Fur4‐mediated uracil‐scavenging to screen for surface protein regulators

Abstract Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil‐scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil‐scavenging.


| INTRODUCTION
Cell surface membrane proteins are regulated by a variety of overlapping and often co-regulated membrane trafficking pathways.
Surface cargoes are co-translationally imported into the endoplasmic reticulum (ER) 1 before transiting the secretory pathway to the plasma membrane (PM). 2 Surface proteins are internalized via clathrinmediated endocytosis, followed by recycling back to the PM or entering the lysosomal degradation pathway. 3 These pathways allow surface localization and activity of myriad proteins to be precisely controlled to meet cellular demands, for example, during the cell cycle or in response to reduced nutrient availability. However, these pathways remain incompletely characterized. The budding yeast system has been useful to discover and define membrane trafficking mechanisms. Yeast cells uptake nutrients from their external environment by various transporters that localize to the PM, such as transporters for sugars, metal ions and vitamins. [4][5][6] Amino acids and nucleobases are also actively transported into yeast cells via permeases, for example, Gap1 broadly uptakes amino acids, 7 Mup1 uptakes methionine 8 and Fur4 uptakes uracil. 9 Permease activity can be controlled by changes in transporter expression, the rate of turnover by ubiquitin-mediated vacuolar degradation, in addition to spatiotemporal control between eisosomes and other regions of the PM. [10][11][12][13][14] To develop an assay that reports on nutrient uptake via surface transporters, we focussed on the uracil permease Fur4, 9 which is controlled by all the abovedescribed trafficking pathways and regulatory mechanisms. For example, the presence of uracil downregulates expression of FUR4 15,16 while also triggering endocytosis and Rsp5-mediated ubiquitination and degradation of Fur4. 17,18 Furthermore, Fur4 activity is regulated in response to metabolic stress via storage in eisosomes. [19][20][21] Beyond this, Fur4-mediated uptake of uracil might be considered particularly important for many lab strains that cannot synthesize uracil biosynthetically, because of disruption of the orotidine-5 0 -phosphate decarboxylase URA3 gene (eg, ura3-52 or ura3Δ), which causes the useful selection characteristics of auxotrophy and resistance to 5-fluroorotic acid. 22 Indeed, many genome-wide libraries [23][24][25][26][27][28][29] have been created from parental ura3Δ strains. 30 We therefore chose Fur4-mediated uptake to develop a simple and cost-effective growth assay that indirectly reports on the surface-mediated uptake of uracil by the Fur4 transporter, which we used to screen a haploid library of deletion mutants for factors that regulate Fur4 trafficking.

| A Fur4-activity-based growth assay
The uracil permease Fur4 is dispensable for growth in rich media but is critically required when uracil-auxotroph cells are grown in synthetic defined media containing replete (4 mg/L) uracil ( Figure 1A-C).
Importantly, the robust Fur4-dependent growth of BY4742 cells, which harbour a ura3Δ mutation, 30 herein referred to as wild type, corresponds to the concentration of available uracil. There is a significantly reduced rate growth when wild-type cells are grown in media containing 0.1 mg/L uracil, but Fur4-dependent uracil-scavenging supports growth ( Figure 1B,C). Fur4 localization has been previously shown to respond to extracellular uracil, 16 and we confirm steady state surface localization of Fur4 tagged with mNeonGreen (mNG) is redistributed to FM4-64 stained vacuoles following 1-hour of uracil addition to the media ( Figure 1D). Collectively these results show that the activity of the uracil-sensitive permease Fur4 correlates with cellular growth in limited uracil conditions. To test if low-uracil-specific growth could be used to screen for membrane trafficking factors that influence Fur4 surface levels, we next compared Fur4-mNG localization in wild-type cells and mutants that mislocalize Fur4 ( Figure 2A). As expected, a temperaturesensitive sec7-1 allele, which disrupts activity of the Sec7 Arfexchange factor required for transit through the Golgi, 31,32 inhibits trafficking of Fur4 through the secretory pathway, with Fur4-mNG accumulating in intracellular puncta instead of the PM. Fur4-mNG localization is also affected in did4Δ (vps2Δ) ESCRT mutants, 33 that do not permit vacuolar sorting and Fur4-mNG instead accumulates in endosomes. However, uracil-scavenging is likely efficient in did4Δ cells as significant Fur4-mNG recycling back to the PM is observed, unlike Mup1-GFP that is trapped by Snf7-oligomers. 34 In contrast, surface levels of Fur4-mNG was greatly reduced in both rcy1Δ or nhx1Δ mutants (Figure 2A), which lack factors required for endosomal recycling. [35][36][37] Therefore, we compared growth of rcy1Δ or nhx1Δ mutants with wild-type cells on plates of varying uracil concentrations. There was no statistically significant difference in growth between wild-type and trafficking mutant cells in the range of 1 to 32 mg/L uracil, but at lower uracil concentrations the rcy1Δ and nhx1Δ mutants, which have reduced surface Fur4, exhibit a low-uracil-specific growth defect (Figures 2B,C and S1a, Supporting Information). Although significant defects were observed at 0.5 and 0.25 mg/L uracil, we selected 0.1 and 0.05 mg/L uracil for scavenging conditions, as wild-type cells grow efficiently but rcy1Δ or F I G U R E 1 Low-uracil growth relies on the Fur4 transporter. (A) Schematic illustration showing uracil-auxotroph cells (ura3Δ) with (left) and without (right) the uracil permease Fur4. (B) Indicated yeast strains were grown to mid-log phase before plating on rich (YPD) and synthetic complete (SC) media containing either 4 or 0.1 mg/L uracil. (C) Quantification of yeast growth from (B), asterisks (*) indicate Student t test comparisons P = <.001. (D) Wild-type cells expressing Fur4-mNeonGreen (Fur4-mNG) from its endogenous promoter were labelled with FM4-64 for 1-hour, grown to mid-log phase in SC-Ura media (upper) or in the presence of 40 μg/mL uracil for 1 hour (lower) prior to confocal microscopy. Arrows indicated plasma membrane (PM), scale bar = 5 μm nhx1Δ both show dramatically reduced growth. Low-uracil-specific growth defects were not observed in did4Δ cells, demonstrating the PM levels observed by microscopy are sufficient to scavenge uracil, or sec7-1 cells at permissive temperature, but lethality at 37 C was confirmed ( Figure S1b). We tested this concept with another surface-localized nutrient transporter, the methionine transporter Mup1 grown in methionine auxotroph (met15Δ) cells but no methionine concentration that supports growth could distinguish trafficking mutants ( Figure S2). We assume differences in steady state surface levels, substrate affinity and uptake pathways 8 account for this.

| A genome-wide screen for uracil-scavenging mutants
We hypothesized that mutants with growth similar to wild-type cells in replete uracil but differences specifically at low uracil could be used to identify mutants from a nonessential haploid deletion library 28 with perturbed surface levels of Fur4 ( Figure 2D). Cultured yeast strains representing 5132 different mutants were diluted and spotted out (16 replicates of each) on to solid agar media containing replete (4 mg/L) and limited (0.1 and 0.05 mg/L) uracil concentrations ( Figure 3A). As expected, mutants with growth differences in uracil-F I G U R E 2 Surface localization of Fur4 is required for growth in low uracil. (A) Vacuoles from indicated strains expressing Fur4-mNeonGreen (mNG) were labelled with FM4-64 prior to confocal imaging. (B) Wild-type, rcy1Δ and nhx1Δ cells grown to mid-log phase were then spotted in a 1 in 10 serial dilution onto plates titrated with indicated concentrations of uracil and grown at 30 C for 3 days. (C) Growth of strains from (B) were quantified, asterisks (*) indicate Student t test comparisons of mutants with wild-type cells, P = <.005. (D) Schematic diagrams showing the predicted effects on Fur4 following different trafficking pathway perturbations. Scale bar = 5 μm replete media were observed, but the screen was specifically focused on differences in growth between high and low uracil concentrations. This is exemplified by the mutants used to calibrate the assay, rcy1Δ and nhx1Δ, which both exhibit growth defects specifically in low uracil when compared with neighbouring mutants ( Figure 3B). A low stringency scoring system was used to identify 208 null mutants for follow-up analysis. Candidates were grown to mid-log phase then serially diluted and spotted on 4, 0.1 and 0.05 mg/L uracil media.
Growth was quantified for the 208 mutant strains compared to a wild-type control from the same plate, and then values were used to compare growth across uracil concentrations (Table S1). Statistical comparisons of mutants from these optimized growth assays revealed 58 mutants, such as cla4Δ ( Figure 3C), that did not show a significant difference in growth compared to wild type. However, 126 mutants with significant growth defects specifically in uracil-scavenging conditions were identified ( Figure 3D), ranging from relatively subtle defects (eg, vps74Δ at 0.1 mg/L = 0.78 ± 0.04) to extreme (eg, vps3Δ at 0.1 mg/L = 0.004 ± 0.003). Furthermore, although the assay was calibrated for mutants with defective growth in low uracil, the screen identified 24 mutants with enhanced growth compared to wild type.
We note many of these strains exhibit growth defects in uracil replete media, such as ipk1Δ ( Figure 3C), allowing for benefits to be observed at low uracil (Table S1).

| Screen enriched for molecular complexes
A comparative orthologue search 38 of uracil-scavenging mutants identified 94 highly conserved genes, corresponding to 267 human orthologues associated with 87 distinct diseases (Table S2). Gene Ontology (GO) Slim terms were obtained 39 and revealed a number of cellular component enrichments of molecular complexes ( Figure S3), including the GET complex and the prefoldin complex ( Figure 4A). The identification of multiple complex members suggests the screen was stringent and robust. For example, deletion of GET1, GET2, or GET3 results in low-uracil growth defects ( Figure 4B). As the GET complex is required for sorting of tail-anchored single-pass membrane proteins, 40 many of which are essential factors in secretory pathway trafficking, such as SNARE proteins, 41 we presume the role of the GET complex in efficient surface trafficking of Fur4 is via an indirect membrane trafficking mechanism. Various prefoldin complex members were also identified as having defective growth specifically on low uracil ( Figure 4C), implicating it as a potential regulator of Fur4 trafficking. This could be explained by prefoldin-mediated assembly of cytoskeleton proteins. 42,43 Indeed, actin filament structures observed in wild-type cells are absent in prefoldin mutant cells pfd1Δ and pac10Δ ( Figure 4D), indicating impaired cytoskeletal function that could adversely affect correct trafficking of Fur4 to the surface. However, the prefoldin complex is also involved in transcriptional elongation, 44 so its role in uracil-scavenging could be less direct.

| Screen enriched for trafficking and transcriptional machinery
GO enrichments for biological process revealed almost a third (44/ 150) of annotations were for machinery associated with membrane trafficking and signalling ( Figure 5A and Table S3), including the blind identification of rcy1Δ and nhx1Δ mutants that were used to calibrate the assay (Figure 2). The other biological process significantly enriched F I G U R E 3 A genetic screen for mutants that affect uracilscavenging. (A) Yeast strains grown overnight in 96-well plates were diluted 20-fold in water then replicated 16 times onto solid agar plates containing varying uracil concentrations. An example 4 mg/L uracil plate shows identifier wells (yellow), alongside strains with defective (white) and accelerated (blue) growth. (B) Example of screen data showing indicated mutants grown on media containing 4, 0.1 and 0.05 mg/L uracil, including known Fur4 trafficking mutants (red).
(C) The screen identified 208 candidates that were subsequently grown to mid-log phase and spotted out over a 6-step, 10-fold serial dilution onto high (4 mg/L) and lower (0.1 and 0.05 mg/L) uracil containing media. Uracil-related cellular growth relative to wild-type was quantified and categorized as defective (eg, pmp2Δ), advantageous (eg, ipk1Δ) or not significantly altered (eg, cla4Δ). (D) Ratio of relative growth between 4 and 0.1 mg/L uracil from (C) was plotted for all candidates was transcription, including three prefoldin subunit annotations ( Figure 5A and Table S3). Physically interacting transcription factors were identified ( Figure S3), such as members of the Complex Proteins Associated with Set1 (COMPASS) complex, Swd1, Swd3 and Sdc1 45 alongside the Swi3/Snf5 pair 46 among others that gave significant defects in low uracil ( Figure 5B). We reasoned transcriptional regulators could be indirectly involved in Fur4 membrane trafficking, controlling gene expression of either essential genes not tested in the primary screen or mutants identified from the screen itself. To explore transcription factors (TFs) implicated in uracil-scavenging, we assembled genome-wide expression datasets for wild-type cells vs 28 TF-null mutants from a large-scale microarray analyses. 47 A matrix of all mutants showed high correlation of associated factors, such as known complex members ( Figure S4). Cross-referencing expression data for 1183 genes that are essential for viability (Table S4), followed by hierarchical clustering was used to generate a heat map of related gene expression changes ( Figure 5C). Strains, such as ies2Δ, bre1Δ and elf1Δ created distinct expression signatures, but others were similar, such as each of the COMPASS complex mutants swd1Δ, swd3Δ and sdc1Δ. Particularly modulated clusters of genes were identified from this analysis (Table S5), including genes associated with membrane trafficking that we chose for experimental testing (Pma1, Gpi8, Mrs6, Gpi12 and Sec62). To achieve this, we performed uracil-scavenging assays with strains containing Decreased Abundance by mRNA Perturbation (DAmP) cassettes at the 3 0 UTR of each candidate. 48 This analysis revealed sec62-DAmP cells, which have very low protein levels of Sec62, 49 have specific growth defects in low uracil ( Figure 5D).

| Uncharacterized factors are controlled at the transcriptional level
The uracil-scavenging screen identified 10 uncharacterized candidates ( Figure 6A). In an effort to understand whether these were also regulated by the TFs from the screen, we used a similar approach to crossreference gene expression profiles of the TF-deletion strains against the 150 genes identified in the screen. Again, hierarchical clustering revealed many gene profiles share signatures across different TF deletion experiments ( Figure 6B). We were particularly intrigued by ydr222wΔ mutants defective in growth from the screen ( Figure 6A) as YDR222W was greatly downregulated in many of the transcription factor null mutants, including the swi3Δ, spt3Δ and COMPASS complex mutants, but upregulated in other mutants not associated with low-uracil growth (Figures 6C and S5). We find ydr222wΔ mutants ing factor protease (bar1-1). Alpha factor is synthesized and subjected to post-translational modifications through the secretory pathway before being secreted. 53 We hypothesized that mutants with defects in the secretory pathway leading to reduced alpha factor secretion would result in a reduction in the growth arrest response seen when Matα cells are spotted onto a lawn of MatA cells ( Figure 6E).
Conversely, mutants with no halo phenotype could act downstream of the PM, or indirectly, to explain uracil-scavenging phenotypes.
We observed mutants to have a reduction in halo size (eg, bud16Δ) or a halo comparable to WT cells (eg, adk1Δ). Mutants of proteins well characterized to act in the early stages of the secretory pathway such as GET-complex nulls (discussed above) and apl2Δ 40,54 were seen to display a significantly reduced halo phenotype. While mutants of factors acting downstream of the secretory pathway in the endosomal system such as rcy1Δ and nhx1Δ 35,37 were seen to display a halo phenotype not significantly different from WT ( Figure 6F). Interestingly, uncharacterized mutant ydr222wΔ gave a halo type not significantly different to WT cells. It might be the role of Ydr222w, which shares homology to the Svf1 protein implicated in survival pathways, 55 functions at endosomes to regulate nutrient transporters in response to changes in extracellular nutrients.

| Inducible tools to study surface proteins
In addition to fluorescently labelled Fur4-mNG, and other surface cargoes affected by mutants from the screen, like Mup1-GFP and the G-protein coupled receptor Ste3 tagged with mCherry ( Figure S6), for localization experiments to validate trafficking mutants, we created GFP tagged versions of both full length Fur4, or a mutant lacking its N-terminal 60 residues under copper inducible CUP1 promoter, to F I G U R E 7 Legend on next page. allow temporal control of trafficking ( Figure 7A-D). We reasoned Fur4 ΔN -GFP would be a suitable reporter for secretory pathway mutants, as it cannot be endocytosed, 18 however localization in get1Δ, get2Δ and get3Δ was indistinguishable from wild-type cells ( Figure 7E). This might suggest the GET complex exhibits a distinct function in uracil-uptake. However, given the GET complex is known to be involved in secretory trafficking 40 and in addition to all three members being identified from a blind screen for uracil-scavenging, get1Δ get2Δ and get3Δ cells were also all defective in secretion of mating factor (0.53 ± 0.28, 0.80 ± 0.15 and 0.76 ± 0.24, respectively). Therefore, we favour the explanation that the uracil-scavenging assay is sensitive enough to reveal a phenotype that is not apparent from steady state localization experiments. We did observe small amounts of signal at the ER of the truncated Fur4 ΔN -GFP. To confirm that the peripheral Fur4 ΔN -GFP signal was indeed the PM, and not the cortical ER, we imaged the ER marker Sec63-RFP and GFP labelled versions of nicotinic acid permease Tna1, which traffics through the secretory pathway when tagged at the N-terminus ( Figure S7) but is retained in the ER when tagged at the C-terminus. 26 Unlike these ER proteins, Fur4 ΔN -GFP signal is contiguous at the surface and localizes to eisosomes ( Figure 7F), as previously documented. 21 Furthermore, perinuclear signal of Fur4 ΔN -GFP is very weak compared to the peripheral signal, unlike the ER localised proteins that exhibit the opposite phenotype with an intense signal at the perinuclear ER compared with the cortical ER ( Figure 7G). Finally, we show clear distinction between Fur4 ΔN -GFP at the PM and ER-PM contact sites 56 indicated by Sec63-RFP ( Figure 7H,I).

| SUMMARY
In conclusion, we report a simple growth assay that indirectly reports on surface protein trafficking via nutrient transporter activity of uracil auxotroph yeast strains. The assay relies on comparison of growth efficiency of yeast cells on relatively high and low uracil media to infer the capacity of the Fur4 transporter to scavenge uracil required for growth. It is therefore cheap, simple and easy to perform at high throughput, as demonstrated by testing a haploid deletion library of over 5000 yeast strains. This genetic screen identified many novel candidates as potential Fur4 regulators and was particularly enriched for membrane trafficking and transcriptional machinery. By crossreferencing essential genes and factors identified from the screen, with genome-wide expression patterns in most of these transcriptional regulators, we were able to identify connections between TFs and the genes they regulate, both of which relate to uracil-scavenging.
As an example, our bioinformatics identified the essential gene SEC62 and the uncharacterized gene YDR222W, as repressed in many of the TFs mutants identified from the screen. As proof of principle, we show experimentally that decreased expression of SEC62 does, as  Table S7 and   plasmids used are itemized in Table S8.

| Confocal microscopy
Yeast cells expressing GFP and/or mCherry tagged proteins were grown to mid-log phase and then viewed in kill-buffer (50 mM Tris pH 8.0, 10 mM NaN 3 , 10 mM NaF) or water (dH 2 O) at room temperature on Zeiss laser scanning confocal instruments (LSM710 or LSM880 equipped with Airyscan) with a Plan-Apochromat 63Â/1.4 Differential Interference Contrast (DIC) objective lens. Images were captured using Zen Black Imaging software and modified using Image J (NIH). Yeast vacuoles were labelled with 1.6 μM FM4-64 (ThermoFisher) in YPD media, followed by 3Â washes with SC media and further growth in SC media for 1-hour prior to imaging. 4.4 | α-factor-induced arrest "halo" assay Wild-type and mutant Matα cells were grown to saturation overnight and then diluted back and grown for 4 to 6 hours in YPD media.
Equivalent volumes of cells were harvested and spun down and brought up in 50 μL sterile water before spotting on low density lawns of MatA bar1-1 cells created from mid-log phase cells on YPD plates.
Plates were incubated at 30 overnight. Halos of growth inhibition for mutants relative to wild type were determined using ImageJ (NIH).

| Gene ontology analyses
All GO enrichments were acquired using the GO Term Finder v0.86, 39 using the default settings. The default background was used for all analyses except those for clusters derived from essential gene expression analysis, which instead used a background of essential genes listed in Table S4.

| String analysis
Interactome analysis of physical interactions was carried out using STRING software. 62

| Statistical analysis
Statistical significance for experimental conditions was calculated using a Student t test/Bonferroni-Dunn method in GraphPad prism v8. Asterisks were used to denote significance on scatterplot histograms with P values documented in Tables S1 and S9.

ACKNOWLEDGMENTS
We would like to thank Luke Mackinder, Jared Cartwright and the Protein Production Laboratory at the York Bioscience Technology Facility (BTF) for access and assistance with the Rotor HDA robotics, and the Imaging and Cytometry Core of the York BTF for technical support with microscopy. Thanks to Daphne Ezer for assistance with bioinformatics, and to Maya Schuldiner (Weizmann Institute of Science, Israel) for providing DAmP-cassette integration yeast strains.
We are grateful to Kamilla Laidlaw and other members of the lab for useful comments and assistance with the project.