In vivo transport of folded EGFP by the  pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Abstract

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.

Differential Coloring Reveals That Plastids Do Not Form Networks for Exchanging Macromolecules

“…For labeling the plastid stroma with mEosFP, the enhanced GFP (EGFP) coding sequence of the previously reported tpFNR:EGFP fusion (Marques et al, 2003(Marques et al, , 2004 was replaced by mEosFP and placed in the binary T-DNA vector pCAMBIA1300 (http:// www.cambia.org.au). Standard molecular biology protocols were followed for the cloning (Sambrook and Russell, 2001).…”

“…A novel probe was created for highlighting plastids by fusing the Nterminal transit peptide sequence of plastid ferredoxin NADP(H) oxidoreductase (Marques et al, 2003(Marques et al, , 2004 to mEosFP and driving its expression under a cauliflower mosaic virus 35S promoter. Agroinfiltration of Nicotiana benthamiana leaves with tpFNR:mEosFP showed that each of the photoconvertible probes efficiently labeled their target organelle in a bright fluorescent green color.…”

“…Plants were stably transformed with tpFNR:mEosFP using the floral dip transformation method (Clough and Bent, 1998). tpFNR:EGFP was introduced in the arc5, arc6-1, and pgm-1 mutants by crossing with FNR:EGFP plants (Marques et al, 2004).…”

The Arabidopsis thaliana Type I Isopentenyl Diphosphate Isomerases Are Targeted to Multiple Subcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

“…First, we cloned the eGFP gene into the same eGFP-ready Gateway plant transformation vector (pB7FWG2) (Karimi et al, 2002), producing an eGFP dimer lacking any transit peptide, which served as a control for cytosolic localization. Second, we used Arabidopsis plants transformed with a signal peptide fused to eGFP, for which plastid localization of the eGFP fusion protein had already been confirmed (Marques et al, 2004).…”

Stromuling when stressed!

“…A transgenic Nicotiana benthamiana line, which is available to highlight the plastid stroma and stromules, expresses a GFP fusion protein with ferredoxin NADP(H) oxidoreductase (FNR), FNR::eGFP [19][20][21]. Other constructs have also been reported to visualize stromules, e.g.…”