To ensure proper cellular function, proteins need to reach their appropriate subcellular destination. This is achieved by the accurate and faithful targeting of newly synthesized proteins to subcellular organelles. We are focusing on one fundamental pathway of co-translational protein targeting mediated by the signal recognition particle (SRP). We combine biochemical, structural, and in vitro single-molecule approaches to break the SRP targeting reaction into individual steps (for example, SRP interacting with ribosomes, or SRP in complex with the SRP receptor interacting with the membrane translocon). The ultimate goal is to reconstitute all of the individual steps to obtain a “molecular movie” of the entire targeting reaction. In recent work, we observed for the first time SRP binding events to actively translating ribosomes. These studies will provide a mechanistic framework for how cells process thousands of newly synthesized proteins and partition them to where they need to go in the cell.
An additional critical aspect of achieving appropriate protein localization is the ability to sense and degrade proteins that are not effectively targeted to their intended subcellular location and are instead inserted into inappropriate organelles. We recently discovered a novel quality control pathway that recognizes the mistargeting of a unique class of membrane proteins known as tail-anchored (TA) proteins that can be inappropriately inserted into the outer mitochondrial membrane (OMM). This pathway uses the conserved AAA-ATPase Msp1 to sense mistargeting events in the OMM. In a nucleotide-hydrolysis dependent manner, Msp1 then extracts mistargeted substrate, and channels it to destruction in the proteasome. In this way, Msp1 reinforces the appropriate localization of TA proteins in the cell. We are using a combination of yeast genetics and biochemical approaches to identify additional players in this pathway and are reconstituting the process in vitro to decipher the molecular mechanism by which Msp1 extracts TA proteins from the lipid bilayer of the OMM.