Abstract

Chloroplasts are the characteristic organelle in cells of plants and algae. They host numerous essential metabolic pathways, including photosynthesis. Many processes are required for the biogenesis and homeostasis of the chloroplast. This thesis will look at the spatial organization of some of these processes in the chloroplast of Chlamydomonas reinhardtii. This unicellular alga is a model organism for chloroplast biology. The diverse functions of chloroplasts depend on the translation of thousands of different proteins. Most of these proteins are encoded in the nucleus, translated in the cytoplasm, and imported into the organelle post-translationally. Translation by organelle-bound ribosomes and co-translational import have been described for the ER and mitochondria, but not for the chloroplast. Chapter 2 describes evidence of localized translation at the chloroplast in Chlamydomonas and addresses the questions of ribosome docking on the chloroplast and co-translational import of chloroplast proteins. The results reveal a domain of the chloroplast envelope which is bound by translating cytoplasmic ribosomes and is a specialized location of co-translational protein import. These ribosomes are retained by chloroplasts, during their purification from other cellular organelles, can be visualized on the outer chloroplast envelope with immunofluorescence microscopy (IF) and high-resolution electron tomography, and are translationally active. Co-translational protein import is supported by results of fluorescence in situ hybridization (FISH) showing that mRNAs encoding chloroplast proteins, but not an mRNA encoding a non-chloroplast protein, are retained by this domain during chloroplast purification. This envelope domain is spatially aligned with regions of envelope that were shown previously to be enriched in the protein translocons of the inner and outer membrane of the chloroplast envelope (TIC and TOC) and presumed to be specialized locations of protein import. Inside the chloroplast and adjacent to the envelope domain with translating cytoplasmic ribosomes is the chloroplast translation zone (T-zone). This intraorganellar compartment is where plastid ribosomes translate subunits of photosystem I (PSI) and photosystem II (PSII) of the photosynthetic electron transport chain. Together, these results reveal a complex spatial coordination of translation by cytoplasmic and chloroplast ribosomes for protein targeting and biogenesis of photosynthesis complexes in this semi-autonomous organelle.
Chapter 3 describes new insights into the spatial organization of the Calvin Benson Cycle, the pathway that converts CO2 to carbohydrates in photosynthesis. Using fluorescence microscopy, I show that, under certain conditions, enzymes and related proteins in this pathway are localized to a specific compartment in the chloroplast. This compartment is different from the widely accepted locations of these enzymes and suggest some intriguing possibilities and resolutions to current problems in the field. Together, these findings support the highly compartmentalized nature of chloroplasts in Chlamydomonas reinhardtii and highlight the potentials of this model organism for studying chloroplast biogenesis and addressing fundamental cell biology questions. Moreover, they raise the possibility that similar organizations can occur in more complex photosynthetic organism such as higher plants.