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.) storage protein gamma-gliadin were expressed in transgenic tobacco (Nicotiana tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous targeting signals are present in the wild-type gamma-gliadin protein. The mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the addition of a C-terminal propeptide from barley lectin which has been shown to be necessary and sufficient for targeting to the vacuole. Only modified forms of the protein accumulated in leaves of transgenic tobacco, although the transcript levels were similar for all the constructs. Pulse-chase analysis indicated that whereas the wild-type gamma-gliadin was rapidly turned over in tobacco leaves, KDEL and HDEL forms were highly stable. The vacuolar-signal mutant protein accumulated in tobacco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuolar-signal mutant protein was turned over slowly in tobacco, perhaps indicating a poor level of transport competence. When pulse-chase analysis was carried out on protoplasts isolated from tobacco plants expressing 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 plants and does not contain any structural determinants which confer retention in the endoplasmic reticulum.
Napier
J A
JA
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, UK. jon.napier@bbsrc.ac.uk
Richard
G
G
Turner
M F
MF
Shewry
P R
PR
D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005903:Gliadin / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D014908:Triticum
D003517:Cyclopentanes; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; C077480:histidyl-aspartyl-glutamyl-leucine; D020126:Brefeldin A; D005903:Gliadin