc48/p97, moreover, causes the neuro-degenerative syndromes Inclusion Body Myopathy with Paget disease and Fronto-temporal Dementia (IBMPFD), and some cases of Amyotrophic Lateral Sclerosis (ALS). These rare, monogenic diseases portend a broader role for the RQC in both inherited and sporadic cases of neurodegeneration. We propose to determine the structural mechanisms by which the RQC recognizes stalled ribosomes and extracts defective translation products for degradation. Our proposal to combine structural, biochemical and genetic studies of the RQC complex will lead to a greater understanding of this newly-discovered pathway and inform efforts to enhance its protective function in aging people.
other research interests
Our limited knowledge of membrane-associated machines stems from the inherent difficulties in studying multi-component complexes that only assemble on the surface of phospholipid bilayers. To overcome these challenges we are developing genetic, biochemical and structural approaches that will allow us to discover and characterize membrane-associated machines in molecular detail and to learn how they function within complex cellular pathways. We are 1) building genome-wide genetic interaction maps to identify multi-component complexes and to annotate their functions; and 2) exploiting advances in cryo-electron microscopy and image analysis to resolve the molecular structures of these machines in native, membrane-associated states. These two arms of our undertaking are distinct technologically but mutually reinforcing: genetic studies define functional complexes for in vitro studies and then, when we solve a molecular structure by cryoEM, we test the functional predictions of our model with loss-of-function phenotypes and genetic suppression assays. Together these orthogonal methods provide synergistic observations of the mechanisms that underlie in vivo functions.
We are focusing initially on molecular machines that remodel cellular membranes during organelle biogenesis, organelle homeostasis, and cytokinesis. Our analyses of genetic interaction networks from multiple model organisms and work by our collaborators have allowed us to identify four membrane-associated machines for in depth study: 1) The regulatory complexes formed with dynamin-family GTPases, which control endocytosis and mitochondrial fragmentation; 2) The ESCRT-III and VPS4 protein complexes, which catalyze membrane fission in diverse cellular contexts; 3) A complex containing a novel transmembrane protein, a BEACH-domain protein, and a Rab GTPase we discovered that appears to measure lysosome size and to promote lysosome fragmentation; and 4) the STRIPAK complex, which we discovered bridges Golgi and nuclear membranes and mediates targeting of Golgi fragments to mitotic centrosomes and Golgi reassembly following cytokinesis.
In parallel to our cell biological studies, we are developing new analysis methods for studying membrane-associated complexes. For example, we have developed algorithms for correcting a common aberration in electron micrographs of phospholipid bilayers. The aberration arises from Fresnel diffraction along the strong scattering edges of phosphate-rich lipid headgroups and, when uncorrected, this Fresnel fringe obscures the signal from membrane-bound and transmembrane proteins in real-space reconstruction procedures. In conjunction with advances in electron optics and electron detectors, we expect our analysis to enable molecular and even near-atomic resolution 3D reconstructions of membrane-associated complexes — structures that cannot be solved by any other technique. By validating our structures with other forms of dynamic microscopy and genetic assays, we hope to close the gap in structural biology where membranes and membrane-associated molecules have remained out of sight.