Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.
Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.
Wandelt
C I
CI
CSIRO, Division of Plant Industry, Canberra, Australia.
Khan
M R
MR
Craig
S
S
Schroeder
H E
HE
Spencer
D
D
Higgins
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002451:Cell Compartmentation / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D007887:Fabaceae / Q000235:genetics*; D006207:Half-Life; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D014674:Plant Proteins, Dietary / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics; D010946:Plants, Medicinal; D010947:Plants, Toxic; D014176:Protein Biosynthesis; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D055314:Seed Storage Proteins; D014018:Tissue Distribution; D014026:Nicotiana / Q000235:genetics* / Q000648:ultrastructure; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D010940:Plant Proteins; D014674:Plant Proteins, Dietary; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C015575:vicilin protein, plant