Transmembrane Nox4 topology revealed by topological determination by Ubiquitin Fusion Assay, a novel method to uncover membrane protein topology.
The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available. To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins. Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.