Seed storage proteins of the prolamin class form very large insoluble polymers, termed protein bodies (PB), which often accumulate in the endoplasmic reticulum (ER) through molecular interactions that are still poorly understood. We have previously shown that a fusion between the vacuolar 7S storage protein phaseolin and the N-terminal half of the maize prolamin gamma-zein forms ER-located PBs with the morphological and solubility characteristics of gamma-zein PBs. Zeolin has six out of the fifteen cysteine residues of the maize prolamin and, like .-zein, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying the in vivo effects of the reducing agent 2-mercaptoethanol and by mutagenesis of zeolin. We show that in tobacco protoplasts 2-mercaptoethanol enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits secretory traffic of proteins that have or do not have disulfide bonds. This indicates that the plant secretory mechanism is negatively affected, unlike the situation in mammalian and yeast cells where reducing agents only inhibit traffic of disulfide-bonded proteins. In spite of this general inhibition, 2-mercaptoethanol 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 secretory traffic also in the absence of 2-mercaptoethanol treatment. We conclude that inter-chain disulfide bonds are a determinant for efficient PB-mediated protein accumulation in the ER.
Retention of a bean phaseolin/maize gamma-zein fusion in the endoplasmic reticulum depends on disulfide bond formation
Pompa A;Vitale;
2006
Abstract
Seed storage proteins of the prolamin class form very large insoluble polymers, termed protein bodies (PB), which often accumulate in the endoplasmic reticulum (ER) through molecular interactions that are still poorly understood. We have previously shown that a fusion between the vacuolar 7S storage protein phaseolin and the N-terminal half of the maize prolamin gamma-zein forms ER-located PBs with the morphological and solubility characteristics of gamma-zein PBs. Zeolin has six out of the fifteen cysteine residues of the maize prolamin and, like .-zein, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying the in vivo effects of the reducing agent 2-mercaptoethanol and by mutagenesis of zeolin. We show that in tobacco protoplasts 2-mercaptoethanol enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits secretory traffic of proteins that have or do not have disulfide bonds. This indicates that the plant secretory mechanism is negatively affected, unlike the situation in mammalian and yeast cells where reducing agents only inhibit traffic of disulfide-bonded proteins. In spite of this general inhibition, 2-mercaptoethanol 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 secretory traffic also in the absence of 2-mercaptoethanol treatment. We conclude that inter-chain disulfide bonds are a determinant for efficient PB-mediated protein accumulation in the ER.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
