Ubiquitin‐dependent trafficking of Arn1, the ferrichrome transporter of Saccharomyces cerevisiae

The intracellular trafficking of Arn1, a ferrichrome (FC) transporter in budding yeast, is controlled in part by the binding of FC to the transporter. In the absence of FC, Arn1 is sorted directly from the Golgi to endosomes. FC binding triggers the movement of Arn1 from endosomes to the plasma membrane, while FC transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. Here we report that ubiquitination by the Rsp5 ubiquitin ligase is required for specific steps in the trafficking of Arn1. In the absence of FC, Arn1 was sorted from endosomes to the vacuole for degradation, and trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5. In the absence of clathrin binding protein Gga2p, Arn1 was missorted to the plasma membrane. At low concentrations of FC, inactivation of Rsp5 did not inhibit plasma membrane accumulation of Arn1. High concentrations of FC, which stimulate FC transport and endocytosis of Arn1p, led to higher levels of ubiquitination of Arn1, but did not induce degradation. In the absence of this ubiquitination, Arn1 failed to undergo endocytosis and remained on the plasma membrane, where it was active for transport. Arn1 was primarily modified with poly‐ubiquitin chains coupled via the lysine 63 residue of ubiquitin, and a cluster of lysine residues at the amino terminus of Arn1 was preferentially targeted for ubiquitination.