Ratiometric Fluorescence‐Imaging Assays of Plant Membrane Traffic Using Polyproteins

Abstract

Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the aminoterminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…This approach appears to be more reliable in comparison to systems based on expression of two proteins transcribed divergently from the same cauliflower mosaic virus 35S enhancer elements. An analysis of the expression of two fluorescent ER luminal proteins, ssYFP-HDEL and ssGFP-HDEL, in these conditions showed a considerable variation in the relative expression of the two proteins (Samalova et al, 2006). GFP and its spectral variants have also been useful as reporters for biochemical analyses on protein traffic and membrane protein orientation.…”

“…To overcome these limitations, Samalova et al (2006) have developed a polyprotein-based system for ratiometric fluorescence imaging assays. In this approach, the authors have used the 20-residue, self-cleaving 2A peptide from the foot and mouth disease virus to generate polyproteins that express a fluorescent trafficked marker in fixed stoichiometry with a reference fluorescent protein in a different cellular compartment.…”

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

“…To this end, another notable observation from this study is that some of the RFP Strep released from the ND‐4 polyprotein accumulated inside the vacuole (Figure 7d, ND‐4) although we did also detect extracellular RFP (Figure 7b). In an earlier study of plant membrane traffic using polyproteins (Samalova et al ., 2006), secretion of a monomeric RFP (a DsRed variant (Campbell et al ., 2002) very similar to the mCherry RFP used in the present study) into the apoplastic space of N. benthamiana leaf was noted when an N‐terminal secretory signal peptide was incorporated. Therefore, the C‐terminal Strep Tag appended to the RFP in ND‐4 may be recognized as a putative vacuole sorting determinant.…”

“…In the event the C‐terminal F2A sequence remained attached to an ER‐targeted protein, it might cause erroneous protein sorting (François et al ., 2004; Samalova et al ., 2006). To this end, we investigated tobacco NT‐1 cells expressing the N(‐)‐2 polyprotein (Figure 1) that consists of a secretory GFP 172 and a cytosolic RFP Strep , separated by a mutated IntF2A with abolished N‐terminal autocleavage activity, that is Int(N‐)F2A (containing a C1A mutation in the intein).…”

“…The vacuole targeted GFP 172 ‐Int(N‐)F2A was partially degraded, probably due to hydrolysis by proteinases that reside in the secretory pathway or the vacuole, as multiple protein bands were detected by Western blot analysis (Figure S3a). The undesired vacuole sorting mediated by the F2A sequence has been reported in earlier studies that the secretory protein anterior to the F2A peptide is prone to accumulation in the vacuole (François et al ., 2004; Samalova et al ., 2006). Conversely, IntF2A with an active intein (e.g.…”