Extracellular domain 2 of TSPAN4 governs its functions.

Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions.