The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferaseI are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells

In order to investigate sequences of tobacco N-acetylglucosaminyltransferas e I (GnTI), involved in targeting to and retention in the plant Golgi appar atus the cytoplasmic transmembrane stem (CTS) region of the enzyme was clon ed in frame with the cDNA of the green fluorescent protein (gfp) and subseq uently transiently expressed in Nicotiana benthamiana plants using a tobacc o mosaic virus (TMV) based expression vector. Confocal laser scanning micro scopy 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 bq an antibody specific for the Golgi located plant Lewis a epitop e, Furthermore, treatment with brefeldin A, a Golgi specific drug, resulted in the formation of large fluorescent vesiculated areas. These results str ongly suggest a Golgi location for CTS-gfp and as a consequence our finding s 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 reco mbinant proteins to the secretory pathway, (C) 1999 Federation of European Biochemical Societies.