title: Ypt31/32 GTPases and their F-Box effector Rcy1 regulate ubiquitination of recycling proteins.
abstract: 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.
authors.affiliation: Department of Biological Sciences; Laboratory for Molecular Biology; University of Illinois at Chicago; Chicago, IL USA.
authors.forename: Shu H
authors.initials: SH
authors.lastname: Chen
authors.forename: Ankur H
authors.initials: AH
authors.lastname: Shah
authors.forename: Nava
authors.initials: N
authors.lastname: Segev