TRAFFICKING OF WHEAT GLUTEN PROTEINS IN TRANSGENIC TOBACCO PLANTS - GAMMA-GLIADIN DOES NOT CONTAIN AN ENDOPLASMIC-RETICULUM RETENTION SIGNAL

Wild-type and mutated forms of the wheat (Triticum aestivum L.) storag e protein gamma-gliadin were expressed in transgenic tobacco (Nicotian a tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous t argeting signals are present in the wild-type gamma-gliadin protein. T he mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the add ition of a C-terminal propeptide from barley lectin which has been sho wn to be necessary and sufficient for targeting to the vacuole. Only m odified forms of the protein accumulated in leaves of transgenic tobac co, although the transcript levels were similar for all the constructs . Pulse-chase analysis indicated that whereas the wild-type gamma-glia din was rapidly turned over in tobacco leaves, KDEL and HDEL forms wer e highly stable. The vacuolar-signal mutant protein accumulated in tob acco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide ge l electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuola r-signal mutant protein was turned over slowly in tobacco, perhaps ind icating a poor level of transport competence. When pulse-chase analysi s was carried out on protoplasts isolated from tobacco plants expressi ng wild-type gamma-gliadin, but in the presence of Brefeldin A, gamma- gliadin was seen to accumulate. Taken together, these results indicate that gamma-gliadin is targeted to the vacuole in transgenic tobacco p lants and does not contain any structural determinants which confer re tention in the endoplasmic reticulum.