RESUMEN
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.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Western Blotting , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Peroxinas , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/química , Peroxisomas/metabolismo , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Proteínas Represoras/química , Proteínas de Saccharomyces cerevisiae/química , Transducción de Señal , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
We have cloned and characterized the Hansenula polymorpha PEX19 gene. In cells of a pex19 disruption strain (Hppex19), induced on methanol, peroxisome structures were not detectable; peroxisomal matrix proteins accumulated in the cytosol, whereas peroxisomal membrane proteins (PMPs) were mislocalized to the cytosol (Pex3p) and mitochondria (Pex14p) or strongly reduced to undetectable levels (Pex10p). The defect in peroxisome formation in Hppex19 cells was largely suppressed upon overproduction of HpPex3p or a fusion protein that consisted of the first 50 N-terminal amino acids of Pex3p and GFP. In these cells PMPs were again correctly sorted to peroxisomal structures, which also harbored peroxisomal matrix proteins. In Saccharomyces cerevisiae pex19 cells overproduction of ScPex3p led to the formation of numerous vesicles that contained PMPs but lacked the major matrix protein thiolase. Taken together, our data are consistent with a function of Pex19p in membrane protein assembly and function.