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

Abstract

In order to investigate sequences of tobacco Nacetylglucosaminyltransferase 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.z 1999 Federation of European Biochemical Societies.

Structural requirements for Arabidopsisβ1,2‐xylosyltransferase activity and targeting to the Golgi

“…Furthermore, the immunolocalization of this fusion protein gave a similar pattern as observed after immunostaining for plant Le a structures. These glycoepitopes are acknowledged plant Golgi markers (Evans et al, 1997;Essl et al, 1999;Fitchette et al, 1999;Saint-Jore et al, 2002). Moreover and as previously described for other Golgi-specific labelings in plant cells, BFA induces a redistribution of XylT36::GFP from Golgi membranes to the ER (Driouich et al, 1993;Boevink et al, 1998;Pagny et al, 2000;SaintJore et al, 2002).…”

“…Besides those indirect approaches, the recent cloning of several glycosyltransferases from plants (Leiter et al, 1999;Strasser et al, 1999Strasser et al, , 2000Faik et al, 2000;Bakker et al, 2001;Wilson et al, 2001;Faik et al, 2002;Leonard et al, 2002) and the identification of about 250 putative plant glycosyltransferases in Arabidopsis thaliana genome (Henrissat et al, 2001) has paved the way for a detailed characterization of plant glycosyltransferases. This was illustrated in a recent paper providing preliminary indications on the signal responsible for localization of N-acetylglucosaminyltransferase I in the Golgi apparatus of tobacco cells (Essl et al, 1999).…”

“…The signals that control retention of glycosyltransferases in the plant Golgi apparatus are still unknown apart from a large 77 amino acid domain of the tobacco GnTI that covers the cytosolic tail, TMD, and most of the stem and is sufficient for the specific localization of GFP to the plant Golgi apparatus (Essl et al, 1999).…”

ENHANCED DISEASE RESISTANCE4 Associates with CLATHRIN HEAVY CHAIN2 and Modulates Plant Immunity by Regulating Relocation of EDR1 in Arabidopsis

“…At 48 h after inoculation (HAI) with G. cichoracearum, EDR4-GFP accumulated under the penetration peg at the infection site in epidermal cells ( Figure 4B), but overall, the EDR4-GFP protein levels decreased at 72 HAI with G. cichoracearum ( Figure 4C). To further confirm the accumulation of EDR4-GFP at the site of infection, we also examined EDR4-GFP localization at 24 HAI, with the cis-Golgi-localized protein N-acetylglucosaminyltransferase I (NAG) fused to GFP (Essl et al, 1999) as a control. Fungal structures were labeled with propidium iodide.…”

Cell biology of the plant Golgi apparatus

“…It was shown that the signal anchor sequence of a rat sialyl transferase (52 amino acids of the transmembrane domain and the cytoplasmic tail) and the equivalent sequence from a human galactosyl transferase was sufficient to target GFP to the Golgi stacks in tobacco leaf cells and BY2 suspension culture cells ( Boevink et al, 1998;Saint-Jore et al, 2002). Subsequently the equivalent sequences from recently cloned plant glycosyl transferases gave the same result ( Essl et al, 1999;Dirnberger et al, 2002;Pagny et al, 2003). These results suggest that the mechanisms of targeting and retention of glycosyl transferases may be conserved across kingdoms.…”