Green fluorescent protein as a scaffold for intracellular presentation of peptides

Abedi, Majid; Caponigro, Giordano; Kamb, Alexander

doi:10.1093/nar/26.2.623

Cited by 145 publications

(106 citation statements)

References 18 publications

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“…We know that CCVs are used by the receptor in plants to shuttle between the Golgi and an acidic compartment, but we do not know in which direction of transport. This model is based on our demonstration of VSR PS-1 function in yeast and on previously published characteristics (Kirsch et al, 1994;Paris et al, 1997). reporter GFP can efficiently expose N-terminal, C-terminal, and even internal peptides (Abedi et al, 1998) and remains fluorescent. Therefore, we can study the interaction between potential signals and VSR PS-1 in a more realistic context than with synthetic peptides linked to an affinity column.…”

Section: Discussionmentioning

confidence: 99%

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Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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BP-80, later renamed VSR PS-1 , is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR PS-1 characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR PS-1 together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR PS-1 is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR PS-1 therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR PS-1 receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent. INTRODUCTIONThe plant secretory system is far less easy to study than its yeast counterpart, mainly due to the lack of an equivalent mutant collection. Plant and yeast cells, in opposition to mammalian cells, do not use the mannose 6-phosphatebased lysosomal sorting tag (Kornfeld, 1992); instead, the sorting information is carried by a peptidic sequence. In yeast, the two vacuolar proteins carboxypeptidase Y (CPY; Johnson et al., 1987) and proteinase A (Klionsky and Emr, 1989) both are produced with an N-terminal propeptide responsible for the vacuolar location of the mature protein. Almost 50 mutants defective for vacuolar protein sorting ( vps ) have allowed the identification of proteins involved in the yeast vacuolar sorting pathway. One of these proteins, Vps10p, plays a central role because it is the vacuolar receptor for CPY (Marcusson et al., 1994). Vps10p also is able to send foreign or malfolded proteins to the vacuole for degradation in a process that is believed to be independent of any sorting signal (Hong et al., 1996).In plants, two main types of vacuolar sorting determinants (VSDs) have been well studied and are believed to correspond to two separate vacuolar sorting pathways. The first type could be defined as a sequence-specific VSD (ssVSD) and has been well studied for barley aleurain (Holwerda et al., 1992) and sweet potato sporamin A (Matsuoka and Nakamura, 1991). For these two examples, the VSD is located within an N-terminal propeptide and contains a conserved tetrapeptide NPIR, the Ile being essential (Matsuoka and Nakamura, 1999). The position of this category of VSD seems to be less important than its sequence specificity, as shown with sporamin A (Koide et al., 1997). The second type of VSD is found in C-terminal propeptides (Ct-VSD); it is rather short with no obvious sequence conservation but needs to be accessibl...

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Section: Discussionmentioning

confidence: 99%

“…reporter GFP can efficiently expose N-terminal, C-terminal, and even internal peptides (Abedi et al, 1998) and remains fluorescent. Therefore, we can study the interaction between potential signals and VSR PS-1 in a more realistic context than with synthetic peptides linked to an affinity column.…”

Section: A Plant Receptor Functions In Yeast 789mentioning

confidence: 99%

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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“…Both N-and C-termini are on the same side of the barrel. We selected three positions exposed on external loops either on the same side as the termini (gl80) or on the opposite side (gl133 and gl172), which had been described as tolerant to insertion (Abedi et al 1998). The glycosylation site was engineered in the sequence of a secreted GFP, secGFP (Leucci et al 2007) producing the following changed sequences: -MKNHTFF-for gl80, -KENGSIL-for gl133 and -IENDSGG-for gl172.…”

Section: Preparation Of Glycosylated Gfps As Reporters Of Processing mentioning

confidence: 99%

Expression of a glycosylated GFP as a bivalent reporter in exocytosis

et al. 2009

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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.

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“…The scaffold should preferable be small, composed of a single chain, and with a highly stable structure [61]. The choice between different scaffolds such as thioredoxin A (TrxA) [62], staphylococcus nuclease [63], human stefin A [64], and green fluorescent protein [65] is made by taking into account the intended use of the peptide [59]. Scaffold structures restrict the conformation of the peptide such that the loop can only adopt a discrete shape from the conformational space available to it.…”

Section: Peptide Aptamersmentioning

confidence: 99%