Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae).

The general amino acid permease ('Gap') system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after 'pools' of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.     

Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1

In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939-43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.     

Changes in the activity of the general amino acid permease from Saccharomyces cerevisiae var. ellipsoideus during fermentation

The evolution of the activity of the general amino acid permease and ethanol and glucose concentrations in the medium were studied in a mild fermentation process carried out by a wine strain of Saccharomyces cerevisiae var. ellipsoideus isolated from grape musts in spontaneous fermentation. The cells displayed a reduction in the activity of the general amino acid permease parallel to the increase of ethanol in the medium. This ethanol increase was not enough to promote a substantial inhibition on the total polypeptide synthesis measured as polyuridylic-acid-directed polyphenylalanine synthesis.     

Transport activity-dependent intracellular sorting of the yeast general amino acid permease

Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1p(A297V), which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.     

The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease

Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH(4)(+), a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(ts) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH(4)(+) is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH(4)(+). Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.     

Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.     

Brefeldin a decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae

Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.