Vesicle transport in chloroplasts with emphasis on Rab proteins

Sammanfattning: Chloroplasts perform photosynthesis using PSI and PSII during its light-dependent phase. Inside the chloroplast there is a membrane called thylakoid. The thylakoid membranes are an internal system of interconnected membranes that carry out the light reactions of photosynthesis. The thylakoid membranes do not produce their own lipids or proteins, so they are mainly transported from the envelope to the thylakoid for maintenance of e.g. the photosynthetic apparatus. An aqueous stroma made hinder between the envelope and thylakoid for the lipids to move between the two compartments. Vesicle transport is suggested to transport lipids to thylakoids supported by biochemical and ultra-structural data. Proteins could potentially also be transported by vesicles as cargo but this is not supported yet experimentally. However, proteins targeted to thylakoids are mediated by four pathways so far identified but it has been proposed that a vesicle transport could exist for proteins targeted to thylakoids as well similar to the cytosolic vesicle transport system. This thesis revealed similarity of vesicle transport inside the chloroplast to the cytosolic system. A novel Rab protein CPRabA5E (CP= chloroplast localized) was shown in Arabidopsis to be chloroplast localized and characterized to be important for thylakoid structure, plant development, and oxidative stress response. Moreover, CPRabA5e complemented the yeast homologues being involved in vesicle transport, and the cprabA5e mutants were affected for vesicle formation in the chloroplasts. Another Rab (CPRabF1) was also identified inside the chloroplast and could possibly play a role in vesicle transport. Interestingly, CPRabF1 has previously been characterized for its involvement in vesicle transport in the cytosol and thus its localization in chloroplasts might indicate dual targeting of CPRabF1. No phenotype was observed despite usage of several applied factors e.g. high light and osmotic stress. A previous bioinformatics study predicted several Rab related proteins inside chloroplasts linked to a suggested COPII vesicle transport system. We analyzed the gene expression for the Rab related genes at several developmental stages covering the life span of Arabidopsis e.g. from seedlings to senescence. The data indicated a rather homogenous expression pattern among the genes studied being around 20-60% expressed for all developmental stages except for senescence were the expression pattern was more discrete. At senescence chloroplast degradation occurs indicating less need for vesicle components. The idea of a COPII vesicle system inside chloroplast raised the question of a COPI or clathrin coated vesicle (CCV) system in chloroplasts. Through a bioinformatics approach we found several homologues of cytosolic COPI and CCV related vesicle transport components inside chloroplast. However, many of them already had a clear function other than vesicle transport or were having an unknown function. Moreover, many necessary subunits to build a functional COPI and CCV system were not even identified to be chloroplast localized and so we concluded that vesicle transport in chloroplast do not have strong similarities with a COPI or CCV system, rather being more linked to a COPII system as recently suggested.