EMBO/SourceData huggingface dataset:
Figure 1Chromatin-enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2/3 antibody.except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.Relative DPC levels in cells treated were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments).HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies.using HeLa cells transfected with non-targeting control (CTRL) or SPRTN siRNAs.Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies.
Figure 2HeLa cells were transfected with non-targeting control (CTRL) or DNMT1 siRNAs, exposed or not to 5-azadC and collected 2 h later. Chromatin-enriched fractions were immunoblotted with antibodies to SUMO2/3, DNMT1 and MCM6 (loading control).Representative images of HeLa cells that were treated and co-immunostained with antibodies to DNMT1, SUMO2/3 and PCNA. Scale bar, 10 μm.Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with indicated siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).Extracts of HeLa/GFP-DNMT1 cells treated with 5-azadC for the indicated times were subjected to GFP immunoprecipitation (IP) under denaturing conditions followed by immunoblotting with SUMO2/3 and GFP antibodies.HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive (CI) GFP-DNMT1. Cells were then left untreated or incubated with 5-azadC for 2 h, and SUMOylation of GFP-DNMT1 was analyzed Asterisk denotes unmodified GFP-DNMT1 recognized by the SUMO2/3 antibody due to weak cross-reactivity.Mass spectrometry analysis of 5-azadC-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to 5-azadC were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. Proteins displaying significantly altered SUMOylation in response to 5-azadC treatment relative to the control condition were visualized through two-sample t-testing using permutation-based FDR to achieve q-values of <0.05SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.
Figure 3Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayediPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.
Figure 4Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.Quantification of data showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).
Figure 5HeLa cells transfected with plasmids encoding GFP alone, GFP-ACRC or GFP-tagged C. elegans GCNA-1 were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO23-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.GFP expression driven by the gcna-1 promoter was observed in germ cells, proliferating embryos and young larvae but not in post-mitotic tissues in the head Scale bars, 50 µm.Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).
Figure 1A Detection of the RBPJ/L3MBTL3 interaction using the yeast two-hybrid (Y2H) assay. In this Y2H experiment, RBPJ is fused to the GAL4 DNA-binding (DB) domain and L3MBTL3 is fused to the GAL4 activation domain (AD). The DB-RBPJ and AD-L3MBTL3 fusion proteins interact with each other, leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine. The six Y2H controls were previously described (Dreze et al, 2010). The experiment was independently replicated thrice.B Endogenous L3MBTL3 co-purifies specifically with HA-RBPJ but not with HA-EGFP, HA-TBL1X or HA-HEY2. Immuno-precipitation (IP) of HA-tagged RBPJ, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or L3MBTL3 antibody. The experiment was independently replicated twice. C Endogenous RBPJ co-purifies specifically with HA-L3MBTL3 but not with HA-EGFP, HA-TBL1X or HA-HEY2. IPs of HA-tagged L3MBTL3, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or RBPJ antibody. The experiment was independently replicated twice. EV: Empty Vector control; WB: Western blot; IP: immuno-precipitation.
Figure 2B L3MBTL3-Δ(1-64) does not interact with RBPJ. IP of HA-FLAG-tagged RBPJ in the presence of FLAG-tagged L3MBTL3 (WT or deletion mutants) in HEK293T cells followed by Western blotting using FLAG antibody. The experiment was independently replicated twice.D Deletion of the BTD domain impairs the RBPJ/L3MBTL3 interaction. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and deletion mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. The experiment was independently replicated twice. E RBPJF261R point mutant does not interact with L3MBTL3. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and point mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3. The experiment was independently replicated twice. WB: Western blot; IP: immuno-precipitation.
Figure 3A Thermodynamic characterization of the RBPJ/L3MBTL3 interaction. Representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for L3MBTL3-(31-70) binding to RBPJ-(53-474).B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (B) SBP-FLAG-RBPJ and HA-L3MBTL3-Δ(SAM) in the presence of an increasing amount of HA-NOTCH1 ICD. The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (C) SBP-FLAG-RBPJ and HA-NOTCH1 ICD in the presence of an increasing amount of HA-L3MBTL3-Δ(SAM). The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.
Figure 4A De-repression of Notch target genes upon RBPJ knockdown. Shown are means ±s.d. of quadruplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data. Inset: Western blot analysis validates the shRNA-mediated depletion of RBPJ.B De-repression of Notch target genes in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of quadruplicate experiments. [**] P<0.01, NS: Not Significant; two-sample T-test on log-transformed data. Inset: Western blot analysis validates the CRISPR/Cas9-mediated KO of L3MBTL3.C RBPJ and L3MBTL3 co-localize at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes decreases upon RBPJ knockdown. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).E The repressive activity of L3MBTL3 at Notch target genes is RBPJ-dependent. Expression analysis of Notch target genes upon RBPJ knockdown and/or overexpression of L3MBTL3. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA model on log-transformed data where the difference of differences was tested, which is equivalent to testing the interaction in a two-way ANOVA model. Western blot analysis validates the overexpression of L3MBTL3 and the shRNA-mediated depletion of RBPJ (Appendix Fig S3E). Gene expression analyses of OCT4 was performed as control (Appendix Fig S3F).F L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on its RBPJ interaction domain. ChIP analyses of HA-L3MBTL3 WT and HA-L3MBTL3-Δ(1-64) occupancy at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of duplicate experiments measured twice each.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).G The L3MBTL3-(1-64) domain is required for the downregulation of HES1 and HEY2 in U87-MG cells. Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT, L3MBTL3-Δ(1-64) or LacZ control (Control). Shown are means ±s.d. of triplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data.
Figure 5A The RBPJ/KDM1A interaction is indirect and occurs via L3MBTL3. IP of HA-KDM1A in the presence of overexpressed V5-L3MBTL3 or V5-L3MBTL3-Δ(1-64) in L3MBTL3 KO U87-MG cells. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to overexpress the L3MBTL3 proteins. The experiment was independently replicated twice.B KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is L3MBTL3-dependent. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).C KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells upon overexpression of L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM). Control: empty vector. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), leads to decreasing H3K4me2 at the proximal Notch-responsive element of HES1. ChIP analysis of H3K4me2 at the proximal Notch-responsive element of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each. P values were estimated via a one-way ANOVA on log-transformed data.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).E L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), represses HES1. Expression analysis of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) mutants in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA on log-transformed data. NS: Not Significant. WB: Western blot; IP: immuno-precipitation. We note that in the context of this experiment, i.e., in the absence of endogenous L3MBTL3, the overexpression of L3MBTL3-Δ(1-64) does not result in the increased expression of HES1, contrasting with the result obtained in Fig 4G, i.e., in the presence of endogenous L3MBTL3. Indeed, as expected, the dominant negative effect of L3MBTL3-(1-64) on endogenous WT L3MBTL3's ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed.
Figure 6A GST pulldown showing that dL(3)mbt, the Drosophila homolog of L3MBTL3, directly interacts with Su(H), the Drosophila homolog of RBPJ. In vitro transcribed and translated dL(3)mbt or dNotch ICD (dNotch ICD fragment containing the RAM domain and ANK repeats), as positive control, was incubated with bacterially purified GST-Su(H) or GST alone pre-bound to GSH beads. Proteins were resolved via SDS-PAGE and signals were acquired via X-ray exposure. The experiment was independently replicated four times.C Snapshot showing the co-localization of dL(3)mbt and Su(H) at the dNotch (N) locus.D In the wing imaginal disc, dL(3)mbt overexpression in the dorso-ventral (D-V) boundary results in the downregulation of the Notch target gene cut. Wing discs expressing UAS-GFP (top panels) or UAS-HA-dL(3)mbt;UAS-GFP (bottom panels) under the vg-Gal4 driver at 25°C were stained for cut and HA. GFP marks the vg-Gal4 expression domain. Insets below each panel show a closer view of the D-V boundary with yellow arrows marking the regions where HA-dL(3)mbt is expressed and cut is downregulated. At least 20 discs for each genotype were analyzed. Representative images are shown. Scale bars: 100μm.E The vg-Gal4-driven HA-dL(3)mbt overexpression causes a serrated wing (wing notching) phenotype. Flies expressing either UAS-GFP or UAS-HA-dL(3)mbt;UAS-GFP under vg-Gal4 were reared to adulthood at 25°C. P values were estimated by comparing the proportions via a two-proportion Z-test. Scale bars: 200μm.F Functional interaction between lag-1/RBPJ and lin-61/L3MBTL3 during C. elegans vulva development. Proportion of animals (n ≥100) displaying a protruding vulva (Pvl) phenotype after RNAi treatment for two generations. Worms were grown at 25°C. Shown are means ±s.d of duplicate experiments. EV: Empty Vector control.
Figure 1A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.
Figure 2A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.
Figure 3A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.
Figure 4A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B). D Hyperactive PKA promotes enrichment of all HXTs after MAT.E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions