The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila.

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

Introduction

Gustatory Receptors 64 (Gr64s) are a group of 6 tandem gustatory receptor genes (a through f) involved in mediating sensation of sugars, fatty acids, and glycerol in the adult nervous system [1-4]. Gr64s are thought to sense distinct ligands via distinct mechanisms: Gr64e, for example, is reported to act as a ligand gated ion channel in response to glycerol binding, whereas it acts downstream of phospholipase C in fatty acid sensation [4]. Gr64s are thought to function as heterodimers with each other and alternate gustatory receptors, as, for example, both Gr64a and Gr64f are required to mediate responses to certain sugars [5].

Mutations in ribosomal proteins or ribosome biogenesis factors can result in a class of disorders known as ribosomopathies [6-8]. While roles have been established for nucleolar stress, p53 activation, and translational defects in disease progression, ribosomopathy etiology remains poorly understood [8]. Ribosome protein (Rp) mutations are well studied in Drosophila, wherein the majority of cytosolic Rp encoding genes yield the so-called “Minute” phenotype when heterozygous mutant [9]. Rp/+ flies are viable and fertile but exhibit a developmental delay [10]. Epithelial cells in Rp/+ larvae exhibit reduced translation rates, increased cell-autonomous apoptosis, and stress pathway activation, including, JNK, JAK/STAT, Toll/IMD signalling, and the oxidative stress response [11-15]. Rp/+ cells also undergo cell competition and are eliminated from the tissue when confronted with wild-type cells in mosaic tissues [16,17]. Rp/+ cells are therefore said to behave as “losers” relative to wild-type “winners.”

Rp/+ cells have recently been shown to suffer from chronic proteotoxic stress. They exhibit proteasome and autophagy defects, activation of the integrated stress response (ISR), a stoichiometric imbalance of large and small ribosomal subunit proteins and an accumulation of intracellular protein aggregates [18-21]. Importantly, boosting proteostasis rescues stress pathway activation, cell autonomous apoptosis, and competitive elimination [19,20]. These findings point to proteotoxic stress as a potential driver of the pathologies associated with ribosomopathies.

By characterising the biology of Rp/+ cells, we identify a new function for Gr64 genes in maintaining proteostasis in epithelial cells. We find that Gr64 genes, whose expression is normally observed in neuronal cells, become up-regulated in wing disc epithelial cells upon Rp/+ mutation. Loss of Gr64 drives substantial apoptosis in noncompeting and competing Rp/+ cells and exacerbates stress pathway activation. Furthermore, loss of Gr64 exacerbates proteotoxic stress in Rp/+ cells, by reducing proteasome and autophagy function. Calcium imaging reveals reduced calcium activity in Rp/+ cells upon Gr64 down-regulation (as measured by the frequency of calcium flashes, [22]), suggesting that Gr64’s proteostasis promoting effects might be mediated by calcium signalling.

Results and discussion

Mining the list of genes differentially expressed in cells heterozygous mutant for the ribosomal protein RpS3, we observed up-regulation of all 6 Gr64 gustatory receptors relative to wild-type cells (Fig 1A and [11]). Gr64s were also up-regulated in cells mutant in mahjong (mahj), an E3 ubiquitin ligase, whose mutation also leads to proteotoxic stress and cell competition [11,19,23,24]. This was conspicuous, as Gr64s have no known nonneuronal nor larval function. In order to explore the role of Gr64s in cells affected by proteotoxic stress, we tested the effect of removing one copy of the Gr64 locus on RpS3 larvae, using a deficiency spanning the Gr64 locus, along with rescuing constructs of other affected genes (ΔGr64) [1]. ΔGr64/ΔGr64 flies present with no known phenotypes other than deficient gustatory responses [1]. RpS3, ΔGr64 wing discs, however, exhibited a marked increase in apoptosis over levels seen in wing discs carrying either mutation alone (S1A–S1D Fig), indicating that RpS3 cells are acutely reliant on Gr64s for their survival. We confirmed this result using a precise CRISPR/Cas-9 deletion of the Gr64 cluster, Gr64af [4] (Fig 1B and 1C). Gr64 did not contribute to survival in the non-Minute context, as non-Minute wing discs homozygous null for Gr64 exhibited no increase in apoptosis relative to the wild type (S1E and S1F Fig). This is consistent with the fact that Gr64s appear to be only minimally expressed in wing discs (Flybase and [11]). Importantly, Gr64 mutations also led to increased apoptosis in wing discs heterozygous mutant for RpS17 or RpS23, 2 other Rp genes (S1G–S1J Fig), indicating a general requirement for Gr64 for multiple Rp mutants. RpS23 discs homozygous mutant for Gr64 further showed morphological defects with a loss of the characteristic pouch and hinge folds observed in wing discs (S1I and S1J Fig). To confirm that the effect observed was specifically caused by the Gr64 mutations, we sought to rescue the effect by overexpressing Gr64 in RpS3, Gr64 mutants using a UAS construct driving expression of 5 of the 6 Gr64 genes (Gr64abcd-GFP-f) [1]. Overexpression of Gr64 in the P compartment resulted in toxicity and wiped out the compartment (Fig 1D), probably due to unphysiologically high expression levels. However, this specific UAS-Gr64 transgene has been shown to provide rescuing activity in the absence of Gal4 drivers [1]. Accordingly, we tested whether we could similarly observe rescue of Gr64 induced death by using the rescuing UAS-Gr64 transgene in the absence of Gal4. Indeed, UAS-Gr64 substantially rescued Gr64 induced death in RpS3 cells, confirming that the observed effect is due to the Gr64 mutation (Fig 1E and 1F).

Fig 1

Noncompeting RpS3 cells depend on Gr64 for their survival.

(A) Fold change differences in Gr64 transcript expression relative to WT in wing discs heterozygous mutant for RpS3 (as detected in 2 separate mutant alleles: RpS3{Plac92} or RpS3*) or homozygous mutant for mahjong (mahj). Numbers and p-values are derived from [11]. (B) Wing discs heterozygous for a precise deletion of the Gr64 cluster (Gr64af) (left panel), heterozygous mutant for RpS3 (middle panel), or heterozygous mutant for both (right panel), assessed for cell death via immunostaining for cleaved-Dcp1 (red). (C) Quantification of cell death from wing discs of the same genotypes as in (B) (nGr64 = 9, nRpS3 = 10, nRpS3,Gr64 = 9, 2-sided Mann–Whitney U test). (D) Representative image of a wing disc heterozygous mutant for RpS3 and Gr64 expressing the UAS-Gr64abcd-GFP-f construct driven by hhGal4 and stained with anti-Ci (cyan) to label the anterior compartment, and with anti-cleaved-Dcp1 (red). (E) Wing discs heterozygous mutant for RpS3 (left panel), heterozygous for both RpS3 and Gr64 (middle panel), or heterozygous for both and expressing the UAS-Gr64abcd-GFP-f construct, without Gal4 driver (right panel), assessed for cell death by immunostaining for cleaved-Dcp1 (red). (F) Quantification of cell death from wing discs of the same genotypes as in (E) (nRpS3 = 10, nRpS3,Gr64 = 10, nRpS3,Gr64,UAS-Gr64 = 15, 2-sided Mann–Whitney U test). (G) WT (left panel) or RpS3 wing discs (right panel) expressing Gr64f-RNAi in the posterior compartment driven by hh-Gal4 and assessed for cell death with a staining for cleaved caspase3 (red). (H) Quantification of cell death from wing discs of the same genotype as in (G, right panel) (n = 12, 2-sided Wilcoxon signed rank test). Horizontal lines in C and F indicates the median. For this and all other figures, scale bars correspond to 50 μm, and white dashed lines denote compartment boundaries, where the anterior compartment is shown on the left side of the image and ventral is up. Numerical data can be found in the “Fig 1” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein; WT, wild-type.

To determine whether the survival function of Gr64 reflects a systemic or cell autonomous role, we knocked down Gr64 specifically in the posterior compartment, using the hedgehog-Gal4 driver. We used a RNA interference (RNAi) line against Gr64f, which, given that the Gr64 cluster locus is polycistronic [1], is likely to silence multiple Gr64s. Gr64f-RNAi expression in wild-type discs yielded no appreciable change in levels of apoptosis (Fig 1G), whereas expression of Gr64f-RNAi in RpS3 wing discs caused a strong increase in apoptosis, specifically in the RNAi expressing cells (Fig 1G and 1H). A modest level of cell death was also observed in the non-RNAi compartment, perhaps due to apoptosis-induced apoptosis or to a systemic effect of Gr64 silencing. Expression of Gr64f-RNAi in clones in RpS3 discs yielded a similar result (S2A–S2C Fig). These data argue that RpS3 cells are cell autonomously dependent on Gr64 for their survival, although an additional non-cell autonomous contribution cannot be ruled out.

Next, we asked whether the requirement of Gr64 in Rp/+ cells is also observed in other imaginal discs. Both haltere (S2D–S2F Fig) and leg discs (S2G–S2I Fig) showed increased death when Gr64 was knocked down. This effect, however, was less pronounced than in wing discs. Eye discs instead did not appear to be affected by single copy removal of Gr64 (S2J–S2L Fig). Thus, Gr64 is required for RpS3 survival across many but not all imaginal discs.

Having established a prosurvival role for Gr64 in noncompeting RpS3 cells, we then tested whether Gr64s contribute to the survival of competing RpS3 losers. We therefore generated RpS3 losers competing against wild-type winners in wing discs carrying heterozygous mutations in one of any of the 6 Gr64 genes [25] (Fig 2). Strikingly, heterozygosity for any Gr64 yielded a substantial increase in RpS3 loser cell death at the winner/loser interface (Fig 2A–2H). Furthermore, loser cell clones were smaller in wing discs carrying mutations in Gr64b, Gr64c, Gr64d, Gr64e, or Gr64f, but not Gr64a (Fig 2I). RpS3 loser cells are therefore dependent on Gr64s in both noncompetitive and competitive conditions. While these results might suggest that each specific Gr64 protein contributes to RpS3 survival and these constructs have been used successfully in screens of Gr64 gustatory function [2], because of the polycistronic nature of the locus, it is difficult to draw conclusions on the requirements of individual Gr64 isoforms here, as mutations in one coding sequence might affect the regulation and behaviour of the others.

Fig 2

Heterozygosity at Gr64 loci exacerbates competitive RpS3 loser cell elimination.

(A–G) Representative images of wing discs containing RpS3 loser cells (green) competing against wild-type winners (unlabelled) and stained for cleaved-Dcp1 (red). RpS3 clones were generated in a wild-type background (A) or in wing discs heterozygous for any one of the Gr64 genes a through f (mutations used were Gr64a, Gr64b, Gr64c, Gr64d, Gr64e, and Gr64f) (B–G). (H) Quantification of the percentage of cells undergoing apoptosis at loser clone borders in wing discs of genotypes as shown in (A–G). Statistics reflect multiple logistic regression across 3 replicates (details provided in Materials and methods). (I) Quantification of loser cell growth in wing discs of the genotypes shown in (A–G), as measured by the percent loser clone coverage of the pouch. Statistics reflect Student t test with FDR p-correction. ncontrol = 16, nGr64aGAL4 = 15, nGr64bLEXA = 15, nGr64cLEXA = 11, nGr64d[] = 12, nGr64eLEXA = 9, nGr64fLEXA = 8. For all quantifications, the horizontal line indicates the mean. Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 2” sheet of S1 Data. FDR, false discovery rate; Gr64, Gustatory Receptor 64; Rp, ribosome protein.

Rp/+ mutations cause stress pathway activation and cellular malfunctions, many of which are linked with the loser status [11,12,18-21,26]. To evaluate how Gr64 loss affects cellular stress responses, we identified milder expression conditions for Gr64f-RNAi, using the posterior compartment-specific engrailed-Gal4 driver. With these conditions, Gr64f knockdown in noncompeting RpS3 cells exhibited a comparatively mild increase in apoptosis (Fig 3A and 3B), allowing us to investigate Gr64 function without widespread cell death. Gr64f-RNAi yielded increased activation of the oxidative stress response, as measured by GstD1-GFP reporter expression [27] (Fig 3C and 3D). Gr64f-RNAi did not yield an increase in GstD1-GFP signal in wild-type wing discs (S3A and S3B Fig), demonstrating that this result is specific to the RpS3 context. Consistent with these results, an increase in GstD1-GFP expression was also observed in RpS3 wing discs heterozygous for ΔGr64 (S3C and S3D Fig). Furthermore, Gr64f-RNAi expression in RpS3 discs resulted in a mild increase in JNK pathway and in the ISR, as measured by immunostaining for phosphorylated JNK (Fig 3E and 3F) and phosphorylated eIF2α (Fig 3G and 3H), respectively. No differences in phospho-JNK (S4A and S4B Fig) or phospho-eIF2α (S4C–S4D Fig) levels were seen in the posterior compartments of otherwise genetically identical wing discs lacking the Gr64f-RNAi, confirming that this result is due to RNAi expression. These data indicate that loss of Gr64 exacerbates stress responses seen in Rp/+ cells.

Fig 3

Loss of Gr64 worsens stress pathway activation in RpS3.

(A, B) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment stained for cell death, as detected by cleaved Dcp1 (green) (A) along with quantification in (B) (n = 7, 2-sided Wilcoxon signed rank test). (C, D) GstD1-GFP reporter expression (green) in RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment (C) along with quantification in (D) (n = 6, 2-sided paired t test). (E, F) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and stained for phosphorylated JNK (green) (E) along with quantification in (F) (n = 7, 2-sided paired t test). (G, H) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and stained for phosphorylated eIF2α (green) (G) along with quantification in (H) (n = 7, 2-sided paired t test). Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 3” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein.

We next investigated how Gr64 alleviates stress signalling and improves viability of Rp/+ cells. We previously reported that the ISR and oxidative stress response in RpS3 cells are driven by accumulation of protein aggregates, accompanied by reduced degradation of proteins by the proteasome and by autophagy, leading to sustained proteotoxic stress [19]. Thus, we tested the effect of Gr64 reduction on proteostasis pathways using ProteoFlux and ReFlux, genetically encoded reporters of proteasome and autophagy function, respectively, which we previously developed [19]. In these reporters, GFP is tagged for degradation through the proteasome or the autophagosome by fusion to CL1 or ref(2)P, respectively. These GFP fusions are expressed under the control of a heat shock promoter, which enables heat-shock induced pulse-chase experiments, whereby decline in GFP signal is a quantitative measure of proteasomal or autophagosomal degradation rates [19]. Using these tools, we found that Gr64f-RNAi causes a further reduction in proteasomal (Fig 4A–4C) and autophagosomal (Fig 4D–4F) degradation rates, beyond the defects already present in RpS3. These data indicate that loss of Gr64 yields a substantial impairment in cellular proteolytic pathways. Furthermore, Gr64f-RNAi yields an increase in ref(2)P-positive foci (Fig 4G), structures that we have previously shown to colocalise with poly-ubiquitinylated aggregates in RpS3 cells [19]. We conclude that Gr64 loss causes increased stress signalling and cell death by exacerbating proteotoxic stress.

Fig 4

Loss of Gr64 exacerbates proteostasis defects in RpS3.

(A–C) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment, marked with RFP (red) and expressing the CL1-GFP/ProteoFlux construct (green), 0 hours (A) or 4 hours (B) after heat shock along with quantification in (C) (n0hr = 3, n2hr = 14, n4hr = 5, 2-sided paired t test, the horizontal line indicates the mean and the whiskers reflect the 95% confidence interval). (D–F) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment, marked with RFP (red) and expressing the ref(2)P-GFP/ReFlux construct (green), 0 hours (E) or 6 hours (F) after heat shock along with quantification in (D) (n0hr = 4, n3hr = 3, n6hr = 6, 2-sided paired t test, the horizontal line indicates the mean, and the whiskers reflect the 95% confidence interval). (G) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment stained for ref(2)P (green). (H, I) RpS3 wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and assessed for translation through an OPP translation reporter assay (grey) (H) along with quantification in (I) (n = 8, 2-sided paired t test). (J) Quantification of the number of calcium flashes in anterior and posterior compartments of wing discs heterozygous for RpS3 that also express Gr64f-RNAi in the posterior compartment with enGal4 (nAnt = 30, nPost = 30, 2-sided Wilcoxon signed rank test; the horizontal line indicates the median and the whiskers the interquartile range). (K) Proposed model: heterozygous mutation in a ribosomal protein mutant gene triggers proteotoxic stress, making Rp/+ cells highly dependent upon the autophagosome and proteasome for proteostasis. Gr64 gene cluster is up-regulated in Rp/+ cells, where it supports cell survival and inhibits proteotoxic stress by promoting autophagy and proteasome functions, possibly via calcium signalling. Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 4” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein.

Worsening of proteotoxic stress in ribosome mutants could derive from an inhibition of protein catabolic processes or from an increase in protein translation, which, by producing more proteins, could cause a further burden on proteostasis. Interestingly, OPP, a global translation reporter, revealed a mild but statistically significant decrease in translation upon expression of Gr64f-RNAi (Fig 4H and 4I). This is consistent with the observed increase in eIF2α-phosphorylation (Fig 3G and 3H), which is a translation inhibitor as well as a stress pathway marker [28]. Thus, the increase in proteotoxic stress observed upon Gr64 reduction is not due to increased translation but rather to a failure to clear defective proteins.

How could a family of taste receptors play a role in proteostasis? Gr64 proteins are sweet tastant receptors that cause an increase in cytoplasmic calcium upon ligand binding [25]. Calcium is known to play a role in modulating proteostatic pathways, [29,30] suggesting that Gr64 could ameliorate proteostasis by modifying calcium signalling. We therefore investigated what effect loss of Gr64 might have on calcium signalling using a GCaMP fluorescent calcium reporter [31].Consistent with this hypothesis, we found that silencing Gr64 in the posterior compartment of RpS3 wing discs reduced the frequency of calcium flashes in posterior compartment cells, relative to reference RpS3 anterior cells, which served as internal control (Fig 4J, S1 Movie).

In this study, we have identified Gr64 taste receptors as novel players in proteostasis control and as cytoprotective regulators in epithelial cells affected by proteotoxic stress. Our data suggest that Gr64s contribute to proteostasis by promoting protein catabolism rather than by inhibiting translation (Fig 4K). This is potentially mediated by calcium signalling, as Gr64 activity typically induces calcium release [25], and calcium is involved in protein folding, the ISR, proteasome, and autophagy function [29,32-34]. Indeed, consistent with this hypothesis, our data indicate that removal of Gr64 modifies Calcium signalling in Rp/+ cells. It remains to be determined, however, whether this phenotype is causal or a consequence of the proteostasis defects observed in Gr64 knockdown conditions, and thus further work must be done to elucidate Gr64’s mechanism of action.

Taste receptors have been implicated in nontaste-related chemosensation in neuronal and neuroendocrine cells both in flies and in mammals [2,35-37]. However, a role in proteostasis and a function in epithelia have not previously been described for taste receptors. Interestingly, dysregulation of olfactory and gustatory receptors has been observed in nonolfactory human brain tissue from individuals suffering from protein-aggregate driven neurodegenerative disorders, including Alzheimer disease, Parkinson disease, and Creutzfeld–Jacob disease as well as in a mouse model of Alzheimer disease [38], and olfactory receptors are expressed near to amyloid plaques in a mouse model of Alzheimer disease [39]. It is therefore possible that gustatory and olfactory receptors play a conserved role in promoting proteostasis in both neuronal and nonneuronal cells.

Materials and methods

Fly husbandry and stocks

Drosophila lines were kept in an incubator set to 25°C and reared on food prepared according to the following recipe: 7.5 g/L agar powder, 50 g/L baker’s yeast, 55 g/L glucose, 35 g/L wheat flour, 2.5% nipagin, 0.4% propionic acid, and 1.0% penicillin/streptomycin. All larvae were dissected at the wandering third instar stage. For experiments with heat-shock–induced clones, vials were transferred to a water bath set to 37°C for 25 minutes on day 3 after egg laying. The vials were then immediately returned to the 25°C incubator and allowed to grow as normal for 3 more days prior to dissection. Only female larvae were dissected.

The following Drosophila melanogaster lines were obtained from the Bloomington Drosophila Resource Center: RpS3{Plac92} (Cat#BL5627), RpS3[*] (Cat#BL5699), en-Gal4, UAS-RFP (Cat#BL30557), en-Gal4 (Cat#BL30564), and RpS17 , arm-LacZ, FRT80B/TM6 (BL6358). Gr64f-RNAi and 40D-UAS lines (Cat#v100156 and KK60101, respectively) were obtained from the Vienna Drosophila Resource Centre. hs-CL1-GFP (ProteoFlux), hs-ref(2)P-GFP (ReFlux) and hs-FLP, UAS-CD8-GFP;; FRT82B, RpS3{Plac92}, act>RpS3>Gal4/TM6b were reported in [19]. The following lines were kindly provided by Hubert Amrein: R1; R2; ΔGr64 (R1 and R2 are constructs rescuing all other genes flanking the Gr64 locus affected by the deletion) and UAS-Gr64abcd-GFP-f [1]. Gr64a, Gr64b, Gr64c, Gr64d1, Gr64e, and Gr64f. Gr64-Gal4/lexA insertions are both expression reporters and validated null mutants [2,25]. The hs-FLP;; FRT82B and y[ lines were provided by Daniel St. Johnston. The hh-Gal4/TM6B and RpS23 were provided by Jean-Paul Vincent [20]. The GstD1-GFP line was described in [27]. The Gr64af deletion was described in [4]. The sqh-GCaMP3 stock was described in [31]. The tub>CD2>Gal4, UAS-CD8GFP; tub-Gal80ts stock was provided by Bruce Edgar. The UAS-Gr64abcd-GFP-f, Gr64af recombinant was generated in this study.

Genotypes for all experimental crosses are provided in Table 1 below.

Table 1

Genotypes for all experimental crosses.

Figure/panel	Genotype
Main figures
Fig 1B	Gr64af/+ (left)Frt82B, RpS3{Plac92}, tub-dsRed/+ (middle)Gr64af/ Frt82B, RpS3{Plac92}, tub-dsRed (right)
Fig 1D	FRT82B, RpS3{Plac92}, hh-Gal4/Gr64af, UAS-Gr64abcd-GFP-f
Fig 1E	FRT82B, RpS3{Plac92}, ubi-GFP (left)FRT82B, RpS3{Plac92}, ubi-GFP/Gr64af (middle)FRT82B, RpS3{Plac92}, ubi-GFP/Gr64af, UAS-Gr64abcd-GFP-f (right)
Fig 1G	UAS-Gr64f-RNAi(KK)/GstD1-GFP; hh-Gal4/+ (left)UAS-Gr64f-RNAi(KK)/GstD1-GFP; FRT82B, RpS3{Plac92}, hh-Gal4/+ (right)
Fig 2A	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/+
Fig 2B	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/ Gr64aGAL4
Fig 2C	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/Gr64bLEXA
Fig 2D	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/ Gr64cLEXA
Fig 2E	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/Gr64d1
Fig 2F	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/Gr64eLEXA
Fig 2G	hs-FLP, UAS-CD8-GFP/+;; RpS3{Plac92}, act>RpS3>Gal4/Gr64fLEXA
Fig 3A	hs-FLP; en-Gal4/UAS-Gr64f-RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
Fig 3C	hs-FLP; en-Gal4, GstD1-GFP/ UAS-Gr64f-RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
Fig 3E	hs-FLP; en-Gal4/ UAS-Gr64f-RNAi(KK);FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
Fig 3G	hs-FLP; en-Gal4/ UAS-Gr64f-RNAi(KK);FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
Fig 4A and 4B	hs-CL1-GFP (ProteoFlux)/(+ or y); enGal4, UAS-RFP/ UAS-Gr64f RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/+
Fig 4E and 4F	hs-ref(2)P-GFP (ReFlux)/(+ or y); enGal4, UAS-RFP/ UAS-Gr64f RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/+
Fig 4G and 4H	hs-FLP; en-Gal4/ UAS-Gr64f-RNAi(KK);FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
Fig 4J	enG4/ UAS-Gr64f-RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/sqhGCaMP3
Figure supplements
S1A Fig	FRT82B, RpS3{Plac92}, ubi-GFP/+ (left)R1/+; R2/+; FRT82B, RpS3{Plac92}, ubi-GFP/ΔGr64 (right)
S1C Fig	R1/+; R2/+; FRT82B, ubi-GFP/ΔGr64 (left)R1/+; R2/+; FRT82B, RpS3{Plac92}, ubi-GFP/ΔGr64 (right)
S1E Fig	w1118 (left)Gr64af/Gr64af (right)
S1G Fig	w;; RpS174, arm-LacZ, FRT80B/+ (left)w;; RpS174, arm-LacZ, FRT80B/Gr64af (right)
S1I Fig	RpS23R67K/+ (left)RpS23R67K/+; Gr64af/Gr64af (right)
S2A Fig	hs-FLP; tub>CD2>Gal4, UAS-CD8GFP/ 40D-UAS; tub-Gal80ts/ FRT82B, RpS3{Plac92}, tub-dsRed
S2B Fig	hs-FLP; tub>CD2>Gal4, UAS-CD8GFP/ UAS-Gr64f-RNAi(KK); tub-Gal80ts/ FRT82B, RpS3{Plac92}, tub-dsRed
S2D Fig	40D-UAS/+; FRT82B, RpS3{Plac92}, hh-Gal4/+
S2E Fig	UAS-Gr64f-RNAi(KK)/+; FRT82B, RpS3{Plac92}, hh-Gal4/+
S2G Fig	40D-UAS/+; FRT82B, RpS3{Plac92}, hh-Gal4/+
S2H Fig	UAS-Gr64f-RNAi(KK)/+; FRT82B, RpS3{Plac92}, hh-Gal4/+
S2J Fig	RpS23 R67K /+
S2K Fig	RpS23 R67K /+; Gr64af/+
S3A Fig	UAS-Gr64f-RNAi(KK)/GstD1-GFP; hh-Gal4/+
S3C Fig	GstD1-GFP/+; FRT82B, RpS3{Plac92}, hh-Gal4/+
S3D Fig	R1/+; R2/GstD1-GFP; FRT82B, RpS3{Plac92}, hh-Gal4/ΔGr64
S4A Fig	hs-FLP; en-Gal4/+; FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
S4C Fig	hs-FLP; en-Gal4/+; FRT82B, RpS3{Plac92}, tub-dsRed/Frt82B
S1 Movie	enG4/ UAS-Gr64f-RNAi(KK); FRT82B, RpS3{Plac92}, tub-dsRed/sqhGCaMP3

Immunofluorescence

Larvae at the wandering third instar stage were washed once and then dissected in PBS before being immediately transferred to a pre-chilled vial of PBS. Samples were then fixed in 4% formaldehyde in PBS at room temperature for 20 minutes. Samples were then washed 3 times in PBS and then permeabilised in 0.25% Triton X-100 in PBS (PBST). The PBST was then aspirated and replaced with blocking buffer (4% fetal calf serum in PBST) and incubated for 30 minutes at room temperature. Primary antibodies were diluted in blocking buffer, and primary incubations took place overnight at 4°C on a rocker. Samples were then washed 3 times in PBST at room temperature for 3 minutes, followed by a 1-hour incubation with secondary antibody diluted 1:500 in blocking buffer along with 0.5μg/mL DAPI. Secondary antibodies used were Alexa-Fluor 488, 555, or 633 (Molecular Probes Eugene, Oregon, USA). Samples were then again washed 3 times for 10 minutes in PBST before being mounted in VECTASHIELD (Vector Laboratories Newark, California, USA) on a borosilicate glass slide (number 1.5, VWR International, Radnor, Pennsylvania, USA).

The antibodies used were Rabbit anti-pJNK pTPpY (1:500, Promega, Madison, Wisconsin, USA, Cat#V93B), Rat anti-Ci(1:1,000, DSHB, Iowa City, Iowa, USA, Cat#2A1), Rabbit anti-Ref(2)P (1:2,000, provided by Tor Erik Rusten [40]), Rabbit anti-cleaved Caspase-3 (1:25,000, Abcam, Cambridge, UK, Cat#13847), Rabbit anti-DCP1 (1:2,500, Cell Signalling, Danvers, Massachusetts, USA, Cat#9578S), Rabbit anti-p-eIF2α (1:500, Cell Signalling, Cat#3398T).

ProteoFlux and ReFlux pulse-chase assays

On day 6 after egg laying, vials containing third instar larvae carrying the ProteoFlux or ReFlux constructs were transferred to a water bath set to 37°C for 40 or 45 minutes, respectively. Larvae were then immediately dissected and transferred to ice cold 4% formaldehyde in PBS to act as a zero time point. The vials were then returned to the 25°C incubator and dissected at 2 and 4 hours after heat shock for ProteoFlux and 3 and 6 hours after heat shock for ReFlux. Larvae were then fixed and mounted as normal.

OPP translation assay

Larvae were washed once and then dissected in pre-warmed Schneider’s medium. Hemi-larvae were then transferred to a 1.5 mL Eppendorf tube containing 5 μM OPP (Molecular Probes, Cat#C10456) diluted in Schneider’s medium and placed in a heating block set to 25°C for 15 minutes. Samples were then washed quickly in PBS and then fixed in 4% formaldehyde in PBS for 20 minutes at room temperature, permeabilised for 30 minutes at room temperature in 0.5% PBST and incubated for 30 minutes in blocking buffer. Samples were then washed in PBS, and staining was performed using the Click-iT Plus protocol according to manufacturer’s instructions.

Calcium live imaging

Larvae were dissected in Schneider’s medium, and wing discs were placed on a custom imaging chamber filled with Schneider’s medium. The chamber was made by attaching a glass coverslip to a steel slide with silicone sealant. The other side of the chamber was covered with an oxygen permeable membrane (YSI Membrane Kit Standard, Yellow Springs, Ohio, USA). Wing discs were then live imaged on a Yokogawa CV7000S microscope using a 20× long working distance 0.45 numerical aperture dry objective. The images taken were XYZT stacks consisting of 3 slices over 6-μm depth, and each stack was imaged every 10 seconds for 10 minutes. The images were manually analysed in Fiji. Flashes in the anterior and posterior compartments were counted and the frequency of flashes normalised to posterior and anterior compartment surface area.

Imaging, quantification, and statistical analysis

All images were acquired as z-stacks with 1 μM z-planes on Leica SP5 and SP8 confocal microscopes using a 40× 1.3 numerical aperture PL Apo Oil objective. All images were quantified using the PECAn image and data analysis pipeline [41]. Statistical analysis was performed using Rstudio and Graphpad Prism 8 software. Specific statistical tests and number of replicates performed for each experiment is provided in the statistical source data sheet (S1 Data). The following workflow was performed: If data met parametric assumptions (normality, homogeneity of variance), a t test or paired t test was used. If these criteria were not met, a Mann–Whitney U test or Wilcoxon signed rank test was used. A minimum of 2 independent biological replicates were performed of each experiment. Logistic regression was performed in PECAn. The dependent variable was the number of viable and nonviable RpS3 cells in the loser clone border, as determined via a staining for cleaved DCP1. Predictor variables were determined by running the model with different, noncollinear variables (as determined by variance inflation factor below 5) and scoring by minimising the Akaike information criterion. A false discovery rate (FDR) correction was used to correct for multiple comparisons.

(Related to Fig 1).

Further characterisation of the Gr64 and RpS3 interaction using additional (A) Wing discs heterozygous mutant for RpS3 without (left panel) or with (right panel) a heterozygous deficiency in the Gr64 locus (ΔGr64) and assessed for cell death with a staining for cleaved-caspase3 (red), along with quantification in (B) (n = 6, n = 7, 2-sided Mann–Whitney U test). (C) Wing discs heterozygous for a deficiency spanning the Gr64 locus (ΔGr64) in a wild type (left panel) or RpS3 background (right panel) and assessed for cell death with a staining for cleaved-caspase3 (red), along with quantification in (D) (n ΔGr64 = 10, nRpS3,ΔGr64 = 11, 2-sided Mann–Whitney U test). (E) Wild type (left panel) or Gr64 homozygous null (right panel) wing discs assessed for cell death via a staining for cleaved-Dcp1 (red), along with quantification in (F) (nwt = 8, nGr64 = 10, 2-sided Mann–Whitney U test). (G) Wing discs heterozygous for RpS17 (left panel) or heterozygous for both RpS17 and Gr64 (right panel) assessed for cell death with a staining for cleaved-Dcp1 (red), along with quantification in (H) (nRps17 = 10, nRps17,Gr64 = 10, 2-sided Mann–Whitney U test). (I) Wing discs heterozygous for RpS23 (left panel) or both heterozygous for RpS23 and homozygous null for Gr64 (Gr64af) (right panel) assessed for cell death with a staining for cleaved-Dcp1 (red), along with quantification in (J) (nRps23 = 10, nRps23,Gr64 = 10, 2-sided Mann–Whitney U test). Horizontal lines indicate the mean in S1B, S1D, S1F and the median in S1H and S1J. Numerical data can be found in the “S1 Fig” sheet of S1 Data. Gr64, Gustatory Receptor 64; Rp, ribosome protein.

(TIF)

Click here for additional data file.

Characterisation of the Rps3 and Gr64 interaction in other imaginal discs. (A–C) Representative images of tubulin flp-out clones (green) in an RpS3 heterozygous background expressing a blank UAS control (A) or Gr64f-RNAi (B), assessed for cell death with a staining for cleaved-Dcp1 (magenta) along with quantification in (C) (nUAS-control = 10, nGr64f-RNAi = 10, 2-sided Mann–Whitney U test). (D–F) RpS3 haltere discs expressing a blank UAS control (D) or Gr64f-RNAi (E) in the posterior compartment with hh-Gal4, stained with anti-Ci (cyan) to label the anterior compartment, and assessed for cell death with a staining for cleaved-Dcp1 (red) along with quantification in (F) (nUAS-control = 9, nGr64f-RNAi = 10, 2-sided Wilcoxon signed rank test). (G–I) RpS3 leg discs expressing a blank UAS control (G) or Gr64f-RNAi (H) in the posterior compartment with hh-Gal4, stained with anti-Ci (cyan) to identify the anterior compartment, and assessed for cell death with a staining for cleaved-Dcp1 (red) along with quantification in (I) (nUAS-control = 13, nGr64f-RNAi = 19, 2-sided Wilcoxon signed rank test). (J, K) Eye discs heterozygous for RpS23 (J) or eye discs heterozygous for both RpS23 and Gr64 (K) assessed for cell death with a staining for cleaved-Dcp1 (red) along with quantification in (L) (nRps23 = 11, nRps23,Gr64 = 13, 2-sided Mann–Whitney U test). Horizontal lines indicate the median in all graphs. Numerical data can be found in the “S2 Fig” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein.

(TIF)

Click here for additional data file.

(Related to Fig 3).

Effect of Gr64 inhibition in wild type and . (A, B) GstD1-GFP reporter expression (green) in wild-type wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment, stained with anti-Ci (grey) to label the anterior compartment, along with quantification in (B) (n = 7, 2-sided t test). (C–E) Wing discs heterozygous mutant for RpS3 without (C) or with (D) a heterozygous deficiency in the Gr64 locus (ΔGr64) and assessed for GstD1-GFP reporter expression (green) along with quantification in (E) (nRpS3 = 6, nRpS3,ΔGr64 = 7, 2-sided Student t test, the horizontal line indicates the mean and the whiskers reflect the 95% confidence interval). Numerical data can be found in the “S3 Fig” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein.

(TIF)

Click here for additional data file.

Negative (no RNAi) controls. (A) RpS3 wing discs carrying the enGal4 driver but expressing no RNAi construct in the posterior compartment and stained for phosphorylated JNK (green) with quantification in (B) (n = 11, 2-sided paired t test). (C) RpS3 wing discs carrying the enGal4 driver but expressing no RNAi in the posterior compartment and stained for phosphorylated eIF2α (green) along with quantification in (D) (n = 9, 2-sided paired t test). Numerical data can be found in the “S4 Fig” sheet of S1 Data. RNAi, RNA interference.

(TIF)

Click here for additional data file.

(Related to Fig 4).

Effect of Gr64 on calcium signalling. Wing discs heterozygous for RpS3, expressing the calcium reporter sqh-GCaMP3 ubiquitously and Gr64f-RNAi in the posterior compartment with enGal4. Gr64, Gustatory Receptor 64; RNAi, RNA interference.

(AVI)

Click here for additional data file.

Supporting information file.

Numerical data for all quantifications in the manuscript are organised per figure, where all quantifications relevant to a given figure are arranged in a separate sheet. For every experiment, repeat quantifications and statistical tests performed are included.

(XLSX)

Click here for additional data file.

8 Sep 2021

Submitted filename: Gr64 revision plan.docx

Click here for additional data file.

23 Sep 2021

Dear Dr Piddini,

Thank you for submitting your manuscript entitled "Ribosome protein mutant cells rely on the GR64 cluster of gustatory receptors for survival and proteostasis in Drosophila" for consideration as a Short Report by PLOS Biology.

Your manuscript, reviews from eLife, and revision plan have now been evaluated by the PLOS Biology editorial staff as well as by an Academic Editor with relevant expertise and I am writing to let you know that we would like to consider a revised version of your manuscript that addresses the reviewer comments from eLife.

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25 Sep 2021

Submitted filename: Gr64 revision plan.docx

Click here for additional data file.

27 Sep 2021

Dear Dr Piddini,

Thank you for submitting your manuscript "Ribosome protein mutant cells rely on the GR64 cluster of gustatory receptors for survival and proteostasis in Drosophila" for consideration as a Discovery Report at PLOS Biology. As mentioned in our last email, your manuscript, the reviews from eLife, and your revision plan have been evaluated by the PLOS Biology editors and an Academic Editor with relevant expertise.

In light of the reviews from eLife, which I have appended below, we will not be able to accept the current version of the manuscript. However, we would welcome re-submission of a much-revised version that takes into account the reviewers' comments as outlined in your submission. Having discussed your revision plan with the Academic Editor, we think that it would be particularly important for your revised manuscript to provide data from the homozygous Gr mutants, plus the rescue experiments. Given that the core of the claims rely on genetic interactions, we think that such experiments would strengthen our confidence in the provocative claims reported here.

Additionally, after discussion within the team, we think that the framing of the study might be shifted a bit to be better suited for our Discovery Report format. If you agree, we would suggest the emphasis of the title and manuscript be shifted slightly to highlight the novel finding that GR64 cluster of gustatory receptors regulate proteotoxic stress and survival of non-neuronal cells, instead of presenting the findings as a modifier of the ribosome mutant protein stress phenotype.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers. We will do our best to engage with the same reviewers that initially reviewed the paper at eLife, however this is not always possible, as it depends on the reviewer availability. However, if we deviate from this commitment for external circumstances, we will let you know.

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*****************************************************

REVIEWS FROM eLife:

Reviewer #1 (Public Review):

This paper investigates mechanisms that modulate the phenotype of cells that are heterozygous for ribosomal protein genes (Rp/+). The previously described effect of the Rp/+ or minute genotype in completely heterozygous animals is delayed development and increased levels of proteostasis stress, which correlates with decreased autophagy and proteasome activity. In a clonal situation where Rp/+ cells are juxtaposed to wild type cells, they get eliminated by cell competition and apoptosis.

Based on results of transcriptomic analyses, the authors found that six clustered tasteGr64 receptor genes are more highly expressed in Rp/+ than in wild type cells. In loss of function experiments they show that heterozygous loss of the Gr64 cluster and even loss of heterozygosity for any one of these six genes or RNAi mediated knock down aggravates the stress response in Rp/+ cells and promotes apoptosis when these cells are in competition with neighboring wild type cells. Reporter experiments and immunostaining indicates that the Gr64, Rp double heterozygous cells experience higher levels of oxidative and unfolded protein stress. Heterozygosity or even complete loss Gr64 genes does not cause apoptosis of elevated stress markers in a wild type background.

This paper leaves a lot of questions open: What is the explanation of individual loss of function alleles for all six Gr64 receptors having the same dominant phenotype in an Rp/+ background? What is the molecular function of these receptors that modulates to the minute phenotype? Are any ligands involved? If so, which?

Reviewer #1 (Recommendations for the authors):

Figure 1 and supplemental Fig 1: Why do you show the experiment with the larger deficiency that requires complicated complementation with rescue constructs when you have a precise CRISPR deletion?

Figure 2a: why is no apoptosis detectable at the borders between WT and RpS3/+

clones?

Some minor issues that should be addressed in the paper:

Line 108: "ΔGr64/ΔGr64 flies present with no known phenotypes other than a deficient gustatory responses" should be "than a deficient gustatory response" or "than deficient gustatory responses")

Line 129: "heterozygousity" should be "heterozygosity"

It would be helpful if the figures and supplemental figure were identified by the numbers that refer to them in the text.

Label the genotypes in the figures better, which drivers are used etc.

Fig 1E,F. It is not clear why the paper suggests that knockdown in Hh-expression domain which span most of a parasegment shows a cell-autonomous requirement for Gr64. In fact, there are Caspase positive cells outside the Hh-expression domain. Clonal knock down for example by MARCM and precise analysis at the clonal boundaries would be the accepted method to test cell autonomy.

Reviewer #2 (Public Review):

Baumgartner et al. follow up a curious observation they made previously (Kucinski et al. 2017) , in which they noticed that Drosophila larvae heterozygous for the ribosomal protein Rsp3 show upregulation of sugar taste receptor genes of the Gr64 subfamily (Gr64a-Gr64f). They test a functional involvement of these receptors by creating double

heterozygous Rsp3+/-, Gr64a-f+/-, larvae and analyzing imaginal discs for evidence of increased proteotoxic stress/apoptosis compared to Rsp3+/- imaginal discs and find the former have indeed an increase in expression of caspase3 and DCP1, stress/cell death markers. In a slightly modified experiment (Figure 2), they investigate whether this phenomenon is also seen in discs in which winner (Rsp3+/+) and loser cells Rsp3+/-) are placed adjacent to each other, finding that small groups of DCP1 expressing cells are indeed more frequent at the interface of winner/loser cells in heterozygous Gr64 discs, compared to Gr64+/+ discs. They develop a more nuanced assay, in which they downregulate Gr64s via RNAi, and by expressing this construct in the posterior part of the disc. In this assay, they observe a comparatively milder effect on apoptosis, assayed by several different markers. Employing this more sensitive assay, they explore whether Gr64a contributes to protein aggregation using two reporters that represent proteolytic pathways and poly-ubiquitinylated aggregates.

Reviewer #2 (Recommendations for the authors):

Strengths:

1) A non-canonical role for taste receptors in ribosome biogenesis is a very interesting finding. While no experiments to address the mechanism are presented, the observation presented here open up new lines of investigations.

2) The experiments are well-designed, and appropriate controls are included.

3) The paper is well-written and easy to digest.

Weaknesses:

1) The authors should extend the analysis further, including phenotype assessment of the minute phenotype in hemi- and homozygous Gr64 mutant that are Rsp3+/-. Likewise, why did they never use homozygous Gr64 mutants in any of their experiments? The effects on the observed phenotype would be expected to be even stronger.

2) Interpretation of the data in Figure 2 is difficult. These mutations are mostly gene knock-ins into a locus that is transcribed from a polycistronic message, so any of these

mutations can affect levels of expression of the entire locus. While these knock-ins mostly the purpose to reveal cellular expression but using them as gene knock-outs is fraught with many potential caveats.

3) There are no "rescue" experiments in this paper. It appears that it would be very

valuable to see if the observed phenotypes (Capsase3, DCP1 expression) incurred by heterozygosity of the Gr64 locus can be rescued by expression of individual G64 genes.

4) The authors' previous data indicate that upregulation of Gr64 genes in Rsp3+/- (and

other heterozygous mutants of genes involved in ribosome biogenesis) larvae might be a counter measure by increasing intracellular Ca2+. They do not provide an opinion whether this would be a ligand dependent or independent process. A simple test for

that would be a rescue type of experiments with other Gr genes (see point 4) not related

to sugar receptors)

Minor points:

1) nomenclature for Gr64 mutants is not accurate and confusing (Gr64a-GAL4, Gr64b- LexA, etc). They should refer to the original nomenclature used by Fujii et al. (Gr64aGAL4 etc)

2) The authors initiate the investigation on a possible role of Gr64 genes based on differential expression (i.e. upregulation) of these genes in Rsp3+/- and other mutants. This was a very restricted analysis (wing imaginal disc). Is this phenomenon observed in other discs/tissues that form adult structures?

Reviewer #3 (Public Review):

I found this paper quite satisfying, because it is an excellent example of "following the science" to discover something new and totally unexpected about the way cells work. I thought the story was compelling, the presentation was clear and well written, and that the impact will be significant.

The authors followed up on observations made in a previous paper (Kucinski et al.,

2017), where they compared gene expression in wild type flies versus flies heterozygous for a mutation in a ribosomal protein gene or another gene, mahjong,

associated with proteotoxic stress. The analysis resulted in a long list of differentially expressed genes, some of which they explored in that paper. Here, they explore the reasons for the unusual upregulation of a family of gustatory receptors. Rather than representing a meaningless case of gene misregulation, they found that increased expression of these receptors is an adaptive response to proteotoxic stress. In this way, they implicated the receptors in regulating pathways that no one had suspected them of regulating.

I have no criticisms of the authors' choice of experiments, the methods they used, their interpretations or their presentation. The major strength of this paper is the soundness of the methods and results. It is an exceptionally "clean" and well-constructed report.

For me, the significance of the work relates to the long history of ribosomal protein gene haploinsufficiency in Drosophila and the increasing importance of Drosophila to understanding cellular responses to disrupted protein synthesis. A relatively short time ago, explanations for the Minute syndrome-the phenotypes seen in Drosophila when

one copy of a ribosomal protein gene is eliminated-were speculative. The syndrome was considered an esoteric "fly thing". Over the past decade or so, the phenotypes have been

tied to an assortment of cell stresses. A major challenge now is sorting through all the ways ribosome dysfunction affects cellular pathways. It is a complicated situation, but it mirrors cellular defects in human ribosomopathies and other diseases affecting protein synthesis. This paper is an excellent example of the relevance, importance and productivity of such studies: a new cellular pathway linking gustatory receptors to cellular stress responses has now been discovered from its contribution to the Minute syndrome.

Reviewer #3 (Recommendations for the authors):

After reading this paper, one is left with the impression that what you describe for reduce RpS3 dosage is universal to reduced dosage for all Rp genes. I would like to have seen some statement that interactions between RpS3 and the Gr64 genes might be RpS3 specific. Better yet, I would like to have seen at least some experimental evidence showing the same interactions with another Rp gene. While I understand why you think the effects are probably not RpS3 specific, I urge to be hesitant and modest in generalizing. I recommend including appropriate caveats in the text.

The text is very well written, but I had a hard time understanding the section on "flux" beginning with line 156. "Flux" is not a word that is universally understood. I had to go back to your previous paper to figure out what you were talking about and to understand the purposes of your ProteoFlux and ReFlux tools. For ease of reading, I strongly recommend that you use more familiar phrases such as "import anddegradation", "elimination", etc. and provide better descriptions of the ProteoFlux and

ReFlux tools.

Because an asterisk is used to denote an unknown allele, I wanted to know why you used RpS3[*] in the allele designation in the experiments associated with Figure 1, but I could find no description of the origin of the allele. From context, I suspect it came from Bloomington stock 5699. Please include a note about its origin somewhere (or, better yet, sequence it and the known alleles and identify it definitively).

On line 66, you meant to use "proximate", not "proximal".

You should use FlyBase nomenclature for the genetic elements in your experiments, especially transgenes, at least once so that readers will not have to guess the correspondences between your lab notations and the carefully curated FlyBase entries describing them. Shortened symbols are OK for the text, but standardized symbols should be included in the Materials and Methods. This should be done for the sake of accuracy and reproducibility and to show that you value the efforts of the people who work hard organizing and archiving genetic information for you. Along the same lines, you should acknowledge research resources such as FlyBase that have enabled your work by explicitly citing their grants.

5 Apr 2022

Submitted filename: Baumgartner et al, rebuttal 4.docx

Click here for additional data file.

10 May 2022

Dear Dr Piddini,

Thank you for your patience while we considered your revised manuscript "The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila" for publication as a Discovery Report at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and two of the original reviewers.

The reviews are appended below. As you will see, both reviewers think the findings reported here are interesting and Reviewer 3 is fully satisfied by the revision. However, Reviewer 1 has highlighted a few lingering concerns, including that the study does not fully elucidate the functional basis for the phenomena reported here. Reviewer 1 also highlights that some of the conclusions should be toned down.

Having discussed Reviewer 1’s concerns with the Academic Editor, we think that additional mechanistic studies would be interesting, but are beyond the scope of the current study, which we think fits the criteria for our Discovery Report format. Therefore, we would not require additional experimental data for publication at PLOS Biology. However, before we can accept your study we think it would be important for you to thoroughly address the other concerns raised by Reviewer 1 by toning down claims regarding cell autonomy in the abstract and manuscript and by adding further discussion of caveats, limitations, and future directions.

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Lucas Smith, Ph.D.,

Associate Editor,

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PLOS Biology

REVIEWER COMMENTS

Reviewer #1: Dirk Bohmann (note this reviewer has signed his review)

Review of PBIOLOGY-D-21-02398R2

Baumgartner et al.

This paper describes a previously unrecognized phenotype of Drosophila mutants that are deficient for a certain group of taste receptors. Six Gr64 receptors are expressed from a polycistronic mRNA. Flies that are heterozygous for mutations in one of more of these 6 clustered genes display increased levels of oxidative stress and cell death in tissues that are also minute, i.e. heterozygous for ribosomal protein (rp) gene loss of function mutations. This combined rp+/-, Gr64+/- phenotype is observed regardless of whether the double heterozygous genotype is shared by all cells in the organism or whether the mutants are in a competitive situation next to wild type cells, in which case cell competition is expected.

All three reviewers of the first submission of this manuscript agree that the phenomenon described in is interesting and unexpected. It may give rise to new lines of research that may help to better understand the intriguing biology of minute mutants.

The major concern that reviewer 2 and I pointed out was that the paper describes in interesting observation but does not go very far in providing insight into the functional basis for the unexpected interplay between smell receptors and minute mutants. In the revised version one experiment is added which indicates that in rp/+ imaginal discs cells in which Gr64 expression is knocked down calcium signaling is reduced. Demonstration of a connection between this effect and the observed phenotype cell stress phenotype would require addional experiments. At this stage such a connection is a matter of speculation. So, the question of novelty and general interest remains.

The specific issues that I raised in my previous review included:

It is difficult to interpret the data presented in figure 2 where individual insertion mutants in the each of the six Gr64 receptors are tested for their effect on rp/+ dependent cell death. Reviewer 2 expressed a similar concern. The authors point out, correctly, that it is difficult to know whether mutations in the coding regions of one of the six genes would only affect the expression that particular gene product or also that of some or all of its neighbors. In the end I am still confused about how to interpret this result, beyond: these are six more independent alleles that can be presumed to affect Gr64 function in a somewhat undefined way that support the conclusion of the previous picture.

I also wondered how clear it is that the effect of the rp+/-, Gr64+/- genotype is cell autonomous. There is clearly more cell death in the tissue regions in which Gr64 expression is reduced. But there is also some in adjacent areas. The newly added experiments looking at haltere and leg imaginal discs do not clarify this question. The conclusion of cell autonomy should be qualified.

The other issues I pointed out were mostly suggestions to improve the paper.

Reviewer 2 has some additional technical comments, several of which were satisfactorily addressed:

* Test minute mutations other than Rps3

This is satisfactorily addressed by showing experiments on RpS17 and Rps23 (revised Fig S1)

* Test the interaction of minutes with a Gr64 homozygous loss of function genotype.

This was done, in an experiment with Rps23 (revised Fig S1). The presented results are consistent with the author's model.

* Do a rescue experiment, where expression Gr64 receptors would revert the mutant phenotype.

This was done using a UAS construct that has enough expression without a Gal4 driver. The presented results support the authors conclusion and are responsive to reviewer 2's query.

In conclusion

The revision provides additional lines of experimental evidence in support of the proposed role of Gr64 receptors in the minute phenotype and cell stress responses. It does however not offer more conclusive functional insight into the mechanism underlying this phenomenon.

Reviewer #3: I was asked to review this revised manuscript (The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila) for PLoS Biology after reviewing the original version for eLife. I was Reviewer 3 in the eLife reviews and wrote generally positive comments with only minor suggestions for revision. I was impressed by the original manuscript and see that the revisions in this version have only strengthened the presentation. In my opinion, you have adequately addressed the concerns presented by the eLife reviewers. You have certainly addressed the relatively minor concerns that I had.

To me, this manuscript fulfills all the requirements for publication as a Discovery Article. It presents a significant discovery that will be interesting to a wide range of scientists. It will be particularly interesting to researchers interested in ribosomopathies and cellular stress. The results are novel and they are presented and analyzed with appropriate rigor and clarity. The manuscript is well written and pleasant to read. I see no need for additional experiments or significant revisions.

I am personally intrigued by this line of research. I think the connection you have made between ribosome dysfunction and gustatory receptor signaling is quite interesting. I am impressed that you followed up on your preliminary observations regarding the Gr64 genes in Minute mutants and found a new cellular role for this receptor gene family.

27 May 2022

Submitted filename: Baumgartner et al, rebuttal .pdf

Click here for additional data file.

2 Jun 2022

Dear Dr Piddini,

Thank you again for your submitting a revised version of your manuscript "The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila" for publication as a Discovery Report at PLOS Biology. Your revision has been evaluated by the PLOS Biology editors and the Academic Editor, and we are largely satisfied by the changes made in response to the reviewers and to our previous editorial requests. However, in looking through the most recent revision, the Academic Editor has noticed two issues which we think should be addressed at this stage in order to make sure the manuscript is as solid as possible.

Therefore, before we can accept your study, we request that you address the following two comments from the Academic Editor in another revised manuscript.

COMMENTS FROM THE ACADEMIC EDITOR

1) Regarding my first comment on the previous version about the values in Figure 1A: I was mistaken in stating the "values are actually "log FC"". I meant to write in the Kucinski 2017 study Supp Table 1, the values are "Fold change" (i.e. neither log10 nor log2). The authors should double check this and correct.

2) The phenomenon of calcium flashes (Figure 4J) is intriguing as a potential physiological link between Gr64s and downstream cellular events, but as such flashes have not been described in the literature before - as far as I can see - it would be good to provide some context as to what the authors think they represent (from the video they seem to occur somewhat randomly in different cells/groups of cells in the disc). In addition, could the authors confirm that the small, but significant A-P asymmetry in frequency observed is really due to the Gr64 RNAi in the P compartment and not reflecting an inherent difference in calcium flash frequency in these two different regions of the disc (i.e. without any Gr64 RNAi)? If they do not have such data, they should at least mention this caveat.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

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NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

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*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

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*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Luke

Lucas Smith, Ph.D.

Associate Editor,

lsmith@plos.org,

PLOS Biology

11 Jun 2022

Submitted filename: Baumgartner et al, rebuttal.pdf

Click here for additional data file.

14 Jun 2022

Dear Dr Piddini,

Thank you for the submission of your revised Discovery Report "The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Richard Benton, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

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Sincerely,

Lucas Smith, Ph.D.

Associate Editor

PLOS Biology

lsmith@plos.org