+

WO2017037285A1 - Moyens et procédés pour moduler la croissance de cellules eucaryotes - Google Patents

Moyens et procédés pour moduler la croissance de cellules eucaryotes Download PDF

Info

Publication number
WO2017037285A1
WO2017037285A1 PCT/EP2016/070840 EP2016070840W WO2017037285A1 WO 2017037285 A1 WO2017037285 A1 WO 2017037285A1 EP 2016070840 W EP2016070840 W EP 2016070840W WO 2017037285 A1 WO2017037285 A1 WO 2017037285A1
Authority
WO
WIPO (PCT)
Prior art keywords
elf2
subunit
elf2b
transporter
nutrient
Prior art date
Application number
PCT/EP2016/070840
Other languages
English (en)
Inventor
Johan Thevelein
Michaela CONRAD
Griet VAN ZEEBROECK
Original Assignee
Vib Vzw
Katholieke Universiteit Leuven, K.U.Leuven R&D
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vib Vzw, Katholieke Universiteit Leuven, K.U.Leuven R&D filed Critical Vib Vzw
Publication of WO2017037285A1 publication Critical patent/WO2017037285A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • 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.
  • PKA protein kinase A pathway
  • 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.
  • yeast cells growing in a complete glucose medium are starved for a single essential nutrient they also downregulate PKA activity during growth arrest.
  • 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 (Wiederscene et al., 2000), it remains poorly characterized.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 4 The transceptor/transporter foci and the elF2/elF2B-GFP foci are identical.
  • 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 ⁇ 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.
  • 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.
  • FIG. 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 ⁇ 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 ⁇ ⁇ 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 ⁇ subunit of elF2B (Gcd1 ) tagged with the N-terminal half of citrin were grown at 30°C till exponential phase ( ⁇ ⁇ 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 ⁇ ⁇ 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).
  • 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.
  • 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.
  • 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.
  • observable or detectable characteristic e.g., age, treatment, time of day, etc.
  • Characteristics which are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
  • 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.
  • 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.
  • 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.
  • the present invention provides an isolated complex composed of a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B.
  • 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.
  • 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.
  • '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.
  • the invention provides an in vivo complex composed of a nutrient transporter and a subunit of elF2 and/or a subunit of elF2B.
  • 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.
  • 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.
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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,
  • 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.
  • 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,
  • 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.
  • 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,
  • 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 refers to the modulation of translation (or cell growth). Modulation can refer to either an inhibition or to a stimulation.
  • 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).
  • 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).
  • 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 can but do not necessary comprise a tag.
  • the elements of the system can be a fluorescent tag.
  • the fluorescent tag is a split fluorescent tag.
  • the nutrient transporter can a sugar transporter, a nitrogen transporter, a sulfate transporter, a phosphate transporter, a vitamin transporter or a metal ion transporter.
  • the screening systems and methods comprise high content screening (HCS) of suitable compounds.
  • HCS is a screening method that uses live cells to perform a series of experiments as the basis for high throughput compound discovery.
  • HCS is an automated system to enhance the throughput of the screening process.
  • the present invention is not limited to the speed or automation of the screening process.
  • the HCS assay of the invention provides for a system to generate high quality "hits" identifying compounds that modulate cell growth.
  • the HCS assay provides for a high throughput assay.
  • 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.
  • 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.
  • a hypothetical compound library may contain all known compounds known to bind to a specific binding region.
  • 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.
  • 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.
  • "high-throughput" modalities 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.
  • 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.
  • the screen and method of the present invention comprise a primary screen, one or more counter screens, and one or more secondary screens.
  • 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.
  • the system and methods of the invention comprise a primary screen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell or cell population is modified by administering an expression vector encoding the proteins of interest.
  • 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.
  • 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.
  • 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.
  • 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.
  • the yeast nutrient transporters Gap1 and Mep2 a nitrogen starvation medium is preferably used during the in vivo screening.
  • a sulfate starvation medium is preferably used during the in vivo screening.
  • a phosphate starvation medium is preferably used during the in vivo screening.
  • 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.
  • 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.
  • 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.
  • the system of the invention comprises numerous test compounds screened on cells cultured on a multi-well plate.
  • multi-well plates include a 6-well plate, a 24-well plate, a 96-well plate, and a 384- well plate.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ subunits of elF2B and the ⁇ subunit of elF2 (see Figure 1 ).
  • 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
  • Gcd6 elF2Be
  • 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.
  • Gap1 -elF2/elF2B foci are identical to the previously observed elF2/elF2B-GFP foci
  • 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.
  • Gap1 -foci might be identical to one of the cell organelles of the protein secretion pathway or the ligand-induced endocytosis pathway.
  • 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).
  • 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).
  • deletion of END3 did not affect formation of the Gap1 -Gcd1 (elF2By) foci (see Figure 6).
  • Gap1 -foci After transfer of the cells to nitrogen starvation medium.
  • cells co-expressing split-citrin fusion constructs of Gap1 and elF2By(Gcd1 ) were grown to exponential phase ( ⁇ ⁇ ⁇ .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 ⁇ ⁇ and to follow the appearance of the Gap1 -foci using confocal fluorescence microscopy.
  • 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 ⁇ ⁇ also ceased after about 2 h, hence coinciding with the appearance of the Gap1 -foci (see Figure 7).
  • ⁇ ⁇ continued to increase for another four h and no clear Gap1 -foci appeared as observed in the nitrogen starvation medium.
  • 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).
  • 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.
  • this correlation suggests that the transporter-elF2B foci might play a role in the control of cell proliferation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the transporters 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.
  • 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.
  • 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.
  • 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.
  • PDS pharmacological diversity set
  • 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.
  • 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.
  • 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.
  • the cells were pre-grown on either YPD or synthetic minimal medium and then transferred to the respective starvation media.
  • 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).
  • 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 , 160 ⁇ ⁇ 3 ⁇ 0 4 , ⁇ . ⁇ Kl, 0.7 ⁇ ZnC , 0.7 ⁇ CuCI 2 , 0.3 ⁇ 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).
  • 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 , 160 ⁇ ⁇ 3 ⁇ 0 4 , ⁇ . ⁇ K
  • 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)).
  • 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.
  • 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.
  • 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.
  • E. coli protein extract containing GST-tagged protein was incubated with glutathione sepharose beads (GE Healthcare) to allow binding for 1.5 h.
  • 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
  • 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.
  • Coomassie blue staining solution 0.25% Coomassie Brilliant Blue, 30% v/v methanol, 10% v/v acetic acid
  • 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.
  • 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 ⁇ 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.
  • Gapl -N-Citrin(caURA) Vph1 -RFP Gapl -N-Citrin(caURA) Vph1 -RFP (KanMX6)
  • Gapl -N-Citrin(caURA) Cop1 -RFP Gapl -N-Citrin(caURA) Cop1 -RFP (KanMX6)
  • Gapl -N-Citrin(caURA) Sec13-RFP KanMX6
  • Gapl -N-Citrin(caURA) end3 :: KanMX6
  • Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth. Proceedings of the National
  • elF2B is a decameric guanine nucleotide exchange factor with a gamma2epsilon2 tetrameric core. Nat Commun. 5:3902.
  • VPH1 The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.
  • Van Zeebroeck, G., M. Kimpe, P. Vandormael, and J.M. Thevelein. 201 1 A split-ubiquitin two- hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2.
  • PLoS One. 6:e24275 Van Zeebroeck, G., M. Rubio-Texeira, J. Schothorst, and J.M. Thevelein. 2014. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor. Molecular microbiology. 93:213-233.
  • Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORCI . Science. 347:188-194.
  • the F-box protein Rcyl p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae. J Cell Biol. 149:397-410.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la détection de nutriments et la régulation de la croissance et du développement dans des cellules eucaryotes, telles que des cellules de levure. Plus particulièrement, l'invention fournit des complexes entre des transporteurs de nutriments et des facteurs d'initiation de la synthèse protéique. En outre, l'invention concerne des dosages de criblage utilisant ces complexes pour isoler des composés qui peuvent moduler la croissance et le développement cellulaires.
PCT/EP2016/070840 2015-09-04 2016-09-05 Moyens et procédés pour moduler la croissance de cellules eucaryotes WO2017037285A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP15183895 2015-09-04
EP15183895.0 2015-09-04

Publications (1)

Publication Number Publication Date
WO2017037285A1 true WO2017037285A1 (fr) 2017-03-09

Family

ID=54105628

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/070840 WO2017037285A1 (fr) 2015-09-04 2016-09-05 Moyens et procédés pour moduler la croissance de cellules eucaryotes

Country Status (1)

Country Link
WO (1) WO2017037285A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226522A (zh) * 2017-11-27 2018-06-29 南京天纵易康生物科技股份有限公司 一种基于双分子荧光互补技术的Cys C检测试剂盒、制备及使用方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002095061A1 (fr) * 2001-05-18 2002-11-28 New York University Procede de criblage de substances test destinees a traiter ou prevenir des maladies impliquant un stress oxydatif

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002095061A1 (fr) * 2001-05-18 2002-11-28 New York University Procede de criblage de substances test destinees a traiter ou prevenir des maladies impliquant un stress oxydatif

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
G. LIANG ET AL: "Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2", HUMAN MOLECULAR GENETICS, vol. 17, no. 20, 16 July 2008 (2008-07-16), gb, pages 3254 - 3262, XP055311370, ISSN: 0964-6906, DOI: 10.1093/hmg/ddn221 *
HOLSBEEKS I ET AL: "The eukaryotic plasma membrane as a nutrient-sensing device", TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, HAYWARDS, GB, vol. 29, no. 10, 1 October 2004 (2004-10-01), pages 556 - 564, XP004575218, ISSN: 0968-0004, DOI: 10.1016/J.TIBS.2004.08.010 *
JOEP SCHOTHORST ET AL: "Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters", CURRENT GENETICS, vol. 59, no. 4, 11 October 2013 (2013-10-11), US, pages 197 - 206, XP055310127, ISSN: 0172-8083, DOI: 10.1007/s00294-013-0413-y *
M. C. S. MORAES ET AL: "Novel Membrane-Bound eIF2 Kinase in the Flagellar Pocket of Trypanosoma brucei", EUKARYOTIC CELL, vol. 6, no. 11, 1 November 2007 (2007-11-01), US, pages 1979 - 1991, XP055310659, ISSN: 1535-9778, DOI: 10.1128/EC.00249-07 *
MARTA RUBIO-TEXEIRA ET AL: "Saccharomyces cerevisiae plasma membrane nutrient sensors and their role in PKA signaling", FEMS YEAST RESEARCH, vol. 10, no. 2, 1 March 2010 (2010-03-01), GB, NL, pages 134 - 149, XP055310163, ISSN: 1567-1356, DOI: 10.1111/j.1567-1364.2009.00587.x *
MICHAELA CONRAD ET AL: "Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae", FEMS MICROBIOLOGY REVIEWS, vol. 38, no. 2, 1 March 2014 (2014-03-01), pages 254 - 299, XP055310111, DOI: 10.1111/1574-6976.12065 *
RUBIO-TEXEIRA MARTA ET AL: "Peptides induce persistent signaling from endosomes by a nutrient transceptor.", NATURE CHEMICAL BIOLOGY APR 2012, vol. 8, no. 4, April 2012 (2012-04-01), pages 400 - 408, XP002763068, ISSN: 1552-4469 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226522A (zh) * 2017-11-27 2018-06-29 南京天纵易康生物科技股份有限公司 一种基于双分子荧光互补技术的Cys C检测试剂盒、制备及使用方法

Similar Documents

Publication Publication Date Title
Montoro et al. Vps39 interacts with Tom40 to establish one of two functionally distinct vacuole-mitochondria contact sites
Tang et al. Pan1p, End3p, and Sla1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis
Wu et al. The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis
Saito et al. Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae
Fujita et al. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin
Borgese et al. Targeting pathways of C-tail-anchored proteins
Humphries et al. Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin
Ludwig et al. Molecular composition and ultrastructure of the caveolar coat complex
Chadrin et al. Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution
Del Dedo et al. ORP-mediated ER contact with endocytic sites facilitates actin polymerization
Wang et al. The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis
Sosicka et al. An insight into the orphan nucleotide sugar transporter SLC35A4
Kanneganti et al. Btn3 is a negative regulator of Btn2-mediated endosomal protein trafficking and prion curing in yeast
Newpher et al. Novel function of clathrin light chain in promoting endocytic vesicle formation
Fernández-Pevida et al. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits
Liu et al. Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis
Longo et al. Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis
So et al. Molecular characterization of adenylyl cyclase complex proteins using versatile protein-tagging plasmid systems in Cryptococcus neoformans
Fell et al. Identification of yeast genes involved in K+ homeostasis: Loss of membrane traffic genes affects K+ uptake
Chen et al. Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p
WO2017037285A1 (fr) Moyens et procédés pour moduler la croissance de cellules eucaryotes
Kawamura et al. Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy
Hoban et al. ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants
Manzer et al. The Arf-GAP Age2 localizes to the late-Golgi via a conserved amphipathic helix
Reck et al. Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16763747

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16763747

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载