Principal Investigator Chris Kaiser
In animal cells, some of the most interesting functions of the secretory pathway involve the regulated delivery of particular proteins to the cell surface in response to an environmental signal. We have discovered an analogous regulated secretory process in yeast, in which the integral membrane protein the general amino acid permease (Gap1p) is sorted in the trans-Golgi and endosome in response to the nitrogen source in the growth medium. We have identified a large collection of genes that are required for the proper sorting of Gap1p. These genes have revealed at least two stages for the proper intracellular sorting of Gap1p. In the first stage Gap1p is covalently modified by ubiquitination. The added ubiquitin tag acts to signal Gap1p trafficking to the vacuole; without ubiquitination all of the Gap1p is transported to the plasma membrane. In the second stage Gap1p can either be recycled to the Golgi (this step appears to be regulated by the abundance of amino acids) or Gap1p can enter luminal vesicles of the multivesicular endosome to ultimately be delivered to the interior of the vacuole.
Among the most interesting genes that govern Gap1p sorting are two small GTPases known as GTR1 and GTR2. These proteins, along with three additional polypeptides form a complex that is localized to the cytosolic face of the endosome. Mutations in any one of these genes prevents Gap1p cycling out of the endosome and causes constitutive delivery to the vacuole. Gtr2p can bind to the C-terminal tail of Gap1p and mutations in the tail that prevent Gtr2p binding will cause missorting of Gap1p to the vacuole. Taken together these results indicate that the Gtr proteins form a complex that is either part of a vesicle coat or is responsible for sorting Gap1p into recycling vesicles. We call this the GSE (GTPase containg complex for sorting in the endosome) and we are currently studying its structure and assembly on the membrane.
Many of the mutants that we identified that influence the intracellular sorting of Gap1p do so because they alter the intracellular abundance of amino acids. These mutants have thus provided access to the regulatory networks that respond to the availability of nutrients and control intracellular amino acid abundance by negative feedback. We are currently dissecting all of the different ways that nitrogen-derived signals are generated and how the membrane trafficking machinery responsible for sorting Gap1 decodes these signals.