Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding.

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and ¹5N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

Reconstructing the mitochondrial protein import machinery of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii several nucleus-encoded proteins that participate in the mitochondrial oxidative phosphorylation are targeted to the organelle by unusually long mitochondrial targeting sequences. Here, we explored the components of the mitochondrial import machinery of the green alga. We mined the algal genome, searching for yeast and plant homologs, and reconstructed the mitochondrial import machinery. All the main translocation components were identified in Chlamydomonas as well as in Arabidopsis thaliana and in the recently sequenced moss Physcomitrella patens. Some of these components appear to be duplicated, as is the case of Tim22. In contrast, several yeast components that have relatively large hydrophilic regions exposed to the cytosol or to the intermembrane space seem to be absent in land plants and green algae. If present at all, these components of plants and algae may differ significantly from their yeast counterparts. We propose that long mitochondrial targeting sequences in some Chlamydomonas mitochondrial protein precursors are involved in preventing the aggregation of the hydrophobic proteins they carry.

Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms.

Chlamydomonas reinhardtii is a model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function. It has distinct advantages in comparison to higher plants because it is unicellular, haploid, and amenable to tetrad analysis, and its three genomes are subject to specific transformation. It also has the possibility to grow either photoautotrophically or heterotrophically on acetate, making the assembly of the photosynthetic machinery not essential for cell viability. Methods developed allow the isolation of C. reinhardtii mitochondria free of thylakoid contaminants. We review the general procedures used for the biochemical characterization of mitochondria from this green alga.