We have used a whole-genome approach to identify genes that are involved in the response to iron deprivation and iron overload in the budding yeast, Saccharomyces cerevisiae. Yeast respond to iron deprivation by activating systems of iron uptake, by mobilizing stored iron, and by shifting to iron-independent metabolic pathways. One type of iron uptake system activated during iron deprivation specifically transports iron-siderophore chelates, such as ferrichrome (FC). The intracellular trafficking of Arn1, a FC transporter in Saccharomyces cerevisiae, 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 redistribution of Arn1 to the plasma membrane, while FC transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. We determined that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi to endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Although Gga2 is reported to act as a receptor for ubiquitinated cargo, the binding of ubiquitin residues was not required for Gga2 to mediate trafficking of Arn1. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, and on the capacity of Gga2 to bind ubiquitin. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with poly-ubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter.Trafficking of Arn1 from the TGN to the lumen of the vacuole requires the activity of the clathrin-adaptor protein Gga2, which recognizes cargo proteins at the TGN and sorts them into clathrin-coated vesicles destined for the endosome and vacuole. Although Gga2 has been characterized as a ubiquitin receptor, we have shown that ubiquitin binding by Gga2 is not required for the TGN-to-endosome trafficking of Arn1, but is required for the subsequent sorting of Arn1 into the lumen of the vacuole. The clathrin adaptors Ent3p and Ent4p are also involved in TGN-to-vacuole sorting of Arn1p, and short peptides in the amino terminus of Arn1 are required for these interactions. In order to determine the global cellular requirements for Arn1 trafficking and to identify new cellular components involved in protein trafficking, we have designed a screen to identify all of the open reading frames necessary for proper Arn1 trafficking. In collaboration with Munira Basrai of NCI, we have engineered a GFP-tagged version of Arn1 into the yeast deletion mutant collection, and are using this collection to identify deletion mutants with defects in protein trafficking.
| Guo, Yan; Au, Wei-Chun; Shakoury-Elizeh, Minoo et al. (2010) Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae. J Biol Chem 285:39564-73 |
| Deng, Yi; Guo, Yan; Watson, Hadiya et al. (2009) Gga2 mediates sequential ubiquitin-independent and ubiquitin-dependent steps in the trafficking of ARN1 from the trans-Golgi network to the vacuole. J Biol Chem 284:23830-41 |
