RESUMO
Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.
Assuntos
Proteínas de Arabidopsis , Cloroplastos/metabolismo , Citosol/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Arabidopsis , Ligação Competitiva/efeitos dos fármacos , Imunofluorescência , Substâncias Macromoleculares , Proteínas de Membrana/farmacologia , Pisum sativum , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico/fisiologia , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
Several previous studies of bone repair have shown 2- to 4-fold increases in bone formation following local delivery of exogenous transforming growth factor-beta (TGF-beta). Here, we use quantitative backscatter electron microscopy to test the effect of TGF-beta1 on mineralization of regenerated bone by examining tissue samples from a previously published canine study in which we found increased bone formation. In the experiment, the proximal humeri of 10 male canines were implanted bilaterally for 28 days with porous-coated implants in the presence of a 3 mm gap between the surface of the implant and the host bone. Implants placed in the left humeri were treated with TGF-beta1 at a dose of either 120 microg (n = 5) or 335 microg (n = 5), and the implants placed in the contralateral humeri served as untreated controls. Quantitative backscatter scanning electron microscopy was used to assess the volume fraction of bone and its degree of mineralization in the 3 mm gaps. The calibrated grayscale mean and median values were depressed compared to the controls in the high dose group (p = 0.048 and p = 0.041, respectively), suggesting that high dose TGF-beta delayed or inhibited mineralization of newly formed osteoid.