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Background Bimolecular Fluorescence Complementation (BiFC) assay has proven very useful to detect protein-protein interaction, protein-DNA/RNA interaction and protein-ligand interaction under physiological condition or near-physiological condition, even the interaction is too weak or too transient to be detected by other assays such as Co-IP or Y2H. Here, instead of studying two proteins interaction, we report a novel, intramolecular application of BiFC in studying the topology of proteins with multi transmembrane domains. Polytopic protein Presenilin 1 (PS1) is a critical component of the gamma-secretase complex that is responsible for the intramembranous cleavage of several type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology used to be an issue of strong controversy. According to the Kyte-Doolittle plot, PS1 has ten hydrophobic regions (HR) potentially to be alpha-helical transmembrane domains. Mature PS1 protein undergoes endoproteolysis resulting in stable NH2and COOH-terminal fragments (PS1-NTF and -CTF). All published models agree that the first six HR cross the membrane, implying a consensus for the topology of the PS1-NTF. In contrast, the localization of the last HR caused the most diversity. Most topology models for PS1, especially studies carried out on SEL-12, place its HR 10 in the cytosolic space. However, several recent observations suggest that HR10 may be integrated into the membrane. Method In the experiments presented here, we have investigated the topology of PS1-CTF by using BiFC. The fluorescent protein Venus was split at 173aa to generate two fragments, the NH2-terminal fragment of Venus (VN) and the COOH-terminal fragment of Venus (VC). The VN and VC fragments were fused to the both ends of the test protein respectively. Our hypothesis is that if the test protein span the membrane odd times, due to the barrier of lipid bilayer, the two fragments of Venus are not able to reach each other to accomplish the reconstitution, hence no fluorescent signal will be observed; on the other hand, when the test protein is with even times trans-membrane domains, the VN and VC have the opportunity to be brought together and the fluorescent signal will be detected.

Application of bimolecular fluorescence complementation in studying the topology of polytopic proteins