Endoplasmic reticulum targeting of active modified beta-glucuronidase (GUS) in transgenic tobacco plants.
We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the beta-glucuronidase (GUS) enzyme with the wheat alpha-amylase signal peptide. In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.
Firek
S
S
Department of Botany, University of Leicester, UK.
Whitelam
G C
GC
Draper
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005966:Glucuronidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005966:Glucuronidase