RESUMO
Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.
Assuntos
Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Western Blotting , Núcleo Celular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Peroxinas , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Peroxissomos/metabolismo , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismoRESUMO
Peroxisomes transport folded and oligomeric proteins across their membrane. Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process. While interactions among individual peroxins have been described, their organization into functional units has remained elusive. We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p. Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein. We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane.
