Neuhaus, Jean-Marc

2013, Stigliano, Egidio, Faraco, Marianna, Neuhaus, Jean-Marc, Montefusco, Anna, Dalessandro, Giuseppe, Piro, Gabriella, Di Sansebastiano, Gian-Pietro

Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.

2010, Paris, Nadine, Saint-Jean, Bruno, Faraco, Marianna, Krzeszowiec, Weronika, Dalessandro, Giuseppe, Neuhaus, Jean-Marc, Di Sansebastiano, Gian Pietro

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 α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(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.

2003-06-01, Flückiger, Ricardo, De Caroli, Monica, Piro, Gabriella, Dalessandro, Giuseppe, Neuhaus, Jean-Marc, Di Sansebastiano, Gian-Pietro

Green fluorescent protein (GFP) allows the direct visualization of gene expression and the subcellular localization of fusion proteins in living cells. The localization of different GFP fusion proteins in the secretory system was studied in stably transformed Arabidopsis plants cv. Wassilewskaja. Secreted GFP (SGFP) and GFP retained in the ER (GFP-KDEL) confirmed patterns already known, but two vacuolar GFPs (GFP-Chi and Aleu-GFP) labelled the Arabidopsis vacuolar system for the first time, the organization of which appears to depend on cell differentiation. GFP stability in the vacuoles may depend on pH or degradation, but these vacuolar markers can, nevertheless, be used as a tool for physiological studies making these plants suitable for mutagenesis and gene-tagging experiments.