Studies on the structure and biogenesis of membrane proteins from the green alga Chlamydomonas reinhardtii

Sammanfattning: The green alga unicellular Chlamydomonas reinhardtii has been used for many years as a model system in the area of photosynthesis. Therefore, the C. reinhardtii chloroplast has been the subject of many studies. In contrast, the mitochondria have received less attention. This may have been due to obstacles in the preparation of pure and intact mitochondria from this organism. In this thesis I have presented a method for isolation of functional mitochondria. From the mitochondria we have isolated an active ATP synthase complex with high purity, consisting of 14 different polypeptides. The N-terminal sequences of 4 subunits of the complex were determined and for one of them, the a subunit, we have isolated cDNA clones from a cDNA library and sequenced completely. By theoretical structural analysis, we have shown that the presequence of the a subunit contains regions with potential to form a helices, which is characteristic for mitochondrial targeting peptides (mTPs) and in agreement with previous results from other mTPs in this organism. It has previously been shown that chloroplast transit peptides (cTPs) from this alga appear to have a dual character, containing both putative amphiphilic a helices, as in mTPs, and putative amphiphilic b strands, as in cTPs from higher plants.We have also developed a purification procedure for isolation of plasma membranes from C. reinhardtii, based on breakage of the cells in a Waring Blendor, differential centrifugation and aqueous two-phase partitioning. The procedure results in high yield and purity of plasma membranes. The procedure can be easily scaled up which offers possibilities to study plasma membrane bound proteins in this alga. We have performed a kinetic characterization of the proton pumping ATPase from these membranes.We have established a method for in vitro protein import into C. reinhardtii mitochondria and examined them for both heterologous and homologous import. Our import results show membrane potential dependency for protein translocation as in mitochondria from other organisms. Studies on the requirement for energy (ATP) during protein import indicated that the C. reinhardtii Rieske FeS protein may be imported into the mitochondria in an ATP-independent manner. In the absence of ATP, the protein is not processed, which indicates that it is not accessible for the processing peptidase. Possibly, it may be arrested at an early import stage (e.g. in the intermembrane space), or it may be translocated into the matrix without being released from matrix chaperones. The heterologous protein import confirms the evolutionary conservation of the protein import mechanisms in mitochondria as higher plant precursors are imported into C. reinhardtii mitochondria.We have shown that the chloroplast PsaF protein from C. reinhardtii has unusual properties, as it can be imported into mitochondria from spinach and C. reinhardtii. This import is presequence-independent but membrane-potential-dependent, and may be due to a direct interaction between the mature protein and membrane lipids. By in vitro processing of mitochondrial precursor proteins, we have found the processing activity in the mitochondrial matrix. This indicates that the mitochondrial processing peptidase in C. reinhardtii is a soluble enzyme in the matrix as in fungi and mammals and in contrast to in higher plants, where the processing peptidase is membrane-bound and identical to the core proteins of the bc1 complex.