RESUMO
BACKGROUND: The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. RESULTS: To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. CONCLUSIONS: Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Peroxissomos/genética , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured alpha-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 muM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions.
