The Running of the Buls: Control of Permease Trafficking by α-Arrestins Bul1 and Bul2

How do changes in the environment trigger selective internalization of membrane proteins? This question is of broad biological importance since efficient endocytosis impacts many facets of cell physiology. In this issue of Molecular and Cellular Biology, Merhi and André (11) provide a compelling answer. They define a molecular pathway that leads from nutrient uptake through intracellular signaling to internalization of a specific nutrient permease. Their findings elegantly connect a cast of regulatory characters, including the nutrient-regulated TORC1 complex, a protein kinase (Npr1) that is a TORC1 target, and the 14-3-3 phosphoserine-binding proteins (11). Importantly, they demonstrate that two members of the -arrestin or arrestin-related trafficking adaptor (ART) family, Bul1 and Bul2, serve as linchpins connecting nutrient signaling to protein trafficking (11). The pathway that they describe bears striking similarity to the recently reported control of two other -arrestins (1, 10), suggesting that a conserved mechanism regulates arrestin-mediated trafficking. A Gap in our knowledge. In Saccharomyces cerevisiae, the general amino acid permease Gap1 is a nonspecific transporter of all L-amino acids and many amino acid analogs (5). Since studies of Gap1 began over 40 years ago in the Grenson lab (5), the trafficking of Gap1 has served as a model to identify basic features of signal-induced endocytosis. Subsequent work from many groups, notably the Kaiser and André labs, has demonstrated that Rsp5, now known to be a protein-ubiquitin ligase (the mammalian ortholog is Nedd4) (14), and Npr1, now known to be a protein kinase (as reviewed in reference 9), antagonistically control Gap1 delivery to the plasma membrane in response to changes in the available nitrogen source (6, 17). When cells are grown on a nonpreferred (or poor) nitrogen source, like proline, Gap1 localizes to the plasma membrane, where its ability to transport a broad spectrum of amino acids assists in nitrogen scavenging. Under these conditions, Gap1 is stabilized at the cell surface by active Npr1 kinase (3, 16). Npr1 is negatively regulated by TORC1, which is active when amino acids are abundant (9, 15). Conversely, growth on a preferred (or good) nitrogen source like ammonium promotes Rsp5-mediated ubiquitylation of Gap1 and stimulates its endocytosis (17). Like many membrane proteins, Gap1 lacks the sequence motifs (consensus PPXY or LPXY [14]) needed to bind Rsp5 directly. Instead, Bul1 and Bul2, which have these motifs, likely recruit Rsp5 to stimulate Gap1 ubiquitylation and internalization (6, 13, 17). However, a detailed molecular mechanism connecting nutrient supply to Gap1 trafficking remained elusive. It was not clear how nitrogen quality regulated Gap1 localization or what controlled Bul-mediated ubiquitylation of Gap1. Ammonium ion uptake leads the way. In this work, Merhi and André (11) make extensive use of the well-established nitrogen regulation of Gap1 trafficking: they grow cells on a nonpreferred nitrogen source (proline), where Gap1 is localized to the plasma membrane, and then add NH4 , a preferred nitrogen source, which induces Gap1 ubiquitylation and internalization. They demonstrate, first, that NH4 -induced Gap1 ubiquitylation and endocytosis require uptake through the ammonium ion permeases Mep1, Mep2, and Mep3 and production of glutamate, mainly by glutamate dehydrogenase Gdh1 during growth on glucose (Gdh3 does so under nonfermentative conditions). Although Gdh2 is thought to have mainly a catabolic role (converting glutamate to -ketoglutarate), Mehri and André found that when NH4 is plentiful, it can also contribute to glutamate synthesis (11). Glutamate, in turn, is the nitrogen donor for synthesis of many amino acids. Thus, the authors hypothesize that when NH4 is added to proline-grown cells, it may increase amino acid levels and activate TORC1, which promotes robust growth and proliferation under nutrient-replete conditions. How intracellular amino acids activate TORC1 is not fully understood. Recent work demonstrates that leucine bound to its leucyl-tRNA synthetase (LeuRS) interacts with the Rag GTPase in the yeast EGO complex, which in turn activates TORC1 (2). Perhaps, addition of NH4 and its conversion to glutamate increase leucine levels and/or or other amino acid levels to stimulate TORC1 via a similar mechanism, but this remains to be determined. Active TORC1 destabilizes Gap1 by stimulating endocytosis of the existing permease (as reviewed in reference 9); TORC1 inhibits Npr1 in a switch-like manner by directly phosphorylating negative regulatory sites in Npr1 and concomitantly preventing dephosphorylation of those sites by sequestering the phosphatase needed to dephosphorylate Npr1, Sit4 (as reviewed in reference 9). It was known that loss of Npr1 function causes enhanced ubiquitylation and internalization of Gap1 (3, 16). Here, the authors show that Npr1 remains dephosphorylated, even after NH4 addition, if (i) TORC1 is pharmacologically inhibited with rapamycin or (ii) NH4 uptake is prevented (in mep1 mep2 mep3 triple mutant cells) (11). Thus, the authors begin to reveal Npr1 regulation: NH4 internalization and conversion to glutamate and perhaps other amino acids activate TORC1 through an undefined mechanism (which may be similar to the tRNA-synthetase/EGO activation pathway [2]), and this in turn inhibits Npr1 and promotes Gap1 internalization (Fig. 1). It will be interesting to see in future studies if components of this same signaling pathway are important for Gap1 endocytosis when it is induced by amino acids trans-