Abstract

The cellular trafficking machinery relies on interactions between tethering factors, Rab GTPases and their GEF activators. One well studied multi-subunit tethering factor with recognized GEF activity is the TRAnsport Protein Particle (TRAPP) complex of which mammals have two; the TRAPPII and the TRAPPIII complexes. While TRAPPIII is linked to autophagy, the role of the TRAPPII complex in mammals, although linked to Golgi trafficking, remains elusive. Part of the ambiguity comes from our lack of understanding of which Rab GTPase is activated by TRAPPII. To answer to this, this work attempts to produce the first recombinant mammalian TRAPPII complex using Escherichia coli. The assembly of the complex is then assessed through the co- expression and the co-fractionation of the TRAPPII subunits after affinity purification and gel filtration chromatography. Upon further validation, the produced complex could be used in GEF activity assays and for structural analysis in ways that would further our understanding of the role of the mammalian TRAPPII complex in vesicular trafficking.