Date Published: January 13, 2017
Publisher: Public Library of Science
Author(s): Wenjun Xie, Mads Eggert Nielsen, Carsten Pedersen, Hans Thordal-Christensen, Els J. M. van Damme.
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.
Novel membrane proteins are still being identified in for instance the endoplasmic reticulum (ER) and the plasma membrane (PM) [1–4], and insight in their topology is essential for unraveling their function [1,5]. Several experimental and software-based prediction methods are available for ER as well as other membrane proteins [6–11]. A number of these experimental methods make use of fusing a fluorescent protein to the membrane protein-of-interest. Gross et al.  used reconstitution of split green fluorescent protein (GFP) for topology studies of chloroplastic, mitochondrial and peroxisomal membrane proteins in plant protoplasts. In the present study, we developed an easy-to-use method also using reconstitution of split-GFP for topology studies of membrane proteins in intact plants, as previously described for e.g. ER proteins in a rabbit kidney cell line . These studies benefit from the work of Cabantous et al.  and Cabantous and Walde , who developed a mutant version of GFP that allowed the first ten β-sheets (GFP1-10) and the eleventh β-sheet (GFP11) to self-assemble and fluoresce when expressed separately.
To initially confirm the ER localization of CNXTM, we transiently co-transformed N. benthamiana leaves with the GFP-CNXTM-expression construct and a construct expressing an ER-luminal marker, SP-RFP-HDEL , using agroinfiltration. Confocal microscopic analysis of the epidermal cells 2 days after bacterial infiltration showed overlap between the GFP and the RFP signals. Both signals were cortical network-like as well as perinuclear, but not intranuclear (Fig 1). This signal pattern is indicative of GFP and RFP being at the ER, agreeing with the ER membrane location of CNXTM and the ER-luminal location of RFP-HDEL.