RESUMO
Ypt/Rab GTPases are conserved molecular switches that regulate the different steps of intracellular trafficking pathways. In yeast, the Ypt31/32 GTPases are required for exit from the trans-Golgi and for recycling from the plasma membrane (PM), through early endosomes, to the Golgi. We have previously shown that the recycling function of Ypt31/32 is mediated by an effector called Rcy1. Specifically, both Ypt31/32 and Rcy1 are required for recycling the vSNARE Snc1. Rcy1 contains an F-box domain shared by proteins that act in substrate recognition of ubiquitin ligases. Here, we show that both Ypt31/32 and Rcy1 are important for Snc1 ubiquitination and that such ubiquitination plays a role in Snc1 recycling. Direct interaction between Rcy1 and Snc1 was demonstrated using two independent approaches. In vitro interaction was observed using co-precipitation of recombinant proteins, whereas interaction in yeast cells was observed using bimolecular fluorescence complementation. Ubiquitination of Snc1 in vivo at the K63 position was previously shown in a proteomic study. We show that the Snc1-K63R mutant protein is less ubquitinated than wild-type Snc1 and is defective in endosome-to-Golgi transport. Additionally, wild-type Snc1 is ubiquitinated to a lesser extent in ypt31/32ts and rcy1Δ mutant cells and Snc1 recycling is also blocked in endosomes in these mutants. Therefore, ubiquitination plays a role in the recycling of Snc1 from the PM to the Golgi, and Ypt31/32 and Rcy1 regulate this ubiquitination. Together, these results suggest a new role for ubiquitination in cargo recycling. Moreover, we propose that Ypt/Rabs integrate intra-cellular trafficking with ubiquitination.
RESUMO
Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.