Abstract
The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions betweendifferent NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. Theluciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.
Published Version
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