Deciphering TRAPP complex function in yeast

Abstract

All eukaryotes contain organelles that perform specialized functions essential for life. The Golgi complex, an organelle that is important for transport of cellular material, serves as the “Grand Central Station” of the cell by receiving, then sorting proteins and membranes to various locations. Many steps of vesicular trafficking at the Golgi complex are regulated by several families of small GTPases, which modulate between an active and inactive state. One particularly interesting GTPase is Rab1, which also has an established role in autophagy. Rab1 is activated by a guanine nucleotide exchange factor (GEF), a process that is conserved from mammals to yeast. Discordantly, mammals contain two protein complexes named TRAPPII and TRAPPIII (transport protein particle) which activate Rab1; while in yeast, four (TRAPPI ‐ TRAPPIV) have been implicated as GEFs in Ypt1 (Rab1 homolog) activation. Yeast TRAPP complexes share a core set of six subunits. Additional subunits are thought to regulate the complex by adding substrate and compartmental specificity. TRAPPII contains four more subunits, while TRAPPIII and TRAPPIV include one additional subunit. Importantly, the precise functions of the accessory subunits remain unclear. We demonstrate that TRAPPII and TRAPPIII are the only two complexes present in yeast cells, which is consistent with findings in mammalian cells, by quantifying isolated TRAPP complexes and simultaneously monitoring the localization of all TRAPP complexes in vivo. We also find that, using an in vitro fluorescent nucleotide exchange assay, TRAPPIII activates Ypt1 more effectively than the other complexes. Concurrently, we notice that Trs85 localizes to the Golgi complex and that Ypt1 is mislocalized in vivo in the absence of the TRAPPIII‐specific subunit, Trs85. Additionally, using an in vitro membrane binding assay, we find that Trs85 has an affinity for membranes. These findings suggest that there are only two TRAPP complexes in yeast, and that TRAPPIII is involved in trafficking at the Golgi complex in addition to its established role in autophagy. Taken together, our model suggests that the main role of Trs85 in the TRAPPIII complex is to bring the catalytic core close to a membrane surface, localizing the active site to the right compartment in an orientation that facilitates localization and activation of Ypt1.Support or Funding InformationThis material is based upon work supported by the NSF GRFP under Grant DGE‐1144153, by NIH/NIGMS Grant R01GM116942, and by the Alfred P. Sloan Foundation. Any opinions, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF, NIH, the Sloan Foundation or their trustees, officers, or staff.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.