Field of the invention

The present invention relates to the field of nutrient sensing and control of growth and development in eukaryotic cells such as yeast cells. More particularly the invention provides complexes between nutrient transporters and protein synthesis initiation factors. In addition, the invention provides screening assays using these complexes to isolate compounds which can modulate cell growth and development.

Introduction to the invention In the yeast Saccharomyces cerevisiae, the activity of the protein kinase A (PKA) pathway is closely correlated with growth rate. In rapidly-growing fermenting cells, the status of all well- established targets of the PKA pathway that PKA activity must be high, while in slowly-growing, respiring cells and in stationary-phase cells it indicates that PKA activity must be low. Similarly, when yeast cells growing in a complete glucose medium are starved for a single essential nutrient they also downregulate PKA activity during growth arrest. Re-addition of the missing nutrient causes within minutes dramatic activation of the PKA pathway as is evidenced for instance by the rapid, 5-10 fold increase in trehalase activity, a well-established phosphorylation target of PKA (Schepers et al., 2012). Analysis of the mechanism responsible for this nutrient activation of PKA has identified transporter-receptors or transceptors as the responsible nutrient sensors (Holsbeeks et al., 2004). They include Gap1 for amino acids, Pho84 for phosphate, Mep2 for ammonium and Sul1 ,2 for sulfate (Conrad et al., 2014). All these transceptors are high- affinity transporters strongly induced during starvation for their substrate and rapidly endocytosed and degraded upon re-addition of the substrate. Substrate-induced endocytosis has been studied in greatest detail for the Gap1 amino acid transceptor (Ghaddar et al., 2014). It is initiated through ubiquitination by the ubiquitin ligase Rsp5, after which Gap1 is gathered in endosomes and transported to sorting/early endosomes, the multi-vesicular body, the late endosome and finally to the vacuole/lysosome for proteolytic degradation. Gap1 is also recycled to the plasma membrane from endosomes and other compartments, its trafficking is complex and highly regulated (Lauwers et al., 2010). Plasma membrane components can also undergo constitutive endocytosis to a sorting endosome from which they are recycled to the plasma membrane. Although some components required for this process have been identified (Wiederkehr et al., 2000), it remains poorly characterized.

l Although the function of the transceptors in nutrient activation of PKA is now well established (Kankipati et al., 2015; Popova et al., 2010; Rubio-Texeira et al., 2012; Van Zeebroeck et al., 2009; Van Zeebroeck et al., 2014), the molecular mechanism responsible for transceptor signaling to PKA has remained unclear. As opposed to glucose signaling in glucose-deprived cells, transceptor signaling to PKA in glucose-repressed cells does not involve cAMP as a second messenger (Thevelein and de Winde, 1999). In stationary-phase starved cells, bulk protein synthesis is down-regulated, while induction of growth by re-addition of one or more lacking essential nutrients results in rapid initiation of bulk protein synthesis. The latter process is controlled by the G-protein elF2, which in the active GTP-bound form binds initiator methionyl- tRNA to interact with the 40S ribosomal subunit and mRNA in order to start up protein synthesis at the start codon. Upon AUG recognition, elF2-bound GTP is hydrolyzed to GDP rendering the protein inactive. Activation of elF2 by exchange of GDP for GTP is stimulated by its guanine nucleotide exchange factor, elF2B. Their vital role in the upstart of translation makes elF2 and elF2B major points of regulation (Mohammad-Qureshi et al., 2008), although many questions in this respect remain unresolved. For instance, there are no upstream activators of elF2B known and how nutrient activation of elF2B or elF2 is triggered during nutrient-induced stimulation of protein synthesis and cell growth also remains unknown. In the present invention we surprisingly show that starvation-induced nutrient transceptors as well as constitutively-expressed transporters, physically bind to elF2B/elF2 in vitro. In addition, also bimolecular fluorescence complementation of the physical interaction in vivo revealed a strong fluorescent focus in most cells. Co-localization of marker proteins and mutants in vesicular trafficking indicate that the newly discovered membrane system appears to be a new organelle, which we herein designate as the 'startosome', because of its presumed function in nutrient regulation of the start of protein synthesis. The major implication of our findings is that screening systems can be set up to identify compounds which can modulate the growth and differentiation of eukaryotic cells. Accordingly the present invention provides isolated complexes between transceptors and translation initiation factors and several in vitro and in vivo assays for selecting compounds able to modulate the growth of eukaryotic cells.

Figure legends Figure 1 : GST pull-down assay reveals strong physical interaction between the nutrient transceptors Gap1 (amino acids), Pho84 (phosphate), Mep2 (ammonium) or SuM (sulfate), and subunits of the translation initiation factors elF2 and elF2B. The transceptors were isolated from extracts of cells made 24 h after starvation for their substrate (Gap1 and Mep2: nitrogen starvation; Pho84: phosphate starvation and Sul1 : sulfur starvation). All subunits of elF2 and elF2B were expressed in E. coli. The first lane of each panel shows the transceptor input present in the total yeast extract. The second lane shows the absence of interaction between the transceptor and the GST loaded beads. The next lanes show the level of interaction between the transceptor and the subunits of elF2 or elF2B. The gel below shows a Coomassie staining of the elF2 or elF2B subunits loaded on the beads. The expected size of the proteins is indicated with a white square: elF2a (Sui2) ^« 35 kDa, elF23 (Sui3) ^« 31 kDa, elF2y (Gcd1 1 ) ^« 58 kDa, elF2Ba (Gcn3) ^« 34 kDa, elF2B3 (Gcd7) ^« 43 kDa, elF2By (Gcd1 ) ^« 66kDa, elF2B5 (Gcd2) ^« 71 kDa, elF2Be (Gcd6) ^« 81 kDa.

Figure 2: GST pull-down assay reveals physical interaction between the amino acid transporters, Gnp1 or Hip1 , and subunits of the translation initiation factors elF2 and elF2B. The transporters were isolated from extracts of cells during exponential growth phase. All subunits of elF2 and elF2B were expressed in E. coli. The first lane of each panel shows the transporter input present in the total yeast extract. The second lane shows the absence of interaction between the transporter and the GST loaded beads. The next lanes show the level of interaction between the transporter and the subunits of elF2 or elF2B. The gel below shows a Coomassie staining of the elF2 or elF2B subunits loaded on the beads. The expected size of the proteins is indicated with a white square: elF2a (Sui2) ^« 35 kDa, elF23 (Sui3) ^« 31 kDa, elF2y (Gcd1 1 ) ^« 58 kDa, elF2Ba (Gcn3) ^« 34 kDa, elF2B3 (Gcd7) ^« 43 kDa, elF2By (Gcd1 ) ^« 66kDa, elF2B5 (Gcd2) ^« 71 kDa, elF2Be (Gcd6) = 81 kDa.

Figure 3: A bimolecular fluorescence complementation assay with split citrin reveals in vivo interaction between the nutrient transceptors Gap1 , Pho84, Sul1 , or the amino acid transporter Gnp1 , and the elF2By (Gcd1 ) subunit. The transceptors were induced by starvation for their substrate (as indicated in the Figure 1 legend), while the transporter was present in exponentially growing cells (as indicated in the Figure 2 legend). Each transceptor or transporter was tagged with the C-terminal half of the fluorescent protein citrin. The elF2By (Gcd1 ) subunit was tagged with the N-terminal half of citrin. Co-expression of the two citrin fusion constructs in the same cells resulted in the appearance of usually one, sometimes two, distinct fluorescent foci in the majority of cells. In cells expressing only one of the fusion constructs, foci were never observed.

Figure 4: The transceptor/transporter foci and the elF2/elF2B-GFP foci are identical. (A) In nitrogen-starved cells, the Gap1 -elF2By(Gcd1 ) split citrin focus co-localizes with the elF2Ba(Gcn3)-RFP focus, indicating that the two foci are identical. Gap1 was tagged with the C-terminal half of citrin, while the γ subunit of elF2B (Gcd1 ) was tagged with the N-terminal half of citrin. The a subunit of elF2B (Gcn3) was tagged with the full-length fluorescent protein RFP. Co-expression in the same cells shows that the fluorescent focus caused by the split-citrin interaction between Gap1 and Gcd1 , and the fluorescent focus of Gcn3-RFP, localize exactly at the same position. Pictures were taken sequentially and merged using Fiji. The read-out of the red channel was changed to magenta to improve visibility. (B) The elF2Be(Gcd6)-GFP foci are present during exponential growth and at least several hours into nitrogen starvation. Cells expressing the elF2Be(Gcd6)-GFP fusion protein were grown till Ο βοο 1 .5 and transferred to nitrogen starvation medium. Pictures were taken shortly before transfer (exponential growth) and 4 hours after transfer to nitrogen starvation medium. (C) In exponentially-growing cells, the Gnp1 -elF2By (Gcd1 ) split citrin focus co-localizes with the elF2Ba(Gcn3)-RFP focus, indicating that the two foci are identical. Conditions were the same as in A except that Gnp1 was used instead of Gap1 and exponentially-growing cells instead of nitrogen-starved cells.

Figure 5: Co-expression of the Gap1 -elF2BY(Gcd1 ) split-citrin focus and fluorescently tagged marker proteins for organelles in the secretion or endocytosis pathways, or P-bodies, does not show co-localization. Cells co-expressing the Gap1 -elF2BY(Gcd1 ) split-citrin focus and tagged marker proteins for different organelles as indicated above the pictures, were grown till Ο βοο 1 .5 and then transferred to nitrogen starvation medium. Pictures were taken after 4 hours of incubation in nitrogen starvation medium. All pictures were taken sequentially and merged using Fiji. The read-out of the red channel was changed to magenta to improve visibility.

Figure 6: Formation of the Gap1 -Gcd1 (elF2BY) focus is not affected in deletion mutants in END3, which display compromised endocytosis, nor in mutants in the ESCRT protein encoding genes, VPS25 or VPS36, which display aberrant formation of the multi-vesicular body, also resulting in incomplete endocytosis. Figure 7: The appearance of the Gap1 foci during nitrogen starvation correlates with the arrest of growth. Cells co-expressing Gap1 , tagged with the C-terminal half of citrin, and the γ subunit of elF2B (Gcd1 ), tagged with the N-terminal half of citrin, were grown to exponential phase (OD600 1 .7), spun down and either transferred to fresh growth medium (A) or nitrogen starvation medium (B). Samples of both cultures were observed under the microscope at the indicated time points. At the same time points, the Ο βοο of the culture was determined (C). The number of cells containing foci versus the number of total cells was counted using Imaris software. At least 150 cells were counted per frame in 5 different frames (D).

Figure 8: Disaggregation and recovery of elF2Be(Gcd6)-GFP foci after heat shock correlates with arrest and recovery of growth, respectively. Cells co-expressing Gap1 tagged with the C- terminal half of citrin and the γ subunit of elF2B (Gcd1 ) tagged with the N-terminal half of citrin were grown at 30°C till exponential phase (Ο βοο 1 .0) after which part of the culture was further incubated at 30°C (A) and the other part heat-shocked at 45°C for 20 min and then further incubated at 30°C (B). Pictures were taken and the Ο βοο of the culture determined at the indicated time points (C). The number of cells containing foci versus the total number of cells was visually determined (D).

Detailed description of the invention

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 ^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

Nutrients not only serve as a source of energy and building blocks but also exert important regulatory effects on virtually all aspects of cellular life. It is well known that cells are able to sense the absence or presence of any essential nutrient, triggering either arrest or stimulation of protein synthesis, cell growth and multiplication. The underlying mechanism able to sense all essential nutrients has been elusive. In the present invention we show that nutrient starvation- induced, high-affinity transceptors, display strong physical interaction in vitro and in vivo with subunits of elF2B/elF2, the G-protein system controlling initiation of protein synthesis. Surprisingly by making use of bimolecular fluorescence complementation, exemplified herein by using the split-citrin fluorescence microscopy, a novel membrane system was identified. Colocalization studies with marker proteins and studies with mutants in the protein secretion, endocytosis and vesicle trafficking pathways, indicate that the transceptor-elF2B/elF2 vesicle does not appear to coincide with any known organelle. We designated this apparently new organelle, the 'startosome', since its physical connection with elF2B/elF2 through nutrient transceptors indicates that it mediates nutrient control of the start of protein synthesis. In growing cells, constitutively expressed nutrient transporters interact with elF2B/elF2 to form the startosome and its recovery after heat-shock induced dissociation correlates with resumption of the growth rate, indicating startosome regulation of cell growth and multiplication.

Accordingly the present invention provides in a first embodiment an isolated complex composed of a nutrient transporter and a subunit of elF2 or a subunit of elF2B. In yet another embodiment the present invention provides an isolated complex composed of a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B.

It is understood that a subunit of elF2 can be an alfa, beta or gamma subunit and a subunit of elF2B can be an alfa, beta, gamma, delta or epsilon subunit.

A preferred subunit of elF2 is an alfa subunit or an epsilon subunit. A preferred subunit of elF2B is the beta subunit.

In a particular embodiment the present invention provides an isolated complex composed of a nutrient transporter and an alfa or epsilon subunit of elF2 and/or the beta subunit of elF2B.

The wording 'an isolated complex composed of a nutrient transporter and a subunit of elF2 or a subunit of elF2B' means that a nutrient transporter is bound to a particular subunit of elF2 or to a particular subunit of elF2B. 'Binding' refers to a non-covalent protein-protein interaction.

It is understood that 'binding' occurs in vitro or in vivo.

In yet another embodiment the invention provides an in vivo complex composed of a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B.

In yet another embodiment the invention provides an in vivo complex composed of a nutrient transporter and a subunit of elF2 and a subunit of elF2B. In yet another embodiment the invention provides an in vivo complex composed of a nutrient transporter and a subunit of elF2 or a subunit of elF2B.

In the present invention an in vivo complex refers to a complex which is generated in an eukaryotic cell. A preferred eukaryotic cell is a yeast or plant cell. In vivo complexes of the invention can be identified by generating a tagged nutrient transporter and a tagged subunit of elF2 and/or a tagged subunit of elF2B. Preferred tags are fluorescent tags. In a particular embodiment a fluorescent tag is a split fluorescent tag such as for example split citrin. Bimolecular fluorescence complementation is one example of visualization of the in vivo complexes of the invention. In yet another embodiment the invention provides the use of an isolated complex of the invention to screen for compounds which inhibit elF2.

The wording "to screen for compounds which inhibit elF2" is equivalent with the wording "to screen for compounds which inhibit translation" or 'to screen for compounds which inhibit cell growth". The word "inhibit" is equivalent with the words "prevent" and "reduce". In yet another embodiment the invention provides the use of an isolated complex of the invention to screen for compounds which activate elF2.

The wording "to screen for compounds which activate elF2" is equivalent with the wording "to screen for compounds which activate translation" or 'to screen for compounds which activate cell growth". The word "activate" is equivalent with the words "stimulate" and "enhance". The term "compound" is used herein in the context of a "test compound" or a "drug candidate compound" described in connection with the methods of the present invention. As such, these compounds comprise organic or inorganic compounds, derived synthetically or from natural resources. The compounds include polynucleotides, lipids or hormone analogs that are characterized by low molecular weights. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.

Bimolecular fluorescence complementation as used herein is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest. Other in vivo assays most commonly used to study protein-protein interactions include fluorescence resonance energy transfer (FRET) and yeast two-hybrid (Y2H) assay.

In yet another embodiment the invention provides a method for producing a compound that modulates the activity of elF2 comprising the following steps: i. providing a system comprising a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B,

ii. administering at least one compound to said system,

iii. monitoring the interaction between said nutrient transporter and a subunit of elF2 and/or a subunit of elF2B, wherein compared under the same test conditions in the same system without the administration of a compound, a reduced interaction between the nutrient transporter and a subunit of elF2 and/or a subunit of elF2B produces a compound which inhibits the activity of elF2 and wherein an increased interaction between the nutrient transporter and a subunit of elF2 and/or a subunit of elF2B produces a compound which activates the activity of elF2.

In yet another embodiment the invention provides a method for producing a compound that modulates the activity of elF2 comprising the following steps: i. providing a system comprising a nutrient transporter and a subunit of elF2 or a subunit of elF2B,

ii. administering at least one compound to said system,

iii. monitoring the interaction between said nutrient transporter and a subunit of elF2 and/or a subunit of elF2B, wherein compared under the same test conditions in the same system without the administration of a compound, a reduced interaction between the nutrient transporter and a subunit of elF2 or a subunit of elF2B produces a compound which inhibits the activity of elF2 and wherein an increased interaction between the nutrient transporter and a subunit of elF2 or a subunit of elF2B produces a compound which activates the activity of elF2. In yet another embodiment the invention provides a method for producing a compound that modulates the formation of the startosome in a cell comprising the following steps: i. providing a system comprising a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B,

ii. administering at least one compound to said system,

iii. monitoring the interaction between said nutrient transporter and a subunit of elF2 and/or a subunit of elF2B, wherein compared under the same test conditions in the same system without the administration of a compound, a reduced interaction between the nutrient transporter and a subunit of elF2 and/or a subunit of elF2B produces a compound which inhibits the formation of the startosome in the cell and wherein an increased interaction between the nutrient transporter and a subunit of elF2 and/or a subunit of elF2B produces a compound which activates the formation of the startosome in the cell.

In yet another embodiment the invention provides a method for producing a compound that modulates the formation of the startosome in a cell comprising the following steps: i. providing a system comprising a nutrient transporter and a subunit of elF2 or a subunit of elF2B,

ii. administering at least one compound to said system,

iii. monitoring the interaction between said nutrient transporter and a subunit of elF2 or a subunit of elF2B, wherein compared under the same test conditions in the same system without the administration of a compound, a reduced interaction between the nutrient transporter and a subunit of elF2 or a subunit of elF2B produces a compound which inhibits the formation of the startosome in the cell and wherein an increased interaction between the nutrient transporter and a subunit of elF2 or a subunit of elF2B produces a compound which activates the formation of the startosome in the cell. As used herein a method refers to a screening method or a screening assay. A compound that modulates the activity of elF2 is equivalent to a compound that modulates the activity of elF2B or a compound that modulates the activity of elF2 interacting with elF2B. Modulation of the activity of elF2 (or elF2B or elF2 and elF2B) refers to the modulation of translation (or cell growth). Modulation can refer to either an inhibition or to a stimulation. In a specific embodiment the system is a cell free system. An example of a cell free system is wherein the nutrient transporter and a subunit of elF2 and/or a subunit of elF2B are mixed together (e.g. made in a recombinant way). In a preferred embodiment a cell free system is a an artificial membrane system (such as a liposome or micelle) and the individual components are incorporated into an artificial membrane system (such as a liposome or micelle). In another specific embodiment the system is an in vivo system such as a eukaryotic cell. A preferred eukaryotic cell is a plant or a yeast cell.

The elements of the system (nutrient transporter and a subunit of elF2 and/or a subunit of elF2B) can but do not necessary comprise a tag. When the elements of the system comprise a tag it can be a fluorescent tag. In a specific embodiment the fluorescent tag is a split fluorescent tag.

In the screening methods of the invention the nutrient transporter can a sugar transporter, a nitrogen transporter, a sulfate transporter, a phosphate transporter, a vitamin transporter or a metal ion transporter.

In particular embodiments the screening systems and methods comprise high content screening (HCS) of suitable compounds. In some instances, HCS is a screening method that uses live cells to perform a series of experiments as the basis for high throughput compound discovery. Typically, HCS is an automated system to enhance the throughput of the screening process. However, the present invention is not limited to the speed or automation of the screening process. In one embodiment, the HCS assay of the invention provides for a system to generate high quality "hits" identifying compounds that modulate cell growth.

In another embodiment of the invention, the HCS assay provides for a high throughput assay. Preferably, the assay provides automated screening of thousands of test compounds. Compounds tested in the screening method of the present invention are not limited to the specific type of the compound. In one embodiment, entire compound libraries are screened. Compound libraries are a large collection of stored compounds utilized for high throughput screening. Compounds in a compound library can have no relation to one another, or alternatively have a common characteristic. For example, a hypothetical compound library may contain all known compounds known to bind to a specific binding region. As would be understood by one skilled in the art, the methods of the invention are not limited to the types of compound libraries screened. Non-limiting examples of compound libraries include the sets from LOPAC, Chembridge, Maybridge, LifeChemicals and the NIH Clinical Collection.

The test compound may be added to the assay to be tested by any suitable means. For example, the test compound may be injected into the cells of the assay, or it can be added to the nutrient medium and allowed to diffuse into the cells. In situations where "high-throughput" modalities are preferred, it is typical to that new chemical entities with useful properties are generated by identifying a chemical compound (called a "hit compound") with some desirable property or activity, and evaluating the property of those compounds. A non-limiting example of a high- throughput screening assay is to array the membrane of the invention to 96, 385, 1536, etc. well or slot format to enable a full high throughput screen. In one embodiment, high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "hit compounds" or can themselves be used as potential or actual therapeutics. As further discussed below, in one embodiment, the screen and method of the present invention comprise a primary screen, one or more counter screens, and one or more secondary screens. In one embodiment, one or more of the primary screen, counter screens, and secondary screens is a high throughput screen or high content screen, as described elsewhere herein.

The screening systems and methods of the invention are based upon the detection of the formation of a complex between a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B in an in vitro system or in a living cell. In one embodiment, the system and methods of the invention comprise a primary screen. In one embodiment, the primary screen comprises the acquisition of images of cells to the complex formation. Localization of the complexes is made through the detection of a signal corresponding to a specific nutrient transporter and one or more subunits of elF2 and/or elF2B. In a particular embodiment, the screening methods of the invention can comprise the use of cells that do not natively express a specific nutrient transporter and one or more subunits of elF2 and/or elF2B. In specific embodiments, cells of the screen express a specific nutrient transporter and one or more subunits of elF2 and/or elF2B wherein the specific nutrient transporter and one or more subunits of elF2 and/or elF2B are tagged with a detectable marker, for example a fluorescent tagged nutrient transporter and/or a fluorescent tagged elF2 and/or a fluorescent tagged elF2B. Non-limiting examples of fluorescent tags include green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), eGFP, mCherry, hrGFP, hrGFPII, Alexa 488, Alexa 594, and the like. Fluorescent tags may also be photoconvertable such as for example kindling red fluorescent protein (KFP-red), PS-CFP2, Dendra2, CoralHue Kaede and CoralHue Kikume or photoactivable such as photoactivatable GFP and photoactivatable Cherry and the like. However, the invention should not be limited to a particular label. Rather, any detectable label can be used to tag a specific nutrient transporter and one or more subunits of elF2 and/or elF2B.

In one embodiment, the screen comprises a cell or cell population modified to express a specific nutrient transporter and one or more subunits of elF2 and/or elF2B and/or other proteins of interest. In one embodiment, the cell or cell population is modified by administering an expression vector encoding the proteins of interest. As would be understood by those skilled in the art, the expression vector used to modify the cell or cell population of the screen includes any vector known in the art such as cosmids, plasmids, phagemid, lentiviral vectors, adenoviral vectors, retroviral vectors, adeno-associated vectors, and the like. In one embodiment, the cells of the screen are modified to transiently express a specific nutrient transporter and one or more subunits of elF2 and/or elF2B. In another embodiment, the cells of the screen are modified for the stable expression of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B.

The present invention is related to screening methods comprising the automated detection of the cellular localization of proteins. In one embodiment, the localization of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B, is determined from images taken of cells expressing a specific nutrient transporter and one or more subunits of elF2 and/or elF2B. The localization of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B, may be determined in the live cell of the assay, or alternatively after the cell has been fixed. The present invention is not limited to the type or mode of microscopy utilized in imaging of the cells of the screen. In one embodiment, acquired images obtained through standard fluorescent microscopy techniques known in the art, detects the localization of the fluorescent signal in a cell, thereby detecting the localization of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B within a cell. The present invention shows that under starvation conditions for certain nutrient transporters the startosome is formed more efficiently and hence it is necessary to conduct the screening, when an in vivo screening is applied, in the appropriate medium conditions. For example for the yeast nutrient transporters Gap1 and Mep2 a nitrogen starvation medium is preferably used during the in vivo screening. For the Sul1 nutrient transporter a sulfate starvation medium is preferably used during the in vivo screening. For the Pho84 nutrient transporter a phosphate starvation medium is preferably used during the in vivo screening.

In one embodiment, localization of a complex composed of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B is quantitatively determined by the automated calculation of the proportion of a complex composed of a specific nutrient transporter and one or more subunits of elF2 and/or elF2B at the membrane. In one embodiment, hits are defined as those test compounds that inhibit (or prevent) startosome formation by greater than 20%, 30%, 40%, 50%, 60%, 70%, 90% or even higher with respect to a system where no compound was applied. In another embodiment, hits are defined as those test compounds that stimulate (or enhance) startosome formation by greater than 20%, 30%, 40%, 50%, 60%, 70%, 90% or even higher with respect to a system where no compound was applied.

HCS assays typically comprise automated screening techniques to generate a high level of information from an experiment. In one embodiment, the system of the invention comprises numerous test compounds screened on cells cultured on a multi-well plate. Non-limiting examples of multi-well plates include a 6-well plate, a 24-well plate, a 96-well plate, and a 384- well plate. As such, each well comprises its own individual experiment detecting the response to a single test compound. Statistical analysis performed on the control wells enable the determination of the overall quality of experimentation done on the entire plate. In plates with controls determined to pass a statistical standard, test compounds that reduce (or enhance) the startosome formation by a pre-defined amount relative to the mean of all compounds tested on the plate, that are not acutely cytotoxic and/or fluorescent outliers are flagged as "hits" as inhibitors (or enhancers) of startosome formation. As such, the primary screen of the invention narrows a first population of test compounds into a second, smaller, population of test compounds that retain the ability to inhibit (or enhance) startosome formation.

Examples of assay methods for identifying compounds in the context of the present invention are described in the Example section, without the purpose of being limitative. It should be clear to the skilled artisan that the present screening methods might be based on a combination or a series of measurements, particularly when establishing the link with amyloid beta peptide generation. Also, it should be clear that there is no specific order in performing these measurements while practicing the present invention.

Assays can be performed in eukaryotic cells, advantageously in yeast cells or in plant cells. Appropriate assays can also be performed in reconstituted membranes, and using purified proteins in vitro. The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Examples

1 . Subunits of elF2 and elF2B physically interact with nutrient transceptors and transporters in a GST pull-down assay

Detailed analysis using in vitro GST pull-down co-IP assays with four different nutrient transceptors and all elF2 and elF2B subunits revealed multiple and consistent physical interactions. We have used GST-tagged versions of all elF2 and elF2B subunits and HA-tagged versions of four transceptors. The GST-tagged proteins were expressed in E. coli, while the HA- tagged transceptors were expressed in yeast. The protein samples of the transceptors were taken from cell extracts made after starvation of the cells for the substrate of the transceptor: 24h of nitrogen starvation for Gap1 (amino acids) and Mep2 (ammonium), 48h of phosphate starvation for Pho84 (phosphate) and 48h of sulfur starvation for Sul1 (sulfate). All transceptors are strongly induced under the respective starvation condition. The pull-down assays revealed that all four transceptors displayed strong physical interaction in vitro with selected subunits of elF2 and elF2B. The strongest interaction was generally observed with the a and ε subunits of elF2B and the β subunit of elF2 (see Figure 1 ). Weaker interactions with other subunits were also observed but they may (also) occur indirectly through binding with other elF2 or elF2B subunits. The absence of any interaction with the GST tag alone supports the genuine nature of the interactions observed between the transceptors and the elF2 and elF2B subunits. Coomassie staining of the extracts revealed high levels of protein isolated with the beads for all subunits. In a next step it was tested whether these interactions were also observed with regular transporters expressed during the growth phase and not supposed to have a receptor function. We selected the SPS-controlled amino acid transporters, Gnp1 and Hip1 (Ljungdahl, 2009), which display of all amino acid transporters the highest sequence similarity to Gap1 . They are both highly expressed during exponential growth in nitrogen replete medium and not or poorly- expressed in nitrogen-starvation medium. For both transporters, we also detected physical interaction with elF2 and elF2B subunits, although it seemed to be weaker than with Gap1 , and notably the interaction with the a subunit of elF2B was conspicuously weaker than with Gap1 and the other transceptors (see Figure 2). We subsequently tested two nutrient transporter like proteins, Ssy1 and Snf3, which function as plasma membrane amino acid and glucose sensor, respectively. Since both proteins lack any detectable transport function, we did not expect similar physical interaction with elF2 and elF2B as with genuine nutrient transporters. Indeed, in vitro co-IP experiments with tagged versions of Ssy1 or Snf3 on the one hand and elF2 or elF2B subunits on the other hand, failed to reveal any evidence of physical interaction (results not shown).

2. The subunits of elF2 and elF2B also physically interact in vivo with the transceptors and form distinct and usually single foci within the cells

To examine physical interaction between the transceptors and the elF2/elF2B subunits in vivo, we have used bimolecular fluorescence complementation with split citrin. For that purpose, split citrin fusion proteins were created by tagging elF2/elF2B subunits intragenomically with one half of the citrin protein and the transceptor proteins with the other half. The strains co-expressing one tagged elF2/elF2B subunit and one tagged transceptor were then examined under the confocal microscope both in exponential phase and after starvation for the transceptor substrate to induce maximal transceptor expression. The Gap1 -elF2By(Gcd1 ) split-citrin tagged strain displayed a strong fluorescence focus in nitrogen-starved but not in exponential phase cells (see Figure 3). Gcd1 (elF2Bv) forms together with Gcd6 (elF2Be) the catalytic part of elF2B (Gordiyenko et al., 2014). The citrin fluorescence signal was limited to a small focus in the cell, which was usually visible as one focus per cell in the majority of cells. After 24h of nitrogen starvation, the Gap1 -Gcd1 focus was visible in about 70-80% of the cells. Foci were visible in mother cells and/or in buds, with occasionally two foci in one cell. Very similarfoci were observed with all other subunits of elF2 or elF2B tested in combination with Gap1 : Sui2 (elF2a), Sui3 (elF23) and Gcn3 (elF2Ba) (results not shown). In cells expressing only one of the split-citrin fusion proteins, foci were never detected. We next tested whether a similar fluorescence focus could be observed with the phosphate transceptor Pho84, the sulfate transceptor Sul1 and the ammonium transceptor Mep2 using split-citrin fusion proteins. In the case of Pho84 and elF2By (Gcd1 ), similar foci as with Gap1 were observed after 24 h of phosphate starvation (see Figure 3). In contrast to the Gap1 -foci, however, more cells displayed two foci. The Pho84-foci also appeared to be slightly larger than those formed with Gap1. This could be due to higher expression of Pho84 or to a specific factor induced during phosphate but not during nitrogen starvation. Also with Sul1 and elF2BY(Gcd1 ), similar foci were detected after 24 h of sulfur starvation (see Figure 3). The foci were less distinct than those observed upon nitrogen or phosphate starvation. This may be due to lower expression of Sul1 during sulfur starvation compared to Gap1 and Pho84 under nitrogen and phosphate starvation, respectively. The DNA sequences encoding the citrin tags were inserted directly in the genome adjacent to the ORFs of the elF2 and elF2B subunits, which are nearly all essential proteins. Since the strains did not display any growth defect, the citrin-tagged elF2 and elF2B subunits appeared to have normal functionality. Next, we tested whether the regular amino acid transporter, Gnp1 , also interacts with elF2By(Gcd1 ) in vivo using the same bimolecular fluorescence approach with citrin fusion constructs. The results clearly revealed in cells growing exponentially in nitrogen-replete medium a similarfluorescence focus as observed in nutrient-starved cells for the transceptors (see Figure 3). There was also a single focus present in most cells. In the control cells, expressing only one of the citrin fusion constructs, no foci at all were observed. These results indicate that the formation of the foci is not restricted to nutrient-starvation induced transceptors, but also occurs with regular transporters, suggesting that some of the latter may also have an additional regulatory function for control of elF2/elF2B during exponential growth.

3. The Gap1 -elF2/elF2B foci are identical to the previously observed elF2/elF2B-GFP foci

The group of Ashe previously reported the presence of a similar cytosolic fluorescence focus upon expression of GFP-fusion constructs of all elF2 and elF2B subunits in yeast, whereas expression of GFP-fusion proteins of other translation initiation factors revealed the expected uniform distribution in the whole cytosol (Campbell et al., 2005). To test whether the Gap1 - elF2/elF2B citrin foci and the elF2/elF2B-GFP foci might be related to each other, we co- expressed RFP-tagged Gcn3 (elF2Ba) in the same cells as those expressing the Gap1 - elF2By(Gcd1 ) citrin focus (see Figure 4A). All strains were grown to Ο βοο 1 .5 after which the cells were transferred to nitrogen starvation medium. Samples were taken after two hours and examined under the confocal microscope. We noticed a perfect match in the overlay of the red Gcn3-RFP fluorescence foci and the Gap1 -elF2BY(Gcd1 ) green fluorescence foci, resulting exclusively in yellow fluorescent foci. Hence, the Gap1 -elF2/elF2B foci appear to reveal the same cellular structure as the elF2/elF2B-GFP foci. From these observations, we can conclude that the elF2/elF2B-GFP foci observed by Campbell et al. (2005) do not reveal 'protein bodies' but rather membrane structures or vesicles, since the Gap1 transceptor is an integral membrane protein. The strong and consistent association of the nutrient transceptors with elF2/elF2B in these vesicles is suggestive of a new system with a specific functionality, indicating the possible existence of a novel cell organelle. We further tested whether the elF2B-GFP foci were also visible during nutrient starvation. Both the elF2Be(Gcd6)-GFP foci (see Figure 4B) and elF2By(Gcd1 )-GFP foci (results not shown) could be observed very clearly up to several hours after the onset of nitrogen starvation. Upon long-term starvation the fluorescence of these GFP foci became weaker. This could be due to decreased levels of the elF2B subunits in long-term nitrogen-starved cells. Co-expression of RFP-tagged Gcn3 (elF2Ba) in the same cells as those expressing the Gnp1 -elF2By(Gcd1 ) citrin focus revealed a perfect match between the two foci (see Figure 4C). This indicates that also in exponentially-growing cells the previously observed elF2/elF2B-GFP focus co-localizes with the transporter-el F2/elF2B focus. Hence, both in growing and in starved cells the elF2/elF2B 'protein body' represents a membrane system or vesicle. 4. The foci do not co-localize with any known organelle of the secretion or endocytosis pathways

Next, we examined whether the Gap1 -foci might be identical to one of the cell organelles of the protein secretion pathway or the ligand-induced endocytosis pathway. For that purpose, we performed co-localization studies of the Gap1 - Gcd1 (elF2By) foci with specifically stained organelles or organelles tagged with specific marker proteins. All co-localization pictures were taken sequentially to avoid spectral bleed through. Staining of the nucleus with DAPI revealed no connection with localization of the Gap1 -Gcd1 foci (see Figure 5). Expression of the RFP- tagged ER marker Sec13 (Bruns et al., 201 1 ; Enninga et al., 2003), Golgi marker Sec7 (Bruns et al., 201 1 ; Losev et al., 2006; Richardson et al., 2012) and Golgi transport vesicle marker Cop1 (Gerich et al., 1995) in the Gap1 -Gcd1 foci expressing strain further showed no co-localization with either one of these organelles (see Figure 5). Hence, the Gap1 -foci do not seem to be related to any organelle in the protein secretion pathway. Next, we investigated a possible connection with the ligand-induced endocytosis pathway. Similarly, no co-localization of the Gap1 -Gcd1 foci was found with the RFP-tagged multi-vesicular-body marker Snf7 (Boysen and Mitchell, 2006; Bruns et al., 201 1 ), with the marker for endosomal membranes, FYVE-dsRED (Bruns et al., 201 1 ; Katzmann et al., 2003), the RFP-tagged endocytic vesicle marker Chc1 (Gruenberg and Stenmark, 2004) or the RFP-tagged vacuolar marker Vph1 (Manolson et al., 1992) (see Figure 5). Hence, the Gap1 -foci do not appear to be related to any organelle in the ligand-induced endocytosis pathway. Because the transceptor foci appear upon nutrient starvation and are apparently related to the control of translation, we have examined whether they might be related to P-bodies, which control mRNA fate and can also be induced under nutrient starvation or other stress conditions. However, expression of the P-body marker Edc3 failed to show any co-localization with the Gap1 -Gcd1 foci (see Figure 5). This fits with previous reports that elF2 and elF2B are not present in yeast P-bodies (Buchan et al., 2008).

5. Mutations in the ligand-induced endocytosis pathway do not affect formation of the Gap1 - elF2B foci

Because Gap1 and the other transceptors are well known to undergo substrate-induced endocytosis, we have investigated whether mutations that disrupt the normal functioning of the endocytic pathway, affect the formation of Gap1 -elF2B foci. Deletion of the END3 gene is known to prevent full endocytosis of Gap1 and stimulate its recycling to the plasma membrane (Lauwers et al., 2007). However, deletion of END3 did not affect formation of the Gap1 -Gcd1 (elF2By) foci (see Figure 6). We next tested whether deletion of the genes encoding two ESCRT proteins, Vps25 and Vps36, would affect formation of the Gap1 -elF2B foci. Both proteins are part of the ESCRTII complex and are involved in formation of the multi-vesicular body. In their absence, the multi-vesicular body fails to form correctly and endocytosed proteins get stuck along the endocytic pathway (Babst et al., 2002). The absence of Vps25 or Vps36, however, did not influence the formation of Gap1 -Gcd1 (elF2By) foci significantly (see Figure 6). These results appear to contradict that formation of the Gap1 -elF2B foci is connected to the functioning of the endocytic pathway, and in particular that it is not connected in some way to substrate-induced endocytosis of Gap1 itself. 6. The appearance of the Gap1 -foci correlates with the arrest of growth during nitrogen starvation

Next we have followed the appearance of the Gap1 -foci after transfer of the cells to nitrogen starvation medium. For this purpose, cells co-expressing split-citrin fusion constructs of Gap1 and elF2By(Gcd1 ) were grown to exponential phase (Ο βοο Ι .5) and harvested by centrifugation, after which half of the culture was re-suspended in nitrogen starvation medium and the other half in fresh growth medium. Subsequently, samples were taken every h to follow growth arrest by measuring Ο βοο and to follow the appearance of the Gap1 -foci using confocal fluorescence microscopy. After one h, the first foci could be detected while after two h the foci were present in about 66% of the cells (see Figure 7). The number of cells with foci did not increase much further over the next h, averaging about 70%. The increase in Ο βοο also ceased after about 2 h, hence coinciding with the appearance of the Gap1 -foci (see Figure 7). In the cells re- suspended in fresh growth medium, Ο βοο continued to increase for another four h and no clear Gap1 -foci appeared as observed in the nitrogen starvation medium.

7. Growth recovery after heat shock correlates with the recovery of elF2B-GFP foci The strong binding of the two citrin halves is known to stabilize the interaction between the binding partner proteins after they have brought the citrin halves together (Kodama and Hu, 2012). Moreover, during exponential growth the expression of Gap1 is virtually absent and that of the other transceptors much lower than under conditions of starvation for their substrate. Therefore, we decided to use the elF2B-GFP foci to test for a possible correlation between growth and formation of the foci. During exponential growth, the elF2B-GFP foci might be formed by multiple interactions of elF2B with nutrient transporters expressed under these conditions. We first tested whether the Gcd6(elF2Be)-GFP foci disassemble during heat stress and if so, whether the reappearance of the foci would correlate with the recovery of cell growth. After growing the strain expressing Gcd6-GFP into exponential phase, the culture was split into two. A 20 min heat shock at 45°C was applied to one part, which was then returned to 30°C forfurther incubation. The other part was not heat shocked and immediately returned to 30°C. Samples were taken every 30 min and used for determination of Ο βοο and for observation of the Gcd6- GFP foci with the confocal microscope. The heat shock treatment caused complete dispersal of the Gcd6-GFP foci while there was no effect in the non-heat shocked control culture (see Figure 8). After re-incubation of the heat-shocked cells at 30°C for one h the Gcd6-GFP foci started to reappear, and after two h the number of Gcd6-GFP foci was the same as in the control culture. The heat shock at 45°C also caused a strong, temporary reduction in the growth rate. The recovery of the growth rate coincided with the re-appearance of the Gcd6-GFP foci. Although still preliminary, this correlation suggests that the transporter-elF2B foci might play a role in the control of cell proliferation.

8. Conclusions

The present invention shows that the transceptors show strong physical interaction with elF2 and elF2B, suggesting not only that this interaction may be important for the signaling mechanism, but at the same time revealing a new and straightforward mechanism for nutrient regulation of the initiation of protein synthesis. Second, the transceptors display this physical interaction also in vivo and its manifestation reveals a putative new organelle (herein designated the 'startosome', with a specific composition and function. Third, elF2 and elF2B do not only physically interact with established transceptors but also with transporters expressed during exponential growth and previously not considered to have a nutrient receptorfunction. Moreover, manifestation of this interaction in vivo reveals the same cytosolic focus as evidenced with the transceptors in nutrient-starved cells. Fourth, the foci revealed with bimolecular fluorescence complementation of the transceptors/transporters and elF2/elF2B are identical to the previously observed elF2/elF2B-GFP foci (Campbell et al., 2005), both in exponentially-growing and nutrient-starved cells. This indicates that the previously adjudged protein bodies are actually membranous systems representing a candidate new organelle with a specific composition and function. In the present invention we further characterized the new membranous system revealed by the transceptor-elF2/elF2B interaction. There are three major arguments for a possible role of this entity in nutrient regulation of protein synthesis. First, transporter-el F2/elF2B interaction in exponentially growing cells reveals the same system, suggesting its universal presence. Second, the physical interaction between nutrient transporters and elF2/elF2B itself is strongly suggestive for a role in nutrient regulation of protein synthesis. Third, the transceptor/transporter-elF2/elF2B focus is identical to the elF2/elF2B-GFP focus for which the Ashe group demonstrated a correlation between the cycling of elF2 in and out of the focus, and the rate of protein synthesis. Taken together, the data strongly suggest that the fluorescent focus represents a new cell organelle (herein called the 'startosome') used to assess the nutrient status of the environment in order to regulate the initiation of protein synthesis as a function of nutrient availability. Without being bound by a particular mechanism or action we believe that the startosome is formed by constitutive endocytosis of endosomes from the plasma membrane, which do not only contain a representative sample of plasma membrane nutrient transporters in their membrane but also a representative sample of the nutrient present in the medium in their lumen. In the startosome (and possibly also previously already in the endosomes or even at the level of the plasma membrane) the transporters bind to elF2/elF2B to stimulate their activity as a function of nutrient availability. We suggest that transporters for all nutrients exert this function simultaneously, which implies that many or even most regular nutrient transporters also have an additional nutrient receptor function for regulation of elF2/elF2B activity. When cells are starved for an essential nutrient, a high-affinity transporter for this nutrient is strongly induced by a nutrient-specific sensing mechanism as part of a specific transcriptional adaptation program. This high-affinity transporter starts to predominate the transporter population in the plasma membrane and thus also in the startosome. It may also have an altered affinity for elF2/elF2B, which would help it to sequestrate elF2/elF2B from interaction with the other nutrient transporters. Since it is the only nutrient transporter of which the substrate is lacking, stimulation of elF2/elF2B will be reduced and bulk protein synthesis and cell growth will strongly diminish. We have shown that the startosome disaggregates upon heat-shock induced growth arrest in cells growing exponentially in nutrient-replete medium and that its reformation coincides with the resumption of growth. Similarly, formation of the Gap1 -containing startosome upon nitrogen starvation coincides with the arrest of cell proliferation. These observations are consistent with our hypothesis that active nutrient transporters stimulate protein synthesis, while inactive nutrient transporters downregulate protein synthesis through the startosome.

In addition, we have performed multiple co-localization experiments to investigate whether the startosome could be identical to one of the vesicular components of the secretion and endocytosis pathways. Since all plasma membrane transporters follow the protein secretion pathway for their synthesis and sorting to the plasma membrane and since all previously established transceptors undergo substrate-induced internalization through the endocytic pathway, these proteins at one moment or another reside in all compartments of these pathways. However, we could not detect any co-localization with established markers for vesicular components of these pathways. Also, interference with the ligand-induced endocytic pathway using specific mutations did not interfere with the formation of the startosome. We considered that the transporter/transceptor-containing vesicles might reach the startosome through the constitutive endocytosis pathway. However, deletion of RCY1, which disturbs formation of the early/sorting endosome in the constitutive endocytosis pathway (Wiederkehr et al., 2000), did not disturb formation of the startosome (unpublished results). These results seem to indicate that the startosome is different from any known cellular organelle. This conclusion is further strengthened by our observation that the startosome is identical to the elF2/elF2B-GFP 'protein body', for which no convergence with any cellular organelle has been reported, probably because of the unique nature of the elF2/elF2B aggregate (Campbell et al., 2005). The convergence with the elF2/elF2B-GFP 'protein body' and the physical interaction between the transporters/transceptors and elF2/elF2B suggest a specific function of the startosome in nutrient regulation of protein synthesis. Campbell et al. (2005) showed that the a subunit of elF2 cycles through the foci during exponential growth and that the cycling arrests during amino acid starvation (Campbell et al, 2005). They suggested that the cycling correlates with the activity of protein synthesis and cell growth. Combined with our previous work on transceptor signaling to the PKA pathway, we suggest that the startosome may also play a role in integrating nutrient regulation of protein synthesis with nutrient regulation of protein kinase pathways responsible for control of metabolism and other cellular properties. Hence, we feel that the currently available body of evidence strongly suggests that the startosome is likely a novel cellular organelle with a specific composition and function. 9. Small compounds can modulate (decrease as well as increase) the number of startosomes in yeast cells

A high throughput compound screen was developed using a recombinant yeast strain expressing a split version of citrin attached to Gap1 and to Gcd1 (id est the gamma subunit of the translation initiation factor elF2B). The yeast cells were pre-grown in rich medium and harvested at an OD of 1 .5 and added in highly concentrated form to a 96-well plate containing nitrogen starvation medium and different compounds dissolved in DMSO as well as separate controls for each compound in which only DMSO is added. The compounds were derived from a pharmacological diversity set (PDS) library. This library contains 10240 compounds which are similar to known bioactive compounds, meaning that they have similar size, folding or domains as known bioactive compound. The purpose of this library was to discover new bioactive compounds acting on the formation of startosomes. The plates were inserted into a plate reader which takes two images of each well with about 1 sec interval. The purpose of two pictures is to have two independently counted pictures, therefore reducing error introduced by e.g. uneven distribution of cells as well as increasing the number of cells counted. To be able to process the large number of pictures, a program was set up to automatically count the cells as well as the startosomes. The data obtained from the screen show that compounds can be identified which can either enhance and others which can repress the formation of the startosomes in yeast cells.

Materials and methods

1 . Yeast strains and growth conditions

The strains used in this study together with their genotype are listed in Table 1 . Strains expressing C-terminal tags of GFP, citrin and 3HA were created by homologous recombination using a geneticin marker (Longtine et al., 1998) with exception of the strain JT a.405, which was transformed with plasmid JTPL 40412 containing Gap1 -3HA. Strains were either grown on synthetic minimal medium (0.67% yeast nitrogen base, 2% glucose, amino acids and vitamins as needed) or on YPD (1 % yeast extract, 2% peptone and 2% glucose) medium. Strains were grown at 30°C unless otherwise stated. For nutrient starvation, the cells were pre-grown on either YPD or synthetic minimal medium and then transferred to the respective starvation media. For nitrogen starvation, cells were incubated for 2 or 24 h in nitrogen starvation medium (0.17% w/v yeast nitrogen base w/o amino acids, 4% glucose). For phosphate starvation, cells were incubated for 3 days in 0.57% yeast nitrogen base w/o phosphate, supplemented with the appropriate amino acids and vitamins. The starvation medium was refreshed daily. The same culture conditions were used for sulfate starvation with the following medium (37mM NH4CI, 6.5mM KH ₂ P0 ₄ , 0.7 K ₂ HP0 ₄ , 5mM MgCI ₂ , 1.7mM NaCI, 0.9mM CaCI ₂ , 160μΜ Η ₃ Β0 ₄ , Ο.θμπι Kl, 0.7μΜ ZnC , 0.7 μΜ CuCI ₂ , 0.3μΜ FeCI ₃ , 0.4 mg/L calcium panthotenate, 0.4 mg/L thiamine HCI, 0.4 mg/L pyroxidine HCI, 2mg/L biotin and the appropriate nucleotides and amino acids to complement auxotrophic markers).

2. Plasmids Plasmids yN-URA and yC-HIS5 were provided by Geovani Lopez Ortiz (Mexico City) and used for creating the split citrin cassettes introduced by homologous recombination. pFA6-3HA- KANMX6 was a gift of Mark Longtine (Longtine et al., 1998). pFA6A-mRFP1 -KANMX6 was a gift of Erin O'Shea (Cambridge, USA) and pFA6HA-GFP(S65T)-KANMX6 was a gift of Jurg Bahler and John Pringle (Bahler et al., 1998)). To generate the pGEX-4T-1 plasmids containing the subunits of elF2 and elF2B, the ORF of each gene without the stop codon was amplified by PCR. The ORF was then cloned into the pGEX-4T-1 plasmid using a BamHI and Xhol fragment for GCD11, GCD6, SUI2 and SUI3 and an Xmal and Xhol fragment for GCD1, GCD2, GCD7 and GCN3.

3. Expression and purification of proteins from E. coli E. coli BI21 ^* cells were transformed with the pGEX-4T1 plasmids according to established transformation protocols (Gietz and SchiestI, 2007). One of the resulting colonies was used for a 3 ml overnight pre-culture. The whole pre-culture was used to inoculate 500 ml of LB- ampicilline. The cells were grown till an Οϋβοο θί 0.8 at 37°C, protein expression was induced by adding IPTG and the cells were incubated further for 3 h at 30°C. They were then cooled on ice for 20 min, spun down, washed with ice-cold PBS, flash frozen in liquid nitrogen and stored frozen at -80°C till further processing. For protein extraction, the cells were re-suspended in £. coli lysis buffer (1 x PBS, 0.4% Triton X-100, 2 mM MgCI ₂ , 1 mM EDTA pH 8.0, 2 mM DTT, 0.2 mg/mL lysozyme) and lysed by 3 times 15sec of sonication. The extracts were then spun down at 14,000 rpm for 10 min to remove cell debris. The supernatant was pipetted into a fresh eppendorf tube.

4. Expression and purification of proteins from yeast

Yeast cells were grown in synthetic minimal medium in a way to maximize expression of the protein concerned, either into exponential phase or into starvation. The cells were then pelleted, washed with PBS, flash frozen in liquid nitrogen and stored frozen at -80°C till further processing. For extraction, the cells were left to defrost on ice for 10 min. Yeast lysis buffer (1 x PBS, 0.1 % Triton X-100, 10% glycerol, 2.5 mM MgCI ₂ , 1 mM EDTA, 1 mM DTT, 10 mM NaF, 0.4 mM Na3V04, 0.1 mM beta-glycerophosphatase, protease inhibitor mix EDTA free by Roche, 1 mM PMSF) and glass beads were added, after which the cells were broken by shaking 3 times for 1 min in a fast prep. Cell debris were removed by centrifugation.

5. Co-IP assay

E. coli protein extract containing GST-tagged protein was incubated with glutathione sepharose beads (GE Healthcare) to allow binding for 1.5 h. In the meanwhile yeast protein extract was incubated with glutathione sepharose beads (GE Healthcare) to remove unspecific interacting proteins from the extract. The GST-protein bound beads were then washed 3 times with E. coli wash buffer, before yeast extract was added. The beads were left for 2 h with yeast extract in a roller drum at 4 °C to allow for interaction to occur. The beads were then washed 3 times with PBS-T. Finally, sample buffer was added, the samples heated for 5 min at 65°C and subsequently stored frozen at -20°C

6. Western Blot and Coomassie staining

Samples were heated at 65°C for 5 min and finally loaded on two precast NuPAGE ^® Bis-Tris Mini Gels in NuPAGE ^® MOPS SDS Running buffer. The gels were run at 150 V for ca. 2 h. Proteins were then transferred from one gel onto a nitrocellulose membrane (HybondC extra, GE Healthcare) by blotting using 300 mA for 90 min in blotting buffer (NuPAGE ^® MOPS SDS Running buffer, 20% v/v methanol). After blotting, the membrane was incubated overnight with HA peroxidase. The blot was then layered with Western Bright™Quantum and Western Bright ™ Peroxide for visualization in a LAS 400 mini. The other gel was incubated in Coomassie blue staining solution (0.25% Coomassie Brilliant Blue, 30% v/v methanol, 10% v/v acetic acid) for 1 h. It was then de-stained by washing in de-staining solution (30% v/v methanol, 10% v/v acetic acid) 4 times for 10 min and scanned in a standard office scanner.

7. Microscopy

For the split citrin assay and co-localization experiments, the cells were pre-grown on synthetic minimal medium overnight. They were then centrifuged, re-suspended in the appropriate starvation medium and further incubated for 2 h. The cells were then spotted on slides and immediately examined under the microscope at room temperature. All images were acquired using an Olympus 1X81 FluoviewTM FV1000 microscopy system running FV10-ASW software. Images were analyzed using Fiji.

8. Heat shock of GFP foci

Cells were pre-grown in YPD till an appropriate ODeoo as indicated. They were then incubated for 20 min in a shaking water bath at 30°C, 35°C, 40°C, 45°C or 50°C. Subsequently, 5 μΙ was spotted on a slide and the cells immediately observed under the microscope. The cultures were further incubated at 30°C in a shaking incubator and further samples were taken for microscopic examination as a function of time.

9. FM4-64 staining One ml of a culture with nitrogen-starved cells was centrifuged and after removal of the supernatant the cells were re-suspended in YPD containing 1 μΙ FM4-64. The cells were then incubated for 20 min at 30°C in a shaking incubator, washed with YPD and re-suspended in 4 ml of fresh YPD. Cells were then incubated for 90 min at 30°C in a shaking incubator, after which they were pelleted, washed with sterile distilled water and finally re-suspended in nitrogen starvation medium before being spotted on glass slides and examined under the microscope.

10. Growth curves

Cells were pre-grown in a 3 mL overnight culture. One mL was used to inoculate a 50 mL YPD culture. One mL samples were taken every 30 min or 1 h and the ODeoo was measured using a photometer. Table 1 : Saccharomyces cerevisiae strains used in this study.

BY4741 Mata ura3 his3 Ieu2 met15 (Brachmann et al., 1998)

JT a.405 BY4741 Mata ura3 his3 Ieu2 met15 gaplA (Giaever et al., 2002)

MC100 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(caHIS) This work

Gapl -N-Citrin(caURA)

MC142 BY4742 Mata ura3 is3 Ieu2 lys Pho84-3HA (KanMX6) This work

MC177 BY4742 Mata ura3 is3 Ieu2 lys Gnp1 -3HA (KanMX6) This work

MC179 BY4741 Mata ura3 is3 Ieu2 met15 Pho89-3HA (KanMX6) This work

MC178 BY4742 Mata ura3 is3 Ieu2 lys Hip1 -3HA (KanMX6) This work

MC168 BY4741 Mata ura3 is3 Ieu2 met15 Mep1 -3HA (KanMX6) This work

MC169 BY4741 Mata ura3 is3 Ieu2 met15 Mep2-3HA (KanMX6) This work

MC167 BY4741 Mata ura3 his3 Ieu2 met15 Gapl -N-Citrin(caURA) This work

MC179 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

MC175 BY4741 Mata t/ra3 /?/ ^' s3 /et/2 meii5 Gcd1 -C-Citrin(spHIS) Sul1 - This work

N-Citrin(caURA)

MC176 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Pho84-N-Citrin(caURA) MC88 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work Gapl -N-Citrin(caURA) Snf7-RFP (KanMX6)

MC80 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) Vph1 -RFP (KanMX6)

MC92 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) Cop1 -RFP (KanMX6)

MC89 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) Sec13-RFP (KanMX6)

MC102 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) vps25::KanMX6

MC101 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) vps36::KanMX6

MC99 BY4741 Mata ura3 his3 Ieu2 met15 Gcdl -C-Citrin(spHIS) This work

Gapl -N-Citrin(caURA) end3:: KanMX6

MC182 BY4741 BY4741 Mata ura3 is3 Ieu2 met15 Gcd1 -C- This work

Citrin(spHIS) Gapl -N-Citrin(caURA) caURA::KanMX6

Table 2: Plasmids used in this study.

JTPL 40412 YCplac33 GAP 1 -3 HA This work

JTPL 2413 pRS316-SUL1 -HA This work pTPQ128 pRS415 P _A DHi-SEC7-DsRed (Proszynski et al., 2005) yN-Ura3 pFA6a-link-split-yECitrin-CaURA3 (first 155 amino Geovani Lopez acids of citrin) Ortiz, Mexico

City, Mexico yC-HIS5 pFA6a-link-split-yECitrin-spHIS5 (amino acids 155- Geovani Lopez

238 of citrin) Ortiz, Mexico

City, Mexico pFA6a- pFA6a-3HAX-KanMX6 (Longtine et al.,

3HAX- 1998)

KanMX6

pFA6a- pFA6a-GFP(S65T)-KanMX6 (Bahler et al.,

GFP(S65T)- 1998)

KanMX6 Addgene

plasmid #39292(Bahler et al., 1998)(Bahler et al., 1998) pRP1574 Edc3-RFP (Shah et al.,

2013) pPHY3782 Pbp1 -RFP (Buchan et al.,

2008)

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