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Nutrient sensing and signaling by the yeast Gap1 amino acid transceptor

Final Report Summary - YEAST SENSING (Nutrient sensing and signaling by the yeast Gap1 amino acid transceptor)

Project context and objectives

This project has focused on studying signalling to nutrient regulator pathways via the general amino acid permease (Gap1) from the yeast Saccharomyces cerevisiae. This protein has combined transporter and receptor functions (hence, called transceptor). Previous work from this laboratory has shown that the amino acid transporter Gap1 triggers activation of protein kinase A (PKA) targets when amino acids are added back into cell cultures previously starved for nitrogen.

As explained in the middle term report, part of the project was initially dedicated to investigating the relation between the target of rapamycin (TOR)-controlled protein kinase Npr1 and the PKA pathway. Npr1 kinase activity is necessary to stabilise Gap1 and other nutrient transporters at the plasma membrane. Dephosphorylation and activation of the protein is regulated by TOR, another central regulator of cell growth and division in response to nutrient availability. So far, the kinase that phosphorylates and inactivates Npr1 is yet unidentified. As a result of studies mentioned in the first half period of this grant we have found preliminary evidence that Npr1 could be a substrate for PKA, which could explain its regulatory effect on Gap1.

In the second term of this grant, my work in this project has, however, more particularly focused on the elucidation of the mechanism of action of three specific ?-glutamyl dipeptides (GD) which cause persistent activation of Gap1-mediated PKA signalling after their addition into the medium of nitrogen-starved cells. The main results of this work are gathered in the 2012 article: 'Peptides induce persistent signalling from endosomes by a nutrient transceptor', by Rubio-Texeira M., Van Zeebroeck G. and Thevelein J., accepted for publication in Nature Chemical Biology (impact factor 2010: 15.808) in press and scheduled to appear online on 4 March 2012.

Project results

Normally, amino acids induce a Gap1-dependent transient activation peak of trehalase activity (one of the PKA targets) after their addition to nitrogen-starved cells. This activation reaches a maximum within minutes after the addition of the amino acid, then activity goes back down to basal within a period of one hour. However, when the cells were exposed to each of three ?-glutamyl dipeptides: L-Glu-?(?-Abu), L-Glu-?-L-Ala, or L-Glu-?-Gly, trehalase activity remained high for a period of time superior to three hours (work in collaboration with Dr Griet Van Zeebroeck, GVZ).

We next analysed whether this persistent signalling results from a failure of cells to endocytose Gap1 transceptor during exposure to the dipeptides. We monitored localisation of Gap1 tagged with green fluorescent protein (GFP) and this way we found that upon exposure to the persistent ?-GD, Gap1 was not properly endocytosed and sorted to the vacuole for degradation but instead accumulated in small punctae.

In order to discern which type of organelle these punctae were, we analysed colocalisation of Gap1-GFP with organelle markers for the secretory pathway and endocytic / endosome pathway. After endocytosis proteins are sorted to the vacuole via trafficking vesicles that together constitute an intermediate compartment called endosomes. A possibility was that Gap1-GFP was stuck in these compartments and its displacement to the vacuole impaired. The colocalisation study confirmed that the small punctae where Gap1-GFP accumulated were endosomes.

Western blot analysis of untagged Gap1 with Gap1-specific antibodies in membrane enriched samples subjected to ultracentrifugation in a sucrose density gradient also revealed stabilisation of Gap1 co-fractionating with endosome and plasma membrane markers (not shown here; Rubio-Texeira et al., 2012).

The effect on Gap1 sorting and signalling of these three persistent ?-GD was very unique since a collection of other persistent ?-GD was also tested but none of them caused similar phenotypes (Rubio-Texeira et al., 2012).

We wanted to ascertain that the effect of the persistent ?-GD was specific on Gap1, caused by interaction of the dipeptides with this transceptor. Studies in collaboration with Dr Griet Van Zeebroeck revealed that the three persistent ?-GD are competitive inhibitors of Gap1-mediated amino acid transport and are specifically transported by Gap1 and not by other peptide transporters present in the yeast cells. They could also be used by the cells as only nitrogen source in a Gap1-specific manner.

Next, we wanted to analyse whether the effect on Gap1-defective sorting to the vacuole was specific for this protein or whether exposing cells to the persistent ?-GD was in some way causing a general defect of the endocytic and vacuolar sorting pathways. We did a number of separate analyses. First, we added the lipophilic dye FM4-64 to cells simultaneously exposed to the persistent ?-GD. Under normal trafficking circumstances, FM4-64 is endocytosed and sorted to the vacuolar limiting membrane. Trafficking defects cause its accumulation in endosome vesicles such as observed for Gap1-GFP in the presence of the ?-GD. In this case, however, FM4-64 could reach the vacuolar limiting membrane, indicating that no general trafficking defect is caused by the ?-GD. Second, we tested the vacuolar sorting of several plasma membrane permeases and vacuolar resident proteins in cells exposed to the persistent ?-GD and in none of these cases was their vacuolar sorting affected. Third, missorting of the vacuolar resident protein carboxypeptidase Y (CPY) to the extracellular medium happens when the endocytic and vacuolar sorting pathways are defective. We also tested whether this happened in the presence of ?-GD but the cells did not secrete any CPY to the medium in this condition (Rubio-Texeira et al., 2012).

An interesting phenomenon that immediately called our attention when observing the fate of Gap1-GFP in cells exposed to the persistent ?-GD was the fact that cells rapidly lost vacuolar GFP fluorescence after addition of these compounds to the medium. When a GFP-tagged plasma membrane protein is sorted to the vacuole for degradation, the GFP signal accumulates in the lumen of this compartment, since GFP is usually relatively resistant to the acidic pH and proteases present in this compartment. The vacuolar GFP signal usually takes at least one hour or more to fade. However, upon addition of the persistent ?-GD to the medium the vacuolar GFP signal rapidly disappeared in just matter of minutes. The type of GFP that we used to tag Gap1 for this assay is GFP S65T. This GFP variant is sensitive to acidic pH. Vacuoles are themselves acidic compartments. If a phenomenon occurs that further increases their acidity, it could cause a rapid loss in GFP S65T fluorescence remaining in the vacuole lumen. One possibility was that transport of the dipeptides in symport with protons caused an increased influx of protons inside the cells. We therefore analysed whether exposure of cells to the ?-GD was causing, in some way, intracellular acidification.

To analyse this possibility, we expressed cytosolic pHluorin in the cells. pHluorin is a form of GFP that is ultrasensitive to pH changes. Changes in the fluorescence emitted by this variant are considered a faithful readout to monitor changes in intracellular pH. This way we could verify that exposure of cells to each of the three ?-GD caused substantial intracellular acidification.

This acidification happened in a Gap1 transporting activity-dependent manner, since a mutant form of Gap1 (Y395C) or a mutant deleted in GAP1 gene no longer exhibited intracellular acidification upon exposure to the ?-GD. Intracellular acidification did not happen when cells transported amino acids via Gap1 and other non-persistent ?-GD also did not cause intracellular acidification. Gap1 transport amino acids in symport with protons and is therefore dependent on extracellular pH for its activity, showing an optimum pH of 4-5 for amino acid transport. The transport of the three persistent ?-GD was, however, unusually dependent on low pH and had its optimum at external pH 2. For neutral to basic pH (6 and above) we noticed that the transport of the dipeptides decreased (Rubio-Texeira et al., 2012). For these external pHs, as transporting of the ?-GD by Gap1 was reduced, intracellular acidification due to this transport was also reduced by increasing external pH. Concomitantly, the vacuolar sorting defect of Gap1 was ameliorated (pH 6) although reduced transport also stopped the induction of further Gap1 endocytosis at pH of 7 and above. The vacuolar lumen GFP signal was again stabilised in cells exposed to the ?-GD in external pH 6 and above, confirming that the rapid loss of fluorescence that we had observed was due to intracellular acidification caused by rapid transport of these molecules to the cytosol via Gap1.

Intracellular acidification is known to activate cyclic adenosine monophosphate (cAMP) synthesis and accumulation which in turn activates protein kinase A pathway (Ras-cAMP PKA pathway). Gap1-mediated activation of protein kinase A by amino acids is not mediated via acidification and accumulation of cAMP. However, since the transport of the unusual persistent ?-GD caused intracellular acidification we wondered whether the mechanism by which these molecules induce persistent PKA signalling has to do with this acidification. In collaboration with Dr Griet Van Zeebroeck, we analysed the levels of cAMP in this circumstances and confirmed that cAMP accumulates as a side effect of the intracellular acidification occurring by Gap1-mediated transport of the ?-GD (Rubio-Texeira et al., 2012). In principle we thought that this was indicative that the persistent signalling takes place due to the intracellular acidification. To test whether this was the case we used the mutant deletion strain nha1. Lack of nha1 causes alkalinisation of the cytosol so that it counteracted the hyperacidification caused by transport of ?-GD via Gap1. In this case we no longer observed cAMP accumulation. However, Gap1 transport-dependent persistent signalling still took place (Rubio-Texeira et al., 2012). This indicated that the effect of these molecules on Gap1 mediated PKA signalling is not mediated via intracellular acidification.

It is already known by the work of other laboratories that sudden cytosol acidification causes subsequent acidification of the lumen of compartments such as endosomes and vacuoles. In the case of endosomes, progressive acidification is needed to direct sorting of proteins to the vacuole for degradation. However, excessive intraluminal acidification of these compartments can also lead to a block in the delivery of endosome contents into the vacuole. At the limiting membrane of endosomes and vacuoles, proton pumps exist that control this intraluminal pH. Vacuolar-type ATPase (V-ATPase) pumps protons to the lumen of endosomes and vacuoles. Nhx1 extrudes protons. Equilibrium between the two activities keeps proper luminal pH for proper vacuolar trafficking. Permeases that act in symport like Gap1 may be able to continue their activity after endocytosis and in doing so they can also contribute to changes in the intraluminal pH of endosomes in transit to the vacuole. By using the nha1 mutation, we found out that suppression of the cytosol acidification was not sufficient to completely restore Gap1 vacuolar sorting in the presence of the persistent ?-GD. This suggests that the ?-GD may have a more specific effect on Gap1 after endocytosis that impairs its sorting to the vacuole. Perhaps interaction of Gap1 with the dipeptides continues while Gap1 is located in the membrane of endosomes. If this is the case, Gap1 may continue its proton symporting activity and cause aberrant luminal acidification of the endosome lumen. This, along with the V-ATPase activity my cause an endosome pH that is too low for these endosomes functionally be able to continue their trafficking to deliver cargo in the vacuole. If this is the case, inhibiting V-ATPase activity may partially counteract the effect and restore Gap1 vacuolar sorting. This is indeed what we observed when we exposed cells to concanamycin A, a V-ATPase specific inhibitor. This, along with the fact above that acidification derived accumulation of cAMP was not responsible for the persistent signalling elicited by these three molecules, has led us to postulate that conditions exist where internalised Gap1 in transit to the vacuole is still active and can mediate signalling to the PKA pathway.

In collaboration with Dr Griet Van Zeebroeck, we were able to obtain a separate line of evidence showing that Gap1 can cause persistent signalling to PKA pathway while internalised in endosomes. We checked activation of PKA pathway by the regular amino acid L-citrulline (specifically transported by Gap1 at the concentrations tested) in mutants that block endocytosis or endosome trafficking to the vacuole. In conditions that blocked endocytosis of Gap1 causing its accumulation at the plasma membrane (use of endocytosis mutants, latrunculin A or ubiquitination mutants), exposure to L-citrulline did not cause persistent signalling to PKA but only a transient activation similar to wild type conditions. However, blocking internalised Gap1 within endosomes caused persistent signalling to PKA pathway in a way that was comparable to the use of the three ?-GD described above. We increased ubiquitination of Gap1 by overexpression of the protein BUL1, a component of the polyubiquitinating machinery. This further enhanced endocytosis of Gap1 and accumulation of it in endosomes after exposure to L-citrulline. We confirmed that under these conditions the majority of the Gap1 pool was removed from the plasma membrane and accumulated inside endosomal vesicles. In these conditions we still measured persistent PKA signalling, demonstrating that this phenomenon is not dependent on Gap1 located at the cell surface but in internal endosome pools.

Potential impact

Elucidating how nutrient signals are translated in activation of PKA pathway is important both from industrial and biomedical points of view. Since PKA controls yeast ability to withstand different stresses, modulation of this pathway may improve the performance of industrial strains of Saccharomyces. From the biomedical point of view, malfunction of this pathway, as well as malfunction of plasma membrane receptors, are known to be related to a variety of disorders, from cancer to cardiac diseases to mention only a few. A better understanding of this pathway in a lower eukaryotic system is, thus, crucial in order to further understand its function in mammalian cells.

The results obtained during this project have substantially contributed to a more complete overview at the subcellular level of how the transceptor Gap1 is regulated to transduce a nutrient signal. The results we have obtained by studying the effect of the three ?-GD: L-Glu-?(?-Abu), L-Glu-?-L-Ala, or L-Glu-?-Gly, and the effect of mutants in the vacuolar sorting pathway, have allowed us to demonstrate that signalling by a nutrient transceptor to PKA does not terminate with endocytosis and that a transceptor located in endosomes can still continue signalling. This adds the possibility of a new level of modulation in the response of cells to the presence of nutrients in the environment. For higher eukaryotic systems, numerous examples already exist where receptors have been shown to continue signalling while temporarily stored within endosomes. However this phenomenon had so far not been described to happen to transceptors in yeast. This finding has important implications to better understand the connection between nutrient sensing via the PKA pathway and other nutrient regulator pathways, such as TOR (components of the latter are known to be mainly active in endosome and vacuole membranes).

In the course of this research, we have also uncovered unusual properties for Gap1, usually known as an amino acid transceptor, as the ability to transport particular dipeptides. The acidification caused by transport of these dipeptides inside cells may have potential applications for researchers that wish to modify intracellular pH in a transitory manner. The dipeptides are eventually used by the cells as nitrogen source in such way that their effect is reversible upon their removal from the medium.

Project objectives for the period

To the best of my knowledge we did not receive any particular recommendations after presentation of the middle period report. The main objectives of this project were initially set in Annex I as consisting, in the first place, of investigating the relationship between Gap1 mediated amino acid activation of the PKA pathway and the rapid amino acid induced downregulation of Gap1 by dephosphorylation, ubiquitination, internalization and degradation.

In second place, the work focuses on the elucidation of components in the signaling pathway between Gap1 and PKA.

During the first period we dedicated part of the project to analyse the regulation of Gap1 by phosphorylation, focusing our attention on Npr1. This protein kinase is essential to stabilise active Gap1 at the plasma membrane although the possibility of Gap1 being a direct substrate of phosphorylation by Npr1 has not yet been confirmed. Since transceptors like Gap1 have important effects on PKA activity we hypothesized that either Gap1 and/or its regulator Npr1 may be potential substrates for PKA phosphorylation. As a result of work done in this grant we discovered that Npr1 is a potential substrate for protein kinase A phosphorylation. Further work on this line was discontinued in favour of the other studies described above and below. However, we believe this preliminary work opens the door to further studies that will be important in our lab to establish a more direct correlation between protein kinase A and regulation of the transceptors that activate it.

In the first and second half periods of this grant, we focused our studies in the effect of three ?-glutamyl dipeptides that were initially selected for their unusual properties as persistent agonists of signalling, and competitive inhibitors of amino acid transport via Gap1. By using these molecules we have been able to analyse downstream activities involved in the downregulation of signalling via ubiquitination, endocytosis and vacuolar sorting of Gap1. Under this context as well as under the context of mutant backgrounds which constitutively increase or decrease PKA levels, the downregulation of Gap1 by ubiquitination and vacuolar sorting was analysed and major unprecedented conclusions could be drawn from this study as explained in the previous section of this report.