Chloro-Import · Systematic mapping of the chloroplast protein import system

Protein import into a membrane-bound organelle in eukaryotes has been intensively studied, leading to the discovery of the central intracellular import machineries. Yet, this field has undergone a fundamental paradigm shift following recent discoveries of non-canonical import pathways in the mitochondria and ER. While the chloroplast is still thought to have but one import machinery (TIC/TOC), accumulating evidence indicate it, too, has non-canonical pathways. Technological constraints have so far barred the systematic search for alternative import pathways, as well as the exploration of central aspects relating to this import system: Are there additional import pathways other than the canonical chloroplast translocon apparatus? What ensures correct targeting of proteins in the highly crowded cytosol to the chloroplast? What mediates between quality control processes and protein import?In Chloro-Import, we will go beyond the state-of-the-art by implementing multipronged, genome-wide, unbiased approaches we developed or adopted to elucidate all the targeting pathways en route to the chloroplast in Chlamydomonas. Specifically, we will use advanced genome-wide screening to comprehensively elucidate chloroplast import pathways (Aim1); uncover the functional organization of import pathways using genetic-interaction profiling and protein-protein-interaction mapping (Aim2); and conduct an in-depth biochemical investigation of the function of unstudied/novel import factors (Aim3). To the best of our knowledge, this is the first-ever study of this scale in a photosynthetic eukaryote cell. The outcome will be a complete map of the chloroplast protein-import system (pathways, components, regulators) and a genetic platform for this system’s in-depth characterization. It will further offer insight into basic questions in chloroplast functioning, and may also pave the way to the synthetic engineering of crops that are more efficient for projected climate changes.