A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants.

To understand the function of membrane proteins, it is imperative to know their topology. For such studies, a split green fluorescent protein (GFP) method is useful. GFP is barrel-shaped, consisting of 11 β-sheets. When the first ten β-sheets (GFP1-10) and the 11th β-sheet (GFP11) are expressed from separate genes they will self-assembly and reconstitute a fluorescent GFP protein. However, this will only occur when the two domains co-localize in the same cellular compartment. We have developed an easy-to-use Gateway vector set for determining on which side of the membrane the N- and C-termini are located. Two vectors were designed for making N- and C-terminal fusions between the membrane proteins-of-interest and GFP11, while another three plasmids were designed to express GFP1-10 in either the cytosol, the endoplasmic reticulum (ER) lumen or the apoplast. We tested functionality of the system by applying the vector set for the transmembrane domain, CNXTM, of the ER membrane protein, calnexin, after transient expression in Nicotiana benthamiana leaves. We observed GFP signal from the ER when we reciprocally co-expressed GFP11-CNXTM with GFP1-10-HDEL and CNXTM-GFP with cytosolic GFP1-10. The opposite combinations did not result in GFP signal emission. This test using the calnexin ER-membrane domain demonstrated its C-terminus to be in the cytosol and its N-terminus in the ER lumen. This result confirmed the known topology of calnexin, and we therefore consider this split-GFP system highly useful for ER membrane topology studies. Furthermore, the vector set provided is useful for detecting the topology of proteins on other membranes in the cell, which we confirmed for a plasma membrane syntaxin. The set of five Ti-plasmids are easily and efficiently used for Gateway cloning and transient transformation of N. benthamiana leaves.