GO-PROMTO Illuminates Protein Membrane Topologies of Glycan Biosynthetic Enzymes in the Golgi Apparatus of Living Tissues

Abstract

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.

The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall PolysaccharideO-Acetylation

“…S13). These results are consistent with those reported previously and serve as controls that the assay is working as expected (Søgaard et al, 2012). The AXY9-GFP fusion polypeptide Nelson et al, 2007).…”

“…The topology of the AXY9 protein within the Golgi membrane was determined using both a split yellow fluorescent protein (YFP) assay (Søgaard et al, 2012) and a protease protection assay (Akiyama and Ito, 1987;Boyd et al, 1987). In the split YFP assay, the N-and C-terminal portions of the YFP protein are expressed as separate polypeptides and will form a functional YFP protein only if both peptides are present in the same compartment of the cell.…”

“…In the split YFP assay, the N-and C-terminal portions of the YFP protein are expressed as separate polypeptides and will form a functional YFP protein only if both peptides are present in the same compartment of the cell. Constructs containing a transmembrane domain (TMD) of a truncated sialyltransferase from rat fused to the N-or C-terminal portion of YFP (YFP N or YFP C , respectively) were used as controls (Søgaard et al, 2012). When a YFP domain is fused to the N or C terminus of TMD, the YFP domain localizes outside or inside the Golgi, respectively (Søgaard et al, 2012).…”

“…Constructs containing a transmembrane domain (TMD) of a truncated sialyltransferase from rat fused to the N-or C-terminal portion of YFP (YFP N or YFP C , respectively) were used as controls (Søgaard et al, 2012). When a YFP domain is fused to the N or C terminus of TMD, the YFP domain localizes outside or inside the Golgi, respectively (Søgaard et al, 2012). When the split YFP TMD control constructs were transiently expressed in N. benthamiana, a YFP signal was observed only in the combinations TMD-YFP N with TMD-YFP C and YFP N -TMD with YFP C -TMD (Supplemental Fig.…”

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

“…First, the Golgi Protein Membrane Topology (GO-PROMTO) assay (Søgaard et al, 2012) was used. This system allows the topology of Golgi proteins to be deciphered using a modified bimolecular fluorescence complementation (BiFC) approach and hence is an appropriate choice for CSLF6 and CSLH1 as both proteins locate in part to the Golgi.…”