Targeting of EGFP chimeras within chloroplasts

Abstract

We have tested the potential of EGFP, a derivative of the green fluorescent protein (GFP), as a passenger protein for the analysis of protein transport processes across the thylakoid membranes in chloroplasts. In contrast to the majority of fusion proteins commonly used in such studies, EGFP is not of plant origin and can therefore be assumed to behave like a "neutral" passenger protein that is unaffected by any internal plant regulatory circuits. Our in vitro transport experiments clearly demonstrate that EGFP is a suitable passenger protein that can be correctly targeted either to the stroma or to the thylakoid lumen if fused to the appropriate transit peptide. The transport of EGFP across the thylakoid membrane shows, however, a clear pathway preference. While the protein is efficiently targeted by the deltapH/TAT pathway, transport by the Sec pathway is barely detectable, either with isolated thylakoids or with intact chloroplasts. This pathway specificity suggests that EGFP is folded immediately after import into the chloroplast stroma, thus preventing further translocation across the thylakoid membrane by the Sec translocase. The data obtained provide a good basis for the development of molecular tools for transport studies using EGFP as a passenger protein. Furthermore, plant lines expressing corresponding EGFP chimeras are expected to allow in vivo studies on the transport and sorting mechanisms involved in the biogenesis of the chloroplast.

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.…”

Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity

“…(A) Import of proteins with known localization used as a control for fraction purity: 16/EGFP (a transit peptide of spinach [Spinacia oleracea] OE16 of oxygen evolution system from thylakoid lumen, fused with enhanced GFP) (Marques et al, 2003), LHCP (light-harvesting chlorophyll a/b binding pea protein localized in thylakoid membranes) (Tan et al, 2001), and Toc34 (a component of the protein transport complex from the outer envelope membrane) (Chen and Schnell, 1997).…”

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

“…The pellet fractions were then treated with an alkaline buffer to wash away peripherally associated membrane proteins. The purity of fractions was controlled by import and fractionation analysis of a chloroplast lumen protein, tpsOE16::GFP (Marques et al, 2003), and the integral thylakoid membrane-bound protein, LHCP (Tan et al, 2001). SDS-PAGE analysis of chloroplasts and their fractions indicated that CYP97A4 and CYP97C2 were synthesized as precursors of about 69 and 62 kD and then processed to 64 and 59 kD, respectively.…”