Abstract

In chloroplasts, thylakoid membranes contain the complexes responsible for the light-dependent reactions of photosynthesis. While much is known about their functioning, the biogenesis of these membranes and their different components is less well understood. Previous work using in situ approaches to confocal microscopy found evidence for the existence of a specific zone where the synthesis of thylakoid proteins takes place in the chloroplast of the green alga Chlamydomonas reinhardtii . Building on these findings, I used a modified approach of cellular subfractionation by sucrose-density gradient centrifugation to identify sites of thylakoid biogenesis in the chloroplast of this organism. I found evidence for the existence of distinct membrane fractions that might be involved in thylakoid biogenesis. Named high density membranes (HDM), they are higher in density than thylakoids and fractionate below them on a sucrose gradient. HDM's were found to be qualitatively different from thylakoids and contained markers of protein synthesis. Pulse-labelling experiments did not confirm that these membranes are involved in protein and lipid synthesis; however, these results also showed a departure from current models. Finally, these results suggest a potential role for these membranes in chlorophyll synthesis. We propose that these membranes are at the very least involved in the early steps of synthesis and assembly of thylakoid proteins. They are high in density in conditions of active thylakoid membrane biogenesis, whereas in the absence of thylakoid biogenesis they are not laden with ribosomes, RNA-binding proteins and newly-synthesized proteins and thus are light in density. The significance of my work is that it lays the groundwork for further characterization of HDM's in Chlamydomonas and higher plant species.