The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells.
In order to investigate sequences of tobacco N-acetylglucosaminyltransferase I (GnTI), involved in targeting to and retention in the plant Golgi apparatus the cytoplasmic transmembrane stem (CTS) region of the enzyme was cloned in frame with the cDNA of the green fluorescent protein (gfp) and subsequently transiently expressed in Nicotiana benthamiana plants using a tobacco mosaic virus (TMV) based expression vector. Confocal laser scanning microscopy showed small fluorescent vesicular bodies in CTS-gfp expressing cells, while gfp alone expressed in control plants was uniformly distributed in the cytoplasm. The CTS-gfp fusion protein colocalised with immunolabelling observed by an antibody specific for the Golgi located plant Lewis a epitope. Furthermore, treatment with brefeldin A, a Golgi specific drug, resulted in the formation of large fluorescent vesiculated areas. These results strongly suggest a Golgi location for CTS-gfp and as a consequence our findings reveal that the N-terminal 77 amino acids of tobacco GnTI are sufficient to target to and to retain a reporter protein in the plant Golgi apparatus and that TMV based vectors are suitable vehicles for rapid delivery of recombinant proteins to the secretory pathway.
Zentrum für Angewandte Genetik, Universität für Bodenkultur-Wien, Austria.
D
D
Essl
D
D
Dirnberger
V
V
Gomord
R
R
Strasser
L
L
Faye
J
J
Glössl
H
H
Steinkellner
D001692:Biological Transport; D002451:Cell Compartmentation; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*
D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I