Green Fluorescent Protein variants fold differentially in prokaryotic and eukaryotic cells

Abstract

Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding ef®ciency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3±4 times better than the wtGFP or S65T in E. coli, and 10±20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding ef®ciency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or¯uorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that speci®c GFP variants follow different folding trajectories in mammalian versus bacterial cells. The speci®city of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo.

Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

“…3A) is explained by the spontaneous upregulation of the bacterial chaperone program due to the presence of high concentrations of unfolded YFP in the bacterial cytoplasm (16). It is known that green fluorescent protein folding in vivo involves chaperones and that green fluorescent protein variants follow different folding trajectories (29). The increased dependence of cdYFP folding on chaperone activity was confirmed by the fact that the fluorescence yield was greatly increased by the induction of chaperones in liquid bacterial cultures after cold ethanol shock (42), which gave rise to an ϳ40-fold increase in fluorescence yield (Fig.…”

Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins

“…27). Most probably this observation implies that the Tat-EGFP fusion proteins unfold during membrane translocation, as commonly occurs in this process; once unfolded, an intrinsic property of GFP is to re-fold in an optically active form only at very low efficiency (57). This consideration also implies that the use of the EGFP reporter tag as the sole method by which to study the release of Tat from the cells (another process possibly requiring crossing of a cell membrane), as recently described (58), might possibly bring misleading conclusions.…”

Improperly folded green fluorescent protein is secreted via a non-classical pathway

“…Perhaps the GFP is loosely folded or unfolded as a condition for secretion. Once secreted, GFP does not fold into a fluorescent conformation because of the absence of chaperone proteins (Feilmeier et al, 2000;Sacchetti et al, 2001). By contrast, GFP secreted via the ER-Golgi pathway is fluorescent in the medium because proper folding is maintained during secretion (Laukkanen et al, 1996).…”