The majority of outer membrane proteins (OMPs) from Gramnegative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using selfassembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

BackgroundThe multicellular green alga Volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ECM biogenesis. However, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. So far there are only a limited number of molecular tools available in Volvox.ResultsHere, we show that the promoter of the V. carteri nitrate reductase gene (nitA) is a powerful molecular switch for induction of transgene expression. Strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. We also show that the luciferase (g-luc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga Chlamydomonas reinhardtii, is a suitable reporter gene in V. carteri. Emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. Long-term stability of inducible expression of the chimeric nitA/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays.ConclusionTwo novel molecular tools for genetic engineering of Volvox are now available: the nitrate-inducible nitA promoter of V. carteri and the codon-adapted luciferase reporter gene of G. princeps. These novel tools will be useful for future molecular research in V. carteri.

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