Retention of a bean phaseolin/maize gamma-Zein fusion in the endoplasmic reticulum depends on disulfide bond formation.
Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like gamma-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.
Pompa
Andrea
A
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, 20133 Milano, Italy.
Vitale
Alessandro
A
D001483:Base Sequence; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D016296:Mutagenesis; D010940:Plant Proteins / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D003313:Zea mays / Q000378:metabolism*; D015028:Zein / Q000378:metabolism*
D017931:DNA Primers; D004220:Disulfides; D010940:Plant Proteins; C048826:phaseolin protein, Phaseolus vulgaris; D008623:Mercaptoethanol; D015028:Zein