Structural Lipids Enable the Formation of Functional Oligomers of the Eukaryotic Purine Symporter UapA

Summary The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.


In Brief
We describe the first in-depth analysis of membrane lipid interactions with a eukaryotic transporter using native mass spectrometry. We demonstrate that the binding of structural lipids is essential to maintain the stability of the functional UapA dimer in both the gas phase and in vivo.

INTRODUCTION
Cellular membranes play key roles in determining the structure and function of membrane proteins (Opekarova and Tanner, 2003). Membranes are highly fluid and asymmetrical structures that provide distinct physical environments for the associated proteins (Engelman, 2005). The biophysical properties of the membrane, such as lateral and transverse pressures caused by membrane curvature and lipid packing, directly affect membrane protein folding, structure, and function (Booth and Curnow, 2009;Marsh, 1996; van den Brink-van der Laan et al., 2004). Furthermore, both specific and non-specific protein-lipid interactions can affect transporter conformation, stability, and oligomerization Koshy and Ziegler, 2015;Laganowsky et al., 2014;Martens et al., 2016). For example, specific lipid binding to subunit-subunit interfaces can modulate BetP oligomerization (Koshy et al., 2013). In contrast, bulk or annular lipid interactions can provide structural support to facilitate conformational changes in the transporter NapA by stabilizing the position of a static gate domain while allowing the movement of dynamic core domain to accommodate an elevator-like mechanism . Therefore, it is essential to characterize protein-lipid interactions and the relationship between proteins and the cell membrane to fully understand the structure and function of membrane proteins in vivo.
Traditional structural biology methods, such as cryoelectron microscopy and X-ray crystallography, have offered invaluable information regarding membrane protein structure and function; however, even with these high-resolution methods capturing protein-lipid interactions remains a challenge (Peng et al., 2014). While there has been a marked increase in the number of membrane protein structures with resolved lipids in recent years, only a select few studies have established the structurefunction relationship of protein-lipid interactions (Koshy et al., 2013;Mehmood et al., 2016;Norimatsu et al., 2017). Mass spectrometry (MS) of intact protein complexes has emerged as a key method for detecting, identifying, and characterizing proteinlipid interactions Henrich et al., 2017;Laganowsky et al., 2013;Landreh et al., 2017;Mehmood et al., 2016;Reading et al., 2015;Skruzny et al., 2015;Zhou et al., 2011). Native MS employs nano-electrospray ionization as a ''soft'' ionization technique that preserves the structure, oligomerization, and ligand-lipid interactions of protein complexes (Ahdash et al., 2017;Ashcroft, 2005;Hernandez and Robinson, 2007). During native MS of intact membrane proteins, detergent micelles are removed through collisional activation with inert gas molecules. Careful tuning of the instrument parameters ensures well-resolved spectra, while maintaining the overall protein fold as well as interactions with other proteins, ligands, and/or lipids. Consequently, native MS has emerged as a powerful tool for examining the role of lipid binding on the stability of membrane protein oligomers Reading et al., 2015;Wang et al., 2010).
Recent studies using native MS have demonstrated that protein-lipid interactions play a crucial role in stabilizing the dimer form of prokaryotic transporters Henrich et al., 2017). However, our understanding of the role of lipids in maintaining the quaternary structure of eukaryotic transporters remains limited, mainly due to the relatively poor stability of these proteins in non-native environments. Here, we used native MS to study UapA, a eukaryotic transporter from Aspergillus nidulans belonging to the nucleobase ascorbate transporter (NAT) family of metabolite importers. UapA is responsible for H + -dependent uptake of the purines xanthine and uric acid. The high-resolution structure of a transport inactive, conformationally locked mutant of UapA (G411V D1-11 ) was recently solved, revealing that UapA is formed from two domains, the core domain and gate domain, and is likely to transport via an elevator mechanism (Alguel et al., 2016). The structure also showed that UapA is a homodimer confirming earlier biochemical studies (Martzoukou et al., 2015). Analysis of the structure in combination with mutagenesis data demonstrated that dimer formation was essential for function (Alguel et al., 2016). The structure showed that the extensive ($6,000 A 2 ) dimer interface was mainly mediated by hydrophobic interactions and displayed no electron density consistent with lipid binding (Alguel et al., 2016). We combined native MS with molecular dynamics (MD), mutagenesis, and functional analyses to establish that phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are closely associated with UapA and play a crucial role in stabilizing the functional UapA dimer.

Protein-Lipid Interactions Stabilize the UapA Dimer
We began by subjecting a purified thermostabilized, conformationally locked, inward-facing mutant of UapA (G411V D1-11 ) (MW = 60,859.59 Da) (STAR Methods and Figure S1A) to ion-mobility (IM)-MS (Leung et al., 2013). IM-MS reports the molecular shape of biomolecules by determining their rotationally averaged collisional cross-section (CCS) (Jenner et al., 2011;Jurneczko and Barran, 2011;Konijnenberg et al., 2014;Michaelevski et al., 2010;Ruotolo et al., 2008). We found the experimental CCS of UapAG411V D1-11 (6,117 Å ) to be in agreement with the theoretical CCS of the native-like state, calculated from the The relative abundance of each oligomeric species in (B), (C), and (Dii) was quantified using UniDec software . The mass spectra are representative of three independent experiments carried out under identical conditions. The relative abundance data are the average ± SD (n = 3) and the average values are given above the bars on each chart. crystal structure of UapAG411V D1-11 (6,230 Å ) (PDB: 5I6C), indicating that the protein remains folded in the gas phase ( Figure S1B).
Next, to investigate the oligomeric states of UapA, we performed native MS on UapAG411V D1-11 . Well-resolved spectra showed that the protein exists both as a monomer and a dimer ( Figures 1A and 1B). The relative abundance of monomer/dimer was $5:95, suggesting a strong dimer interface (Yefremova et al., 2017). The spectra also revealed the binding of two adduct molecules (measured mass: 775 ± 50 Da) to the UapAG411V D1-11 dimer. We attributed this to the presence of bound phospholipids; however, the resolution of the mass spectra was insufficient to allow identification of the lipid species. We therefore extracted the lipids from the purified protein and analyzed the lipid extract by liquid chromatography-MS (LC-MS) and tandem MS (LC-MS/MS) experiments. We identified phosphatidylcholine (PC), PE, and PI as the major lipid classes co-purifying with UapAG411V D1-11 (Figures 1C and S2; Table  S1). We verified the presence of the lipids identified by LC-MS in the UapAG411V D1-11 sample by negative-ion native MS (Figure S1C). PE, PC, and PI are all abundant species in the plasma membranes of both the expression host (Saccharomyces cerevisiae) and the native host (A. nidulans). Consequently, their interactions with UapA are likely to be physiologically relevant (Birch et al., 1998).
To study the effect of lipids in the formation of UapA dimers, we delipidated UapAG411V D1-11 and subjected it to native MS. Strikingly, our results showed that the delipidated protein is present primarily in the monomeric form ( Figure 1D), suggesting that the lipids are important in maintaining the physiological dimer, as previously seen for prokaryotic transporters . Loss of peaks in the $750 m/z range of the native MS spectra Lipid binding peaks are highlighted. Lipids were added to delipidated UapAG411V D1-11 at a ratio of 100:1 lipid/UapA. (B) Relative abundances of monomer and dimer species in the presence of PC (upper), PI (middle), and PE (lower) were quantified using UniDec software . The mass spectra are representative of three independent experiments carried out under identical conditions. The relative abundance data are the average ± SD (n = 3) and the average values are given above each bar.
further confirmed almost total removal of lipid from the samples ( Figure S3A). To explore the effect of the different lipids identified in lipidomics on UapA dimerization, we added PI, PE, or PC individually, or in combination, into delipidated UapAG411V D1-11 at a ratio of 1:100 protein/lipid ( Figure 2). Addition of either PE or PI restored the UapA dimer with both lipids displaying similar levels of dimer recovery, yielding 73.1% and 59.7% dimer, respectively (Figure 2). The addition of an equimolar mixture of PI and PE to delipidated UapAG411V D1-11 was more efficient at dimer recovery, yielding 81.5% dimer ( Figure S3C). This implies that the effects of the two lipids are additive. As a negative control we also titrated phosphatidylglycerol (PG), a lipid not usually found in eukaryotic plasma membranes, into delipidated UapAG411V D1-11 (Opekarova and Tanner, 2003). PG did not induce any significant changes in the oligomerization of delipidated UapAG411V D1-11 ( Figure S3B).
In contrast to PI and PE, the addition of PC to delipidated UapAG411V D1-11 failed to mediate the reformation of the UapA dimer. Indeed, adding PC actively induced dissociation of nondelipidated UapAG411V D1-11 ( Figure S3D). Surprisingly, the presence of PC inhibited the dimer-stabilizing effects of PI and PE when mixtures of the three lipids were added to delipidated UapAG411V D1-11 ( Figure S3C). Taken together, these data suggest that the dimer-stabilizing effects are specific to PI and PE lipids.
Next, we investigated the effects of lipid binding on the functionally active wild-type (WT) UapA (MW = 61,972.71 Da). We found the relative abundance of monomer/dimer to be approximately 50:50. This is likely to be due to the reduced stability of WT UapA compared with UapAG411V D1-11 (Leung et al., 2013) ( Figure S4). Delipidation of the WT protein resulted in almost complete loss of the dimer form; this could be partially recovered by addition of PI ( Figure S4).

MD Simulations Predict Lipid Binding Sites at the Dimer Interface
MD simulations were performed to predict likely lipid binding sites. We carried out 5-ms simulations using a model of WT UapA based on the crystal structure of UapAG411V D1-11 (PDB: 5I6C). The structure was centered and embedded into a symmetric membrane containing PC (40%), PE (25%), and PI (35%), reflecting the lipid composition revealed via lipidomics. Due to the long simulation time, we coarse-grained the molecules and used the Martini force field . In these simulations, the inward-facing conformation of UapA was locked using an elastic network model. We analyzed our simulations to predict any likely lipid binding sites near the dimer interface that may play a role in stabilizing the dimer. Our MD simulations predicted that PC and PE interact with the same residues and that PI interacts with UapA residues with greater frequency than either PE or PC ( Figure S5A). No significant enrichment of PC or PE in the annular layer around UapA was observed. However, a number of positively charged Lys and Arg residues located principally on the intracellular side of the protein were predicted to be involved in PI binding (Figure 3A). Of particular interest was one clear PI binding site at the UapA dimer interface, comprising residues Arg287 (at the cytoplasmic end of transmembrane 7 [TM7]), Arg478, and Arg479 (at the cytoplasmic end of TM13) ( Figure 3B).

Mutations to the Putative Lipid Binding Site Cause Loss of UapA Function In Vivo
To explore the role of the putative PI binding site in the structure and function of the UapA dimer, we generated a range of single, double, and triple mutants of the residues (R287A, R478A, R479A, R478A/R479A, R287A/R478A/R479A) with C-terminal GFP tags. We individually transformed these constructs into a strain of A. nidulans (uapAD uapCD azgAD pabaA1 argB2) lacking all endogenous nucleobase transport systems. Transformants expressing the individual UapA mutants were tested for their capacity to grow using uric acid or xanthine as the sole nitrogen source. At 37 C, only the triple R287A/R478A/R479A mutant showed markedly impaired growth on either uric acidor xanthine-supplemented media, indicating a loss of UapA transport activity ( Figure 4A). In contrast, at 25 C, closer to the physiological temperature of A. nidulans, both the triple R287A/R478A/R479A and double R478A/R479A mutants were unable to grow on either uric acid or xanthine. The single R479A mutant also showed reduced growth on both substrates ( Figure 4A).
Fluorescence microscopy carried out at 25 C showed that all of the GFP-tagged UapA mutants localized effectively to the plasma membrane, confirming that loss of transport activity in the mutants is not a result of impaired UapA trafficking (Figure 4B). Further analysis using [ 3 H]xanthine uptake assays (Figures 4C and 4D) confirmed the results from the growth assays and revealed close to WT substrate binding affinity for all mutants. This implies that the R478A/R479A and R287A/R478A/ R479A mutations affect substrate transport rather than substrate binding. We have previously shown that dimer formation is critical for UapA function (Alguel et al., 2016), so in order to examine the oligomeric status of the UapA mutants we carried out bimolecular fluorescence complementation (BiFC) analysis using a split YFP system (Martzoukou et al., 2015). This system uses two copies of the individual mutants co-expressed as fusions with either the N-or C-terminal domains of YFP. In this case, fluorescence is observed upon dimerization of copies of UapA tagged with the different YFP domains. Although the R287A/ R478A/R479A mutant (RRR/A) was found to traffic to the membrane ( Figure 4B), it was much less efficient at reconstituting the YFP ( Figure 5A). This suggested that the RRR/A mutant was principally trafficking as a monomer.
To assess the precise oligomeric status of the RRR/A mutant, we introduced the additional G411V mutation and 11-residue N-terminal truncation to generate the construct UapA RRR/A + G411V D1-11 . Native MS of the purified UapA RRR/A + G411V D1-11 (MW = 60,604.26 Da) ( Figure S1A) showed a dramatic reduction in the abundance of UapA dimer compared with UapAG411V D1-11 (Figures 5C and 5D). In addition, there was almost complete loss of lipid binding. These data strongly suggest that the disruption of the putative lipid binding site, and consequent loss of UapA activity in vivo, is due to the reduction of UapA-lipid interactions that play a key role in stabilizing the functional dimer form. There is evidence of minor lipid Residues with a normalized contact probability with PI higher than 0.8 over the course of the simulation ( Figure S5B) are shown in a space-filling representation in dark blue or dark pink. The region highlighted in the dotted circle indicates those residues predicted to form a lipid binding site at the dimer interface of UapA and is shown in close-up view in (B), where individual amino acid residues are shown in stick representation and labeled with the residue number.
binding peaks to the UapA RRR/A + G411V D1-11 construct and limited dimer reformation after the addition of PI at a ratio of 1:100 protein/lipid, although at a much lower level than that seen for UapA G411V D1-11 . It is possible that some of the lipid binding we observe is due to PI binding to the alternative binding sites in UapA predicted by our MD simulations. Furthermore, it is also possible that lipid can still associate to the altered binding site at the dimer interface but with markedly reduced affinity, explaining why the protein purifies as a monomer.

DISCUSSION
Recent years have seen a growing understanding of the key roles played by membrane lipids in the structure and function of membrane proteins; however, as yet there has been no in-depth study of the effects of lipids on eukaryotic transporters. Here, we explore the roles of lipids in maintaining the functional dimeric form of the eukaryotic UapA transporter. We combine native MS with MD simulations and validate the physiological relevance of their findings using mutagenesis and in vivo functional characterization. Principally, we used the UapAG411V D1-11 construct, showing that it primarily exists as a dimer in the gas phase and that removal of tightly bound lipids causes dissociation into a monomer. The dimer can be recovered by addition of PI or PE. Importantly, we obtained similar results for WT UapA, although the spectra acquired for this protein are of reduced quality compared with the UapAG411V D1-11 construct due to the lower stability of the WT protein. However, this gave us confidence that further studies using UapAG411V D1-11 were representative of the native protein.
As mutagenesis of the putative lipid binding site abolished lipid binding, we speculate that PI and PE have defined binding sites and therefore act as so-called structural lipids, forming integral interactions with UapA (Bechara and Robinson, 2015). The dimer interface of UapA has a substantial surface area of $6,000 A 2 (Alguel et al., 2016), which should render the dimer very stable; thus, the dissociation of the dimer into the monomeric form upon delipidation may be a result of the comparatively harsh treatment of the molecule rather than a direct effect of associated lipids. However, the fact that the dimer can be reformed by the addition of PI, PE, or a combination of both lipids is strongly suggestive that these lipids have a specific stabilizing effect on the oligomeric form of the transporter. There does not seem to be an absolute requirement for either PI or PE, as individually both can recover dimer from delipidated protein to a similar extent. Interestingly, the addition of both PI and PE was more effective at dimer stabilization than the addition of PI or PE individually, suggesting the effects of these two lipids is additive. MD simulations predict that these lipids bind different regions of the protein. Together, this suggests that the dimer can be further stabilized by interactions at an alternative lipid binding site. It is also possible that PE binding induces positive allosteric modulation of PI binding; such effects have been reported previously for AmtB (Patrick et al., 2018). Further research will be required to confirm the location of alternative dimer-stabilizing lipid binding sites. Interestingly, MD simulations predicted that most specific lipid binding sites are found within regions of the protein located on the intracellular side of the membrane and are therefore in contact with lipids in the inner membrane leaflet. Similar predictions have been recently made for NapA  and CitS (Wö hlert et al., 2015) transporters. A high-resolution crystal structure of BetP also revealed that asymmetry of lipids is important in mediating oligomer formation with a preference for lipids in the inner leaflet (Koshy et al., 2013).  Figure 1A for the relative abundances of each oligomer of UapAG411V D1-11 under identical conditions. The relative abundance of each species was quantified using UniDec software . The mass spectrum is representative of three independent experiments carried out under identical conditions. The relative abundance data are the average ± SD, n = 3. Of the three residues comprising the putative lipid binding site at the UapA dimer interface, only R479 is highly conserved among the eukaryotic NAT proteins. Interestingly, the R479A mutation was the only single substitution that had detectable effects on the A. nidulans growth rate. A loss of function observed for the R478A/R479A double mutant at the physiological temperature for A. nidulans, 25 C, and for the R287A/R478A/ R479A triple mutant at both 25 C and 37 C was not due to either a loss of correct trafficking to the membrane or impaired substrate binding. The apparent cryosensitivity of the single (R479A) and double (R478A/R479A) mutants may be due to a change in the A. nidulans lipid composition at the lower temperature. Such temperature-dependent lipid composition changes have been reported for S. cerevisiae (Ejsing et al., 2009).
Native MS analysis of the purified triple mutant in the G411V D1-11 background indicated that this construct is almost exclusively monomeric. Given that the functional form of UapA is the dimer, we reason that the lack of transport function in this protein is due to its inability to bind lipids that promote dimerization. However, it is important to note that both native MS and the BiFC assay indicate that dimer does form in the absence of lipid binding, although to a much lower extent than when the lipid binding site at the dimer interface is intact. However, this form of the protein has lost virtually all transport activity. This indicates that lipid binding is not an absolute requirement for dimer formation but is essential for formation of the functional dimer, which suggests that lipid binding could form part of the regulatory mechanism of UapA. This hypothesis could form the basis of future investigations into the nature of UapA-lipid interactions.
The MD simulations predicted that lipids could also bind to the outermost, membrane-facing regions of the core domains of the UapA dimer (see Figure S1D for domain organization of UapA). Lipid binding to this region has been predicted for NhaA and suggested to be involved in stabilizing the core domain during the conformational transitions required for transport by the elevator mechanism . However, in the case of UapA, removal of the putative lipid binding site at the dimer interface caused almost total loss of lipid binding as revealed by native MS ( Figure 5C). This indicates that binding to the core domain may not be a feature of UapA-lipid interactions. Further research is required to confirm this.
In conclusion, we have performed the first detailed analysis of the role of lipid binding to a eukaryotic transporter UapA, and have shown that specific structural lipids are critical for maintaining the protein in a functional dimeric state. Overall, the combination of approaches used here has clear potential to allow investigation of the function of lipid binding to a range of different membrane proteins.

SIGNIFICANCE
Lipids play an important role in the stability and function of membrane transporters. It has been proposed that the binding of lipids to specific sites on membrane proteins is integral to both protein structure and function. Here, we have characterized structural lipids associated with a eukaryotic transporter, UapA, using native mass spectrometry and in vivo functional analysis. We propose that structural lipids stabilize the dimer interface of UapA and are essential for transport activity. These findings expand our understanding of eukaryotic protein-lipid interactions and have direct implications for the molecular mechanism of UapA transport.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

STAR+METHODS
Tris, pH 7.6, 1 mM EDTA, 0.6 M sorbitol) per L culture. The cell suspension was flash frozen and stored at -80 C. Cells were lysed with a Constant Systems cell disruptor at 4 C. Unbroken cells and aggregates were removed by centrifugation at 4000 g for 10 minutes and membranes were isolated by ultracentrifugation at 100,000 g for 2 hrs. Membranes were resuspended in membrane resuspension buffer (20 mM Tris, pH 7.5, 0.3 M sucrose, 0.1 mM CaCl 2 ), flash frozen, and stored at -80 C.

Native MS
Capillaries for nESI were prepared using a Model P-97 (Sutter Instruments) capillary puller. Capillaries were gold coated using a Q150R S sputter coater (Quorum). Using Micro Bio-Spin 6 columns (Bio-Rad) purified UapA was buffer-exchanged into MS-compatible buffer (250 mM EDDA (pH 6.3), 0.014% DDM LA (v/v), 10 mM L-serine) to a final protein concentration of 15 mM. The MS buffer was supplemented with 10 mM L-serine to improve spectral resolution by minimising salt adducts (Clarke and Campopiano, 2015). Delipidated samples were prepared by incubating purified UapA with 1% DDM LA (v/v) overnight before removing displaced lipids and buffer-exchanging UapA into the MS-compatible buffer using Micro Bio-Spin 6 columns (Bio-Rad). The individual proteins samples were loaded into gold-coated nanoflow capillaries (Hernandez and Robinson, 2007) and introduced into a Synapt G2-Si (Waters) mass spectrometer by nESI. The conditions for maximum peak resolution for UapA were: capillary voltage +1.2-1.6 kV, sampling cone voltage 5 V, trap collision energy (CE) 125-200 V, transfer CE 50-240 V, backing pressure 3.88 mbar, trap and transfer pressure (argon) 1.72e -2 mbar, ion mobility cell pressure (nitrogen) 2.58 mbar. A trap CE of 200 V and a transfer CE of 240 V was used when determining the relative abundances of each oligomeric species. To identify lipids in the protein sample, a negative capillary voltage of -1.1 kV was used. Mass measurements were calibrated using caesium iodide (100 mg/mL). Spectra were recorded and smoothed using Masslynx 4.1 (Waters) software. tag has been shown not to affect UapA localisation, function, or transport kinetics in A. nidulans . Successfully transformed strains of A. nidulans were selected via arginine auxotrophy complementation .
As previously described (Karachaliou et al., 2013), UapA subcellular localisation was measured by visualising GFP-tagged UapA using epifluorescence inverted microscopy (Zeiss Observer Z1/Axiocam HR R3 camera/Zen lite 2012 software). Radiolabelled [ 3 H]-xanthine (22.8 Ci mmol -1 , Moravek Biochemicals, CA, USA) uptake was measured using A. nidulans germinating conidiospores at 37 C or 25 C, pH 6.8, as previously described (Krypotou and Diallinas, 2014). To measure growth of A. nidulans in different nitrogen sources, transformed strains were grown on minimal media supplemented with nitrogen sources (10 mM ammonium tartrate or 0.5 mM uric acid or 0.5 mM xanthine) at 37 C or 25 C, pH 6.8. Dimerisation of mutant UapA was measured via a bimolecular fluorescence complementation assay, as described previously (Martzoukou et al., 2015). In brief, the N-terminal 154 amino acids of YFP or the C-terminal 86 amino acids of YFP were cloned into the pAN510exp or pAN520exp vector at the XbaI site. uapA with the necessary mutations was then cloned into the vector and transformed into A. nidulans. YFP fluorescence was measured using epifluorescence inverted microscopy (Zeiss Observer Z1/Axiocam HR R3 camera/Zen Lite 2012 software). Relative quantification was carried out using the ICY colocaliszation studio plugin (pixel-based method) (http://icy.bioimageanalysis.org/) and statistical analysis (Tukey's Multiple Comparison Test, One-Way ANOVA for n=5 hyphae) of plasma membrane fluorescence intensity of mutants compared to WT UapA+-YFPC/UapA+-YFPN was performed as previously described (Martzoukou et al., 2017).

QUANTIFICATION AND STATISTICAL ANALYSIS
Bar charts throughout show mean ± standard deviation (n=3, where n represents the number of repeats). For the bimolecular fluorescence complementation analysis using a split YFP system ( Figure 5B), statistical analysis (Tukey's Multiple Comparison Test, One-Way ANOVA for n=5 hyphae) of plasma membrane fluorescence intensity of mutants compared to WT UapA+-YFPC/ UapA+-YFPN was performed as previously described (Martzoukou et al., 2017). No additional statistical tests were undertaken.