Expression of a glycosylated GFP as a bivalent reporter in exocytosis

Abstract

The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an a-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a b-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology.

‘2A‐Like’ Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting

“…This distribution is typical of secreted proteins . A very faint mCherry fluorescence was also detectable in the central vacuole; this is sometimes observed with fluorescent proteins directed into the secretory pathway and may be caused by the T2A peptide remaining attached to the C‐terminus of mCherry, as C‐terminal FMDV 2A can act as a vacuolar mis‐targeting signal in N. benthamiana epidermis . To further verify these observations, we colocalized Sp NLR2a6 WT ‐mCherry and Sp NLR2A6 P3A ‐mCherry constructs with GFP targeted to the ER lumen and to the plasma membrane .…”

“…v: central vacuole. E–H) Expression of Sp NLR2A6 WT ‐mCherry and Sp NLR2A6 P3A ‐mCherry, respectively, in transgenic plants expressing GFP targeted to the lumen of the ER . Sp NLR2A6 WT –mCherry fluorescence localizes to the nucleoplasm, which is surrounded by the GFP‐labelled nuclear envelope (E), and does not colocalize with GFP‐labelled tubular cortical ER (G).…”

Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells

“…The potential role of glycosylation in determining a vacuolar route, bypassing or not the Golgi, has been previously discussed (Rayon et al 1998;Paris et al 2010). Previous studies have suggested that the glycosylation process was not required for vacuolar trafficking, but may indirectly impair the regulation of vacuolar trafficking by affecting the processing and transport rates of the cargo proteins (Wilkins et al, 1990).…”

Isolation, Culture, and Transient Transformation of Plant Protoplasts

“…Protoplasts are usually isolated by enzymatic digestion of mesophyll cells from either leaves or young seedlings, as well as from suspension‐cultured cells. Taking advantage of its speed, convenience, and flexibility, protoplast transient gene expression technology has been widely used for various applications, including analysis of promoter and regulatory elements involved in transcription and translation (Jacobsen and Beach, ), induction of gene expression and function by exogenous stimuli (Assmann et al., ), study of plant signaling mechanisms (Worley et al., ; Yanagisawa et al., ; Wang et al., ; Paris et al., ), RNA interference (RNAi; Li et al., ), protein interactions and subcellular localization (Miao et al., ; Wang et al., ; Wang and Jiang, ; Qi et al., ; Shen et al., ,b; Zhuang et al., ), study of protein sorting signals for retention in specific compartments (Cai et al., ; Gao et al., ; Shen et al., ), and analysis of protein recruitment and complex formation in vivo (Ding et al., ).…”