An engineered C-terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal.
Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. 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 interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.
Pompa
Andrea
A
Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche, via della Madonna Alta 130, 06128 Perugia, Italy.
De Marchis
Francesca
F
Vitale
Alessandro
A
Arcioni
Sergio
S
Bellucci
Michele
M
D020816:Amino Acid Motifs; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry*; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism*
D010940:Plant Proteins; D021382:Protein Sorting Signals; C048826:phaseolin protein, Phaseolus vulgaris