{"paper_id":"1dbaed27-3ffe-42da-a1d0-dd97aadfddcb","body_text":"Vacuole Fusion Regulation by PI(3,4,5)P3 \n 1 \nVacuolar Phosphatidylinositol 3,4,5-trisphosphate controls fusion through binding Vam7, \nand membrane microdomain assembly \n \n \nChi Zhang 1,3, Jorge D. Calderin 1,3, Aliasgar Topiwalla  1 , Ved Shah 1, Jahnavi M. Karat 1, \nCharlie T. Knapp 1, Razeen Ahmed 1, Daniel Grudzien 1, Elizabeth Williamson 2, \nand Rutilio A. Fratti 1,2 \n \n1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL \n2. Center for Biophysics & Quantitative Biology., Univ. of Illinois Urbana-Champaign, Urbana, IL \n3. These authors contributed equally  \n \nAddress correspondence to: Rutilio A. Fratti (rfratti@illinois.edu) \n \nRunning title: Vacuole Fusion Regulation by PI(3,4,5)P3. \n \nKeywords: Grp1-PH, Ypt7, SNARE, PIP3, HOPS, Vps33, PTEN, Vps34 \n \nORCID ID: Rutilio Fratti - 0000-0001-9109-6666  \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 2 \n \nSUMMARY  \nMembrane trafficking is regulated by phosphoinositides (PI) and their modification by kinases, \nphosphatases, and phospholipases. The endolysosomal pathway is primarily controlled by \nPI3P, PI(4,5)P2 and PI(3,5)P2, whereas a role for PI(3,4,5)P 3 is less clear. We report that yeast \nvacuoles produce PI(3,4,5)P3 from PI(4,5)P2 through class III PI 3-kinase activity. In vitro assays \nshowed that adding dioctanoyl (C8) PI(3,4,5)P 3 or the PI(3,4,5)P 3-binding domain Grp1-PH \nblocked fusion. Furthermore, modifying endogenous PI(3,4,5)P 3 with the phosphatase PTEN \nalso blocked fusion. Fluorescence microscopy showed that PI(3,4,5)P 3 was enriched at \nmembrane vertex microdomains, which was blocked by PTEN, C8-PI(3,4,5)P 3, and the class III \nPI 3-kinase inhibitor SAR405. Importantly, blocking or eliminating PI(3,4,5)P 3 prevented the \nvertex enrichment of Ypt7 and the HOPS subunit Vps33. Finally, we show that the soluble \nSNARE Vam7 binds PI(3,4,5)P\n3 and that PTEN abolished trans-SNARE pairing between \npartner vesicles. Together these data indicate that vacuolar PI(3,4,5)P 3 coordinates the \nassembly of microdomains and SNARE function.  \n \n \nINTRODUCTION \nMembrane trafficking and fusion are driven by a group of conserved regulatory proteins (e.g., \nSNAREs) and lipids with organelle specificity. Regulatory lipids are relatively low in abundance \nyet carry out critical aspects of membrane trafficking (Krauss & Haucke, 2007; Lemmon, 2008; \nCorvera et al , 1999; Balla, 2013). This group of lipids includes phosphoinositides (PI), \nphosphatidic acid (PA), diacylglycerol (DAG), sterols, and sphingolipids (Balla, 2013; Lingwood \n& Simons, 2010; Tu-Sekine et al , 2015; Xie et al , 2015; Starr & Fratti, 2019). PIs are \nglycerophospholipids with an inositol head group that can be differentially phosphorylated at the \nD-3, D-4 and D-5 positions to generate seven distinct lipids whose primary function is the \nbinding of proteins with domains that recognize specific PIs. Broadly speaking, different \norganelles are marked by a dominant PI. Endosomes are marked by PI3P, and PI(3,5)P\n2, \nwhereas the Golgi contains PI4P, and the plasma membrane is populated by PI(4,5)P 2 and \nPI(3,4,5)P3. While concentrated at characteristic organelles, biologically significant amounts of \nthese lipids can traffic to different membranes where they continue to signal and control \nmembrane function.  \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 3 \nThe yeast vacuole/lysosome collects regulatory lipids from various pathways and continues to \nmodify PIs to control homotypic vacuole fusion and vacuole fission (Starr & Fratti, 2019; \nBonangelino et al, 2002). Homotypic vacuole fusion can be divided into at least six stages each \nof which is driven by a spatiotemporal-specific mixture of regulatory lipids. In a stage we now \ncall pre-priming, Sec18 is sequestered from inactive cis-SNARE complexes by PA (Sasser et al, \n2012; Starr et al , 2016, 2019). Sec18 can transfer to cis-SNARE complexes upon the \nconversion of PA to DAG by the phosphatase Pah1. Once Sec18 can engage SNAREs via its \nadaptor protein Sec17, priming  occurs through ATP hydrolysis, which is dependent on \nergosterol and PI(4,5)P\n2 through an undefined mechanism (Kato & Wickner, 2001; Mayer et al, \n2000). Vacuole tethering occurs through the interaction of the Rab Ypt7 and its effector \ntethering complex HOPS (homotypic fusion and protein sorting) between partner membranes \n(Price et al, 2000b, 2000a; Seals et al, 2000; Zhang et al, 2024). Ypt7 recruitment and activation \nis carried out by the GEF Mon1-Ccz1 bound to PI3P (Lawrence et al , 2014; Cabrera et al , \n2014), while HOPS itself can simultaneously bind several PIs including PI3P, PI4P and \nPI(4,5)P2 (Stroupe et al , 2006). PI3P is also essential for binding the PX domain of the soluble \nQc-SNARE Vam7 and the formation of SNARE complexes during the docking stage \n(Boeddinghaus et al, 2002; Fratti & Wickner, 2007). Between docking and full content mixing, \nvacuoles can undergo hemifusion where only the outer leaflets of vesicles mix (Reese & Mayer, \n2005; Reese et al, 2005). We and others have found that this transition requires DAG (Jun et al, \n2004) and is sensitive exogenously added PI(3,5)P 2 and lysophosphatidylcholine (LPC) (Miner \net al, 2019; Reese & Mayer, 2005). \n \nRoles for the remaining PIs (PI5P, PI(3,4)P 2 and PI(3,4,5)P3) remain to be assigned in vacuole \nhomeostasis. PI(3,4,5)P3 is one of the most studied phosphoinositides that is mostly present at \nthe plasma membrane where it directs many signal transduction pathways too numerous to \nsummarize here (Riehle et al , 2013). PI(3,4,5)P 3 was discovered independently by two groups \nand was found to be made by class I 3-kinases p110 ( α, β, δ, and γ ) and the p85 regulatory \ndomain using PI(4,5)P 2 as a substrate (Whitman et al , 1988; Traynor-Kaplan et al , 1988; \nWhitman et al , 1985). PI(3,4,5)P 3 is transient and its signalling is turned off by the 3’-\nphosphatase PTEN or the 5’-phosphatase SHIP2 (Maehama & Dixon, 1998; Pesesse et al , \n1998). While most PI(3,4,5)P 3 signaling is associated with the plasma membrane a significant \namount is found on internal vesicles including the nuclear envelope and early endosome for \nlocalized activation of Akt (Jethwa et al , 2015). PI(3,4,5)P\n3 is also present on recycling \nendosomes for AP-1B dependent sorting (Fields et al, 2010), lysosomes to activate mTORC1 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 4 \n(Hsu et al , 2000), and trans-Golgi vesicles where PI(3,4,5)P 3 on VLDL binds to the cargo \nreceptor Sortilin/Vps10 (Sparks et al, 2016, 2020). At the plasma membrane itself PI(3,4,5)P 3 \nrecruits the protein kinases Akt and PDK1 to the membrane by binding their PH domains \n(Dieterle et al, 2014; Levina et al, 2022). PDK1 subsequently phosphorylates Akt to propagate \nsignaling.  \n \nBy homology, Saccharomyces cerevisiae  lacks a class I p110 PI 3-kinase homolog, thus \nPI(3,4,5)P3 is thought not to exist in baker’s yeast. However, the fission yeast \nSchizosaccharomyces pombe also lacks a p110 homolog, yet its class III Vps34 homolog was \nfound to make PI(3,4,5)P 3 from PI(4,5)P 2 in addition to its canonical product PI3P (Mitra et al, \n2004). This suggests that a synthesis pathway for PI(3,4,5)P 3 could have evolved prior to rise of \nclass I PI 3-kinases. Using S. cerevisiae  vacuoles we examined a role for PI(3,4,5)P 3 during \nvacuole fusion. This study shows that PI(3,4,5)P3 is made on vacuoles by Vps34 and is required \nfor efficient vacuole fusion. Removing PI(3,4,5)P 3 with PTEN or blocking it with the Grp1-PH \ndomain inhibited Ypt7-mediated vertex domain assembly leading to reduced trans-SNARE \npairing and fusion. Finally, we found that Vam7 binds PI(3,4,5)P 3 and PTEN leads to its release \nfrom membranes.  \n \n \nRESULTS \nShort chain PI(3,4,5)P3 inhibits in vitro vacuole fusion.  \nIn previous studies we have used dioctanoyl (C8) lipids to compete with endogenous sources of \ntheir long chain counterparts and various vacuolar proteins. This approach has revealed that \nC8-PA competes for Sec18 binding during pre-priming (Starr et al , 2016, 2019; Sparks et al , \n2019), and that C8-PI(3,5)P\n2 competes for binding to the V-ATPase subunit Vph1 and \nregulatory factors of Ca 2+ transport after SNARE pairing and before hemifusion (Miner et al , \n2019, 2020; Zhang et al , 2022). In this study we asked whether adding C8-PI(3,4,5) 3 affected \nvacuole fusion. While this lipid has not been detected in Saccharomyces cerevisiae, numerous \nscreening papers have shown that baker’s yeast proteins can bind PI(3,4,5)P 3, including the \nsoluble SNARE Vam7 (Yu & Lemmon, 2001; Gallego et al, 2010; Zhu et al , 2001; Dunn et al, \n2004). This could be due to lack of specificity observed in these assays, or it could indicate that \nthe lipid may exist in small transient pools that have escaped detection.  \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 5 \nHere we added exogenous C8-PI(3,4,5)P 3 to in vitro homotypic vacuole fusion reactions and \nfound that C8-PI(3,4,5)P 3 potently inhibited fusion with an IC 50 value of ~80 µM  (Fig. 1A). This \nshowed that vacuole fusion was more sensitive to C8-PI(3,4,5) 3 compared to C8-PI(3,5)P 2 with \nan IC50 values of ~140 µM or C8-PI3P, which failed to fully inhibit fusion at 500 µM (Miner et al, \n2019). To confirm that the measured impacts were PI-specific and not an artifact of the C8 \nchains, we tested the C8 variants of PC, PE and PS and found that none of the bulk lipids had a \nsignificant effect on fusion (Fig. 1B), signifying that the C8 chains themselves had no effect and \nthat the PI headgroups were responsible for altering vacuole fusion efficiency. While many PI \nbinding domains such as the Plc1 δ PH insert a loop into the membrane to further stabilize their \ninteraction (Herzog et al , 2016; Lemmon, 2008), they can also simply bind headgroups in \nsolution, (Kavran et al, 1998; Ferguson et al, 2000) albeit with different affinities compared to full \nlipids. Based on this we asked if the headgroups alone could interfere with vacuole fusion. We \nadded the PI(3,4,5)P 3 head group Ins(1,3,4,5)P 4 as well as other variants (Ins(1,3,4)P 3, \nIns(1,3,5)P3) to fusion reactions and saw that they had no effect even when at present at 500 \nµM (Fig. 1C). This suggested that the head group alone was insufficient for vacuole fusion \ninterference. \n \nC8-PI(3,4,5)P\n3 can inhibit in vitro vacuole fusion after docking. \nIn order to determine which stage of fusion was affected by C8-PI(3,4,5)P 3 we performed \ntemporal gain of resistance experiments (Mayer et al, 1996; Haas et al , 1995; Ungermann et al, \n1998; Price et al , 2000b; Sasser et al , 2012; Miner et al , 2019). Inhibitors were added at \ndifferent timepoints starting at T=0 min and at 5,10, and 30-min intervals for a total of 120 min. \nAs reactions passed a stage of fusion, e.g. Sec18-mediated priming they became resistant to \ninhibitors of that stage such as antibodies against Sec18 and Sec17, NEM, propranolol, C8-PA \nor the small molecule IPA (inhibitor of priming activity) (Mayer et al, 1996; Sasser et al, 2012; \nStarr et al, 2016; Sparks et al, 2019). In this study we used NEM to mark the priming threshold, \nand GDI to mark the tethering/docking phase (Mayer & Wickner, 1997). Tethering and docking \ncannot be separated by this assay. Individual reactions were treated with buffer alone at 27 °C \nfor the duration of the experiment, while a group of buffer-treated tubes were removed and \nplaced on ICE to mark the maximum amount of fusion for any recorded time point. We added \n150 µM C8-PI(3,4,5)P\n3 to individual reactions at the indicated times to see which stage would \nbe impacted by lipid treatment. We found that the gain of resistance curve for C8-PI(3,4,5)P 3 \nwas shifted to the right of GDI indicating that it continued to affect fusion after Ypt7 mediated \ntethering (Fig. 1D). This was further illustrated when the half-times of resistance were \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 6 \ncalculated. Figure 1E  shows that priming and docking were completed by 5 and 12 min, \nrespectively, whereas the added C8-PI(3,4,5)P 3 had later half-time of ~26 min. This half-time is \nlater than the T1/2 ~15 min we saw with PI(3,5)P 2 (Miner et al , 2019). Importantly, it should be \nnoted that gain of resistance assay only indicates the last step the variable molecule had an \neffect; implying earlier stages could have been impacted as well. \n \n \nPI(3,4,5)P\n3-specific inhibitors block vacuole fusion.  \nThe data presented above suggests that adding C8-PI(3,4,5)P 3 affected fusion by \neither disrupting membrane biophysical properties or specific protein-lipid interactions. In other \nwords, a key PI(3,4,5)P\n3-protein interaction needed for fusion could have been disrupted by \nexogenous amounts of C8-PI(3,4,5)P 3. To further test for a required PI(3,4,5)P 3-protein \ninteraction we used purified GST-Grp1-PH, a PI(3,4,5)P 3-specific binding PH domain from the \nARF GEF Grp1 (Guillou et al, 2007; Chen et al, 2012; Corbin et al , 2004; Dowler et al, 2000). \nSequestering PI(3,4,5)P 3 away from natural binding partners with GST-Grp1-PH potently \ninhibited fusion with an average IC 50 of ~1 µM with batch-to-batch variation. This further \nindicated that the lipid was present on vacuolar membranes (Fig. 2A). We also tested the Grp1-\nPHK273A mutant that has reduced PI(3,4,5)P 3 affinity (Lindsay et al , 2006; Naughton et al , 2016; \nGuillou et al, 2007; Yamamoto et al, 2020). We found that it only interfered with fusion with an \nIC50 of 3.6 µM, which was non-specific as shown below.  \n \nTo further probe for the presence of PI(3,4,5)P 3 on isolated vacuoles we used the lipid \nphosphatase PTEN that converts PI(3,4,5)P 3 to PI(4,5)P 2 (Maehama & Dixon, 1998; Lee et al, \n1999). We added a dose-response curve of purified GST-PTEN to fusion reactions and found \nthat it inhibited with an IC 50 of 60 nM (Fig. 2B) . This further indicated that endogenous vacuolar \nPI(3,4,5)P3 affected homotypic vacuole fusion. While PTEN does have some activity against \nPI(3,4)P2 and PI3P, it is 3-5 times weaker to its activity against PI(3,4,5)P3 (Lee et al, 1999).  \n \nWe then examined if we could block PI(3,4,5)P\n3 indirectly with a competitive inhibitor that \ntargets the binding pocket of interacting proteins. We used the PITenin PIT-1, a small molecule \nthat binds the PI(3,4,5)P\n3 binding pocket of PH domains including those of AKT, PDK1, Grp1 \nand ARNO (Miao et al, 2010). Notably PIT-1 does not interfere with PI(3,4)P 2 interactions with \nTAPP1 or TAPP2 or PI(4,5)P 2 interactions with PLC (ibid). We found that PIT-1 on its own \ninhibited fusion with an estimated IC 50 of ~ 65 µM, which was near the IC 50 for its inhibition of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 7 \nAkt-PH-PI(3,4,5)P3 interactions (ibid) (Fig. 2C) . These data indicated that a native vacuole \nprotein bound to PI(3,4,5)P 3 to affect vacuole fusion. In addition, we used the PIT-1 derivative \n3,5-dimethyl PIT (DMPIT) and observed little interference with fusion (Fig. 2C).  \n \nNext, we asked if PIT-1 could rescue the effects of Grp1-PH and PTEN. Fusion reactions were \nfirst treated with 200 µM PIT for 5 min at 27 °C. Reactions were stopped by placing them on ice \nfollowed by the addition of 2 µM Grp1-PH or 200 nM PTEN. Reactions were further incubated at \n27°C for a total of 90 min. This showed that PIT-1 could reverse the effects of both Grp1-PH \n(Fig. 2D)  and PTEN (Fig. 2E) . Similarly, 3,5-DMPIT rescued Grp1-PH inhibition  (Fig. 2F) . \nInterestingly, 3,5-DMPIT was not able to reverse inhibition by the Grp1-PH K237A mutant further \nindicating that its inhibition was a result of non-specific interactions (Fig 2D, F) . We also \nobserved that 3,5-DMPIT moderately rescued the effect of PTEN (Fig. 2G). \n \nLastly, we tested if PTEN and Grp1 had gain of resistance curves that matched the C8-\nPI(3,4,5)P\n3 curve. Using the assay described above we found that the resistance curves of \nPTEN and Grp1-PH were shifted to the right of the GDI curve with half-times of ~25 min, which \nwas in accord with the C8-PI(3,4,5)P3 data (Fig. 2H-I).  \n \nPI(3,4,5)P\n3 was detected on vacuoles \nThe inhibition of fusion by PTEN and Grp1-PH suggested that PI(3,4,5)P3 was present on \nvacuoles. To visualize where PI(3,4,5)P3 was on docked vacuoles we used subinhibitory \nconcentrations of GST-Grp1-PH (150 nM) and fluorescent (CF488) anti-GST antibody. This \nprevented any interference caused by conjugating primary amines or free surface Cys with \nreactive dyes. Grp1 has key Lysines in the lipid binding pocket and a Cysteine next to a lipid \ninteracting Lys (Cronin et al , 2004; Lai et al , 2013). We found that CF488-Grp1-PH localized to \nvertices of docked vacuoles where essential lipids and proteins accumulate to trigger fusion \n(Fig. 3 A-B)  (Wang et al , 2002; Eitzen et al , 2002; Wang et al , 2003; Fratti et al , 2004; \nKarunakaran et al, 2012; Jun et al , 2006; Miner et al, 2019, 2020; Zhang et al, 2024). Vacuole \nlabeling was significantly reduced with the Class-III PI 3-kinase-specific inhibitor SAR405 \n(Ronan et al, 2014). This indicates that PI(3,4,5)P\n3 production could be Vps34-dependent and \nnot due to an unidentified Class I homolog. This is also in keeping with Vps34 production of \nPI(3,4,5)P3 seen in fission yeast (Mitra et al , 2004). In parallel we treated vacuole with PTEN \nprior to adding CF488-Grp1-PH. In Figure 3C-D we show that PTEN blocked labeling by CF488-\nGrp1-PH showing that PI(3,4,5)P3 was largely eliminated from vacuoles.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 8 \n \nCF488-Grp1-PH labeling of vacuoles was blocked by C8-PI(3,4,5)P3 \nTreating vacuoles with PTEN blocked CF488-Grp1-PH from accumulating on docked vacuoles \nthrough converting PI(3,4,5)P\n3 to PI(4,5)P 2. To confirm that this was not an indirect effect we \nnext tested CF488-Grp1-PH localization by adding subinhibitory levels (300 nM) of C8-\nPI(3,4,5)P\n3 or C8-PI(4,5)P2 as competitive inhibitors. This showed that CF488-Grp1-PH labeling \nwas inhibited by C8-PI(3,4,5)P 3 but not C8-PI(4,5)P 2 (Fig. 4A-B).  This further demonstrated \nCF488-Grp1-PH vertex enrichment was specifically due to PI(3,4,5)P3 binding.  \n \nPI(3,4,5)P\n3 affected GFP-Ypt7 and HOPS enrichment at vertex domains \nWhile examining vacuole docking with inhibitory levels of PTEN, Grp1-PH and C8-PI(3,4,5)P3 \nwe observed a reduction in the vacuole clusters as well the number of vacuoles per cluster. This \nsuggested that PI(3,4,5)P\n3 could affect docking/tethering. For this we used vacuoles harboring \nGFP-Ypt7 and examined its enrichment at vertices when PI(3,4,5)P 3 was sequestered by 2 µM \nGrp1-PH. This showed that sequestering PI(3,4,5)P 3 with Grp1-PH blocked GFP-Ypt7 from \nbecoming enriched at vertex domains (Fig. 6A-B) . GFP-Ypt7 was still visible on Grp1-PH \ntreated vacuoles; however, the intensities at vertex sites were diminished while intensities at the \nouter edges were increased. To confirm that endogenous PI(3,4,5)P 3 availability affected GFP-\nYpt7 distribution we used 250 µM C8-PI(3,4,5)P 3 as a competitive inhibitor. This showed that \nthe presence of C8-PI(3,4,5)P 3 inhibited the vertex enrichment of GFP-Ypt7 at vertex domains \n(Fig. 6C-D) . GFP-Ypt7 was distributed throughout the membranes of docked vacuoles. This \nwas like the effects of Grp1-PH, suggesting that free PI(3,4,5)P 3 was needed for the assembly \nof a fully functioning vertex microdomain.  \n \nTo see if other vertex components were affected by PI(3,4,5)P 3 we used vacuoles that \ncontained Vps33-GFP, a HOPS subunit. Vps33-GFP localization to vertices has been shown to \nbe sensitive to the lipid binding probes FYVE, ENTH, C1b and filipin that bind PI3P, PI(4,5)P 2, \nDAG and ergosterol, respectively (Fratti et al , 2004). Moreover, Vps33 can be released from \nvacuoles when PI3P, PI4P and PI(4,5)P 2 are sequestered (Stroupe et al, 2006). Here we tested \nthe effects of C8-PI(3,4,5)P 3 and Grp1-PH on Vps33-GFP distribution. Untreated vacuoles \ncontained enriched Vps33 at vertex sites as previously reported (Fig. 6A-B). However, Vps33-\nGFP vertex enrichment was sharply reduced by both C8-PI(3,4,5)P 3 and Grp1-PH, indicating \nthat free native PI(3,4,5)P3 was required for normal vertex assembly. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 9 \nPI(3,4,5)P3 affected trans-SNARE pairing.  During the docking stage of vacuole fusion, key \nproteins and lipids become enriched at vertex microdomains. The assembly of these domains \npromotes the optimal format ion of trans-SNARE complexes between membranes (Collins & \nWickner, 2007; Sasser et al, 2012). While PI(3,4,5)P3 played a role in the docking stage the gain \nof resistance experiments indicated that it continued to be important a later stage such as trans-\nSNARE pairing. To examine trans-SNARE pairing we used two types of vacuoles. One type \nlacked the R-SNARE Nyv1 and expressed Vam3 with an internal calmodulin binding peptide \nbetween the N-terminal helical H\nabc domain and the SNARE domain (CBP- VAM3 nyv1Δ). The \nsecond type contained Nyv1 and unmodified Vam3 (VAM3 NVY1) (Collins & Wickner, 2007; Jun \n& Wickner, 2007). The formation of trans-SNARE complexes was detected when Nyv1 co-\nisolated with CBP-Vam3 bound to calmodulin beads. We found that under control conditions \nCBP-Vam3 indeed paired with Nyv1 from partner membranes as well as the HOPS subunits \nVps33 and Vps18 (Fig. 7A & C) . As a negative control we used NEM, which inhibited SNARE \npriming and thus prevented downstream trans-SNARE interactions. As expected, NEM \ntreatment abolished Nyv1 co-isolation with CBP-Vam3 but had no effect on Vam3-HOPS \ninteractions (Fig. 7A lane 8 vs 9, and B) . This was consistent with a report showing that HOPS \nbinds to the H\nabc N-terminal domain of Vam3 (Lürick et al , 2015). Interestingly, Vam7 was \ndepleted in Q-SNARE complexes when NEM was administered. The difference could be linked \nto a pool of free Vam7 bound to lipids and not SNAREs (Thorngren et al , 2004). This could also \nbe due to the effect of NEM on the free cystine in Vam7 where alkylation might prevent full Q-\nSNARE complex formation. However, this is unlikely as the alkylated Vam7 was seen by the \nmobility shift of the band present in the pulldown.  \n \nTo test the role of PI(3,4,5)P\n3 in trans-SNARE pairing we used a concentration curve of PTEN. \nThis showed that increasing amounts of PTEN blocked CBP-Vam3 pairing with Nvy1, thus \ndemonstrating that trans-SNARE complex formation was inhibited by eliminating PI(3,4,5)P\n3. \nThis could be due to two factors that may be linked. First, the Qb-SNARE Vti1 was absent from \nthe CBP-Vam3 complex when PTEN was present at 200 nM even though it was fully present in \nthe input (Fig. 7A, lane 6 vs 12) . Second, Vam7 was significantly depleted in the input \nmembrane fraction and completely absent from CBP-Vam3 complexes when vacuoles were \ntreated with 200 nM PTEN. It is important to note that the first step in trans-SNARE isolation is \npelleting the membranes which separates vacuole-bound from unbound proteins. Thus, a \nrelease of Vam7 would result in its absence from the input blot. This was not due to protease \nactivity as both vacuole populations lacked PEP4. Together, this suggested that Vam7 was \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 10\nreleased from membranes when PI(3,4,5)P 3 was eliminated. Vam7 has been shown by others \nto bind PI(3,4,5)P3 in lipid overlay assays (Yu & Lemmon, 2001). Consequently, it could be that \nVam7 bound to PI(3,4,5)P 3 was needed for Vti1 to bind Vam3 in a 3Q-SNARE complex. We \nmust note that Vti1-Vam3 binding was not affected by eliminating or blocking other PIs including \nPI3P, PI4P, and PI(4,5)P2 (Collins & Wickner, 2007). \n \nIn addition to SNARE complex formation CBP-Vam3 also pulled down the HOPS complex; \nhowever, when vacuoles were treated with PTEN we found that it did not inhibit HOPS co-\nisolating with CBP-Vam3. On the contrary, we found that depleting PI(3,4,5)P 3 enhanced HOPS \nbinding to CBP-Vam3 (Fig. 7A-C) . This could be due to an exchange in HOPS-Vam3 \ninteractions for binding Nyv1.  \n \nGrp1-PH displaced Vam7 from membranes \nTo verify that the loss of Vam7 from vacuoles treated with PTEN was due to eliminating \nPI(3,4,5)P\n3 we tested protein binding in the presence of Grp1-PH. Fusion reactions were treated \nwith buffer alone (0 µM) or GST-Grp1-PH at increasing concentrations and incubated at 27 °C \nfor 60 min. After incubation, vacuole-bound and unbound proteins were separated by \ncentrifugation, and the two fractions were examined by Western blotting. As seen previously, \npopulations of soluble proteins were seen in both pellet (bound) and supernatant (unbound) \nfractions including Vps18 and Vps33, actin, and Vam7 (Fig. 8A-B). In contrast, the membrane-\nanchored proteins Nyv1 and Ypt7 were only seen in the bound fraction. Treatment with GST-\nGrp1 only affected Vam7 binding where significantly more was present in the supernatant and \ndepleted from the bound fraction (Fig. 8 lanes 5 and 11) . This was reminiscent of a previous \nstudy showing that Vam7 was released in the presence of the lipid binding domains FYVE, \nENTH and C1b, which bind PI3P, PI(4,5)P\n2 and DAG, respectively (Fratti et al, 2004). That said, \nHOPS binding was not altered in the presence of Grp1-PH, whereas a previous study showed \nthat Vps33 was released by FYVE, ENTH as well as the PI4P binding domain Fapp1-PH \n(Stroupe et al , 2006). We also tested C8-PI(3,4,5)P\n3 at 250 µM which inhibits fusion. Unlike \nGrp1-PH, C8-PI(3,4,5)P 3 had no effect on Vam7 binding. This could be due to differences in \nbinding affinities where C8-PI(3,4,5)P 3 cannot compete with full length lipid. This also indicated \nthat separate factor required for fusion was blocked by C8-PI(3,4,5)P 3 such as the \nmislocalization of Ypt7 and Vps33. Together these data suggested that Vam7 release could be \ndue to a direct interaction and not to a general blockage of vertex microdomain \nassembly/maintenance. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 11\n \nVam7 binds PI(3,4,5)P3 \nVam7 uses its PX domain to bind the vacuole membrane through its interactions with PI3P, and \nmutating Tyr42 to Ala inhibits binding and fusion though not entirely (Cheever et al , 2001; \nBoeddinghaus et al, 2002; Fratti & Wickner, 2007). The ability of Vam7\nY42A to form complexes \nwith SNAREs and HOPS was attributed to its directly binding to proteins without the aid of PI3P \nbinding. That said, it could be that Vam7 Y42A interacted with other lipids, including PI(3,4,5)P 3. \nPreviously we showed that Vam7 also binds to PA and PI5P by liposome flotation, albeit to a \nlower extent versus PI3P (Miner et al , 2016). We also observed weak PA binding by surface \nplasmon resonance, microscale thermophoresis and bio-layer interferometry (BLI) (Sparks et al, \n2019, 2022; Calderin et al, 2025). This suggests that Vam7 could have a second lipid binding \nsite or promiscuous binding by a single site. The former is in accord a study showing that many \nPX domains have a second lipid binding site (Chandra et al, 2019). \n \nHere we compared PI3P and PI(3,4,5)P 3 binding using streptavidin coated BLI probes bound to \nbiotinylated lipids. These were incubated with GST-Vam7 and GST-Vam7 Y42A at different \nconcentrations to measure binding. Here we show curves of response units versus protein \nconcentration. This illustrated that Vam7 strongly bound to PI3P (K D ~200 nM) whereas \nVam7Y42A failed to bind as expected (Fig. 8C). In comparison, we found that both Vam7 and \nVam7Y42A bound to PI(3,4,5)P 3 with KD values of ~70 and ~210 nM, respectively (Fig. 8F). The \nbinding of Vam7 Y42A to PI(3,4,5)P3 suggested that the PX domain could have a second binding \nsite. Alternatively, other residues in the same binding pocket might engage the D4 and D5 \nphosphates of PI(3,4,5)P\n3, which are not involved in binding with the D3 phosphate of PI3P. It is \nalso possible that the Y42A mutation weakened PI(3,4,5)P 3 binding without abolishing it. Future \ninvestigation will explore these options.  \n \n \nDISCUSSION \nIn this study we asked if there was a role for PI(3,4,5)P 3 in yeast vacuole fusion. In previous \nstudies we have shown that adding C8-PA and C8-PI(3,5)P 2 inhibited fusion at the pre-priming \nstage and between SNARE pairing and hemifusion, respectively (Starr et al, 2016; Miner et al, \n2019). Here we saw that C8-PI(3,4,5)P 3 also inhibited fusion after the GDI-sensitive/Ypt7-\ndependent step. Separately we found that sequestering native PI(3,4,5)P 3 with Grp1-PH or \nmodifying it with PTEN inhibited fusion. Furthermore, blocking PI(3,4,5)P 3 interactions with \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 12\nunknown endogenous binding partner(s) with PIT-1 and DMPIT rescued inhibition by Grp1 and \nPTEN. Together these data indicate that vacuoles have an endogenous pool of PI(3,4,5)P 3 and \nthat it interacts with a least one vacuolar protein to promote optimal fusion.   \n \nStage specific experiments showed that both Ypt7-dependent tethering and trans-SNARE \npairing required PI(3,4,5)P\n3. The Ypt7-dependent effect was shown by the lack of its enrichment \nat vertices. Docked vacuoles form vertex microdomains that become enriched in Ypt7, HOPS, \nSNAREs and actin, as well as PI3P, PI(4,5)P\n2, DAG, and ergosterol (Wang et al , 2002, 2003; \nEitzen et al, 2002; Karunakaran et al, 2012; Fratti et al, 2004). The composition of the vertices is \ncritical, highly regulated, and interdependent, meaning that the enrichment of key proteins and \nlipids will consequently affect t he accumulation of other proteins and lipids at the site. The \ncorrect localization of proteins and lipids at vertex sites promotes trans-SNARE pairing and \nfusion. Thus, it follows that a reduction in functional vertex sites led to inhibiting trans-SNARE \npairing. Interestingly though, the block in trans-SNARE pairing was accompanied by a loss of \nVam7 from the membrane and the loss of Vti1 from the 3Q SNARE bundle.  \n \nVam7 and PI(3,4,5)P\n3 \nThe interaction between Vam7 and PI3P has long been recognized as the mechanism by which \nthis soluble SNARE associates with membranes prior to its interactions with its cognate \nSNAREs and HOPS (Cheever et al, 2001; Boeddinghaus et al, 2002). Its lipid binding capacity \nlies in its N-terminal PX domain and PI3P binding can be largely abolished by mutating Tyr42 to \nAla. That said, Vam7\nY42A can still associate with vacuoles and support fusion albeit at reduced \nlevels (Fratti & Wickner, 2007). This was attributed to its interactions with proteins and not other \nlipids. Here we showed that Vam7 and Vam7 Y42A can both bind to b-PI(3,4,5)P 3 by BLI implying \nthat a second binding site exits as seen with other PX domains (Chandra et al , 2019). An \nalternative explanation could lie in the use of additional residues in the same binding pocket to \naccommodate PI(3,4,5)P3 binding that are not involved in PI3P binding. Thus, mutating Tyr42 to \nAla blocked PI3P binding while only attenuating PI(3,4,5)P3 binding. \n \nAlthough Vam7 strongly bound to PI(3,4,5)P\n3 we showed that it can be released from vacuoles \nwhen native PI(3,4,5)P3 is blocked Grp1-PH or modified with PTEN. This poses the question of \nwhy two lipid interactions are needed? While PI3P is delivered to vacuoles via endolysosomal \ntrafficking it is in low concentration and a second round of PI3P is produced on site (Thorngren \net al , 2004). It is possible that the ebb and flow of PI3P levels could negatively affect Vam7 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 13\nrecruitment to vacuoles and that a second ligand is needed for stable association with the \nmembrane. Originally, we hypothesized that PA could be the initial binding partner and that its \nconversion to DAG by Pah1 would lead to a hand off to PI3P. This was based on the required \nconversion of PA to DAG during pre-priming and the idea that Vam7 only binds one lipid at a \ntime (Sasser et al , 2012; Starr et al , 2019, 2016). Now we must consider that PI(3,4,5)P\n3 could \nbe the binding initiator, and that Vam7 could bind two lipids simultaneously.  \n \nAnother question to consider is whether Vam7-PI(3,4,5)P\n3 interactions affect Ypt7 localization. It \ncould be that changes in Vam7 conformation induced by PI(3,4,5)P 3 binding influences how \nHOPS interacts with Ypt7. This is not unlikely as both Vam7 and Ytp7 interact with the HOPS \ncomplex although not necessarily at the same time as seen by pulldown experiments (Price et \nal, 2000a; Seals et al , 2000; Brett et al, 2008; Stroupe et al , 2006; Collins et al , 2005; Fratti & \nWickner, 2007; Fratti et al, 2007). Thus, blocking PI(3,4,5)P3-Vam7 binding could prevent HOPS \nand Ypt7 retention at vertex sites. A more direct effect would through disrupting direct Vam7-\nYpt7 binding. A yeast-two-hybrid screen has shown that Vam7 and Ypt7 interact directly (Uetz \net al , 2000). This scenario is less likely as the screen did not include PI(3,4,5)P 3 binding. \nFurthermore, Vam7 pulldowns have failed to show Ypt7 (Stroupe et al, 2006).  \n \nFinally, how does PI(3,4,5)P 3 affect 3Q-SNARE complex formation? Our data showed that \nPTEN led to the exclusion of Vti1 and Vam7 from CBP-Vam3 complexes. The absence of Vam7 \nis attributed to its release from the membrane, but Vti1 has a transmembrane domain (TMD) \nand was not displaced from vacuoles. Interestingly, the hydrophilic helical region adjacent to the \nVti1 TMD has a poly basic region (PBR) with 5 Lys and Arg that could participate in binding \nnegatively charged lipids including PI(3,4,5)P\n3, however this remains to be tested. Other \nSNAREs including Syntaxin-1 and Syntaxin-17 have TMD-adjacent PBRs that bind to PIPs to \npromote function in chromaffin granule secretion and autophagy, respectively (Laczkó-Dobos et \nal, 2024; Lam et al, 2008). Future studies will test whether the Vti1 PBR-membrane interaction \npromotes binding to Vam3 followed by Vam7, if Vam7 binds Vam3, or if complex formation is \nconcurrent.  \n \nIn conclusion, this study has shown that PI(3,4,5)P 3 is critical in the regulation of vacuole \nhomotypic fusion at multiple steps. While this study ends at the trans-SNARE complex stage, \nthe gain of resistance data suggest that additional down-stream stages could be affected by \nPI(3,4,5)P\n3. Beyond vacuole fusion it is likely that PI(3,4,5)P 3 signals through the yeast \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 14\nhomologs of PDK1 (Pkh1/2), AKT1 (Sch9), PKC (Pkc1) and MAPK (Fus1, Kss1, Mpk1) to \ninfluence other functions such as autophagy, Ca 2+ transport and actin remodeling (Brady et al, \n2006; Inagaki et al , 1999; Ni et al, 2013; Levina et al , 2022; Asano et al , 2008; Dieterle et al, \n2014; Kassouf et al, 2015; Hsu et al, 2000).  \n \n \n \n \nMATERIALS AND METHODS  \nReagents. Reagents were solubilized in PIPES-Sorbit ol (PS) buffer (20 mM PIPES-KOH, pH \n6.8, 200 mM sorbitol) with 125 mM KCl unless indicated otherwise. PIPES [Piperazine-N-N’-\nbis(2-ethanesulfonic acid)], HEPES [N-(2-Hydroxyethyl)piperazine-N\n′ -(2-ethanesulfonic acid)], \nNEM (N-ethylmaleimide), Coenzyme A (CoA), Creatine kinase, and reduced glutathione were \npurchased from Sigma (St. Louis, MO) and dissolved in PS buffer or DMSO. Sorbitol, ATP, \nYeast extract, Tryptone, Glucose Tris base, Triton X100 and DTT were purchased from RPI \n(Mount Prospect, IL). FM4-64, Goat anti-rabbit IgG (H+L) secondary antibody DyLight 650 \nconjugate, Goat anti-mouse IgG (H+L) secondary antibody DyLight 650 conjugate and \nglutathione agarose were from Thermo-Fisher (Waltham, MA). Fluorescent CF488 goat-anti \nGST was from Biotium (Fremont, CA). Creatine phosphate was from Abcam (Waltham, MA). \nPIT-1, 3,5-dimethyl PIT-1, and SAR405 were from Cayman Chemical and dissolved in DMSO \n(Ann Arbor, MI). C8-PC (1,2-dioctanoyl-phosphatidylcholine), C8-PE (1,2-dioctanoyl-\nphosphatidylethanolamine), C8-PS (1,2-dioctanoyl-phosphatidylserine) were from Avanti \n(Alabaster, AL). C8-PI3P (1,2-dioctanoyl-phosphatidylinositol 3-phosphate), C8-PI(3,5)P\n2 (1,2-\ndioctanoyl-phosphatidylinositol 3,5-bisphosphate), C8-PI(4,5)P 2 (1,2-dioctanoyl-\nphosphatidylinositol 4,5-bisphosphate), C8-PI(3,4,5)P 3 (1,2-dioctanoyl-phosphatidylinositol \n3,4,5-trisphosphate), biotin-PI3P (b-PI3P), b-PI(3,4,5)P 3, Inositol-1,3,4-trisphosphate \n(Ins(1,3,4)P3), Inositol-1,3,5-trisphosphate (Ins(1,3,5)P 3), and Inositol-1,3,4,5-tetraphosphate \n(Ins(1,3,4,5)P4) were from Echelon (Salt Lake City UT). p-nitrophenyl phosphate was from MP \nBiomedicals (Santa Ana, Ca). Calmodulin agarose was Agilent (Santa Clara, CA). Octet \nStreptavidin (SA) biosensors were from Sartorius (Göttingen, Germany). Nitrocellulose was from \nBioRad (Hercules, CA).  \n \nPlasmids and Recombinant proteins \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 15\nRecombinant GST-Vam7, GST-Vam7 Y42A, GDI and Pbi2 (Inhibitor of proteinase B) were \nprepared as described previously (Fratti et al , 2007; Fratti & Wickner, 2007; Starai et al, 2007; \nSlusarewicz et al, 1997; Miner et al, 2016). Plasmids to produce GST-Grp1-PH (Kavran et al , \n1998), GST-PTEN WT, were from Addgene (Watertown, MA). The plasmid to make GST-Grp1-\nPHK273A was a gift from Dr. N. Leslie (Lindsay et al , 2006). Plasmids were transformed into E. \ncoli BL21 DE3 pLysS (New England Biolabs) and induced with 0.1 µM IPTG for 16h at 18° C. \nGST-tagged proteins were isolated using standard methods with glutathione agarose, eluted \nwith reduced glutathione and dialyzed against PS buffer with 125 mM KCl.  \n \n \nStrains and Vacuole isolation and fusion \nYeast strains (Tabel 1)  were grown in YPD (1% yeast extract, 2% peptone, 2% dextrorse) or \nsynthetic drop-out media without Trp. The pH of drop out media was adjusted to 6.0. Vacuoles \nwere isolated as described (Haas et al, 1994). In vitro  fusion reactions (30 µl) contained 3 µg \neach of vacuoles from BJ3505 ( PHO8 pep4Δ ) and DKY6281 ( pho8Δ PEP4) backgrounds, \nreaction buffer 20 mM PIPES-KOH pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl 2), ATP \nregenerating system (1 mM ATP, 0.1 mg/ml creatine kinase, 29 mM creatine phosphate), 10 µM \nCoA, and 283 nM Pbi2 (Protease B inhibitor). Fusion was determined by the processing of pro-\nPho8 (alkaline phosphatase) from BJ3505 by the Pep4 protease from DK6281. Fusion reactions \nwere incubated at 27°C for 90 min and Pho8 activity was measured in 250 mM Tris-HCl pH 8.5, \n0.4% Triton X-100, 10 mM MgCl 2, and 1 mM p-nitrophenyl phosphate. Pho8 activity was \ninhibited after 5 min by addition of 1 M glycine pH 11 and fusion units were measured by \ndetermining the p-nitrophenolate produced by detecting absorbance at 400 nm.  \n \nTrans-SNARE complex isolation.  \nTrans-SNARE pairing was measured as previously described with some modifications (Collins \n& Wickner, 2007; Jun et al, 2007; Qiu & Fratti, 2010; Sasser et al, 2012, 2013). Large scale 15X \n(450 µL) fusion reactions containing 45 µg of BJ3505 vacuoles ( VAM3 NYV1 ) and 45 µg of \nBJ3505 vacuoles that lacked NYV1 and contained Vam3 tagged with an internal calmodulin \nbinding peptide (CBP) between the H\nabc and SNARE domains ( CBP-VAM3 nyv1Δ). Reactions \nwere treated with buffer alone or 2 mM NEM as a negative control to prevent SNARE activation. \nSeparate reactions were treated with 50, 100, 150 or 200 nM GST-PTEN. Reactions were \nincubated for 60 min at 27 °C then placed on ice for 5 min before centrifugation (13,000 g, 15 \nmin, 4°C) to pellet membranes. The supernatants were carefully removed, and the pellets were \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 16\ncarefully overlaid and resuspended with 200 µL cold solubilization buffer (SB: 20 mM Tris-Cl, pH \n7.5, 150 mM NaCl, 1 mM MgCl 2, 0.5% Nonidet P-40 alternative, 10% glycerol) with protease \ninhibitors (1 mM PMSF and 1 cOmplete TM EDTA-free protease inhibitor tablet per 10 ml SB). \nReactions were brought up to 600 µL with additional SB and nutated at 4 °C for 20 min. \nInsoluble debris was removed by centrifugation (16,000 g, 20 min, 4 °C). Solubilized (580 µL) \nmaterial was transferred to pre-chilled tubes and 58 µL was removed from each reaction as \n10% of the total reactions. CaCl\n2 was added to each sample to a final concentration of 2 mM. \nNext, 50 µL of calmodulin beads equilibrated with SB. Mixtures were nutated overnight at 4 °C. \nCBP-Vam3 complexes bound material was collected by centrifugation (1,310 rpm, 2 min, 4 °C) \nand washed 4 times with 600 µL fresh ice-cold SB. Calmodulin bound material was eluted with \n1X SDS loading buffer containing 5 mM EGTA and boiled for 5 min. Samples were resolved by \nSDS-PAGE, transferred to nitrocellulose and probed with antibodies against Vam3, Vam7, \nNyv1, Vti1, Vps33 and Vps18. Bound primary antibodies were visualized with DyLight 650-Goat \nanti-rabbit IgG (H+L). \n \nFluorescence microscopy and Vertex microdomain formation \nIsolated vacuoles were subjected to docking assays as previously described (Fratti et al, 2004; \nWang et al , 2002) with slight modifications. Reactions (30 \nμ L) contained 6 μ g of vacuoles \nisolated from the indicated strains in fusion reaction buffer modified for docking conditions (PS \nbuffer, 100 mM KCl, 0.5 mM MgCl\n2, 0.33 mM ATP, 13 mM creatine phosphate, 33 μ g/mL \ncreatine kinase, 10 µM coenzyme A, and 280 nM IB 2). Measuring the vertex enrichment of \nfactors during tethering and docking was performed with vacuoles from cells expressing GFP \nfusion proteins or labeled with lipid binding probes. To track GFP-Ypt7 and Vps33 localization \nreactions were incubated under docking conditions as described above and stained with 4 μ M \nFM4-64 prior to examination (Wang et al, 2003; Fratti et al , 2004). To localize the distribution of \nPI(3,4,5)P3 on vacuoles, reactions were incubated with the PI(3,4,5)P 3 binding PH domain from \nGrp1. GST was then visualized with fluorescent (CF488) goat-anti-GST antibody. Briefly, \nreactions were treated with PS buffer, DMSO, PTEN or SAR405 for 5 min, followed by the \naddition of 150 nM GST-Grp1-PH for 5 min. Next, CF488-anti-GST antibody was added to each \nreaction and further incubated for 20 min. Following incubation at 27°C for 20 min, reactions \nwere mixed with 20 \nμ L of 0.6% low melt agarose in PS buffer melted at 50°C and cooled to prior \nto mixing with vacuoles. Next, 20 μ L aliquots were mounted on pr e-chilled slides and observed \nby fluorescence microscopy. Images were acquired using a Zeiss Axio Observer Z1 inverted \nmicroscope equipped with an X-Cite 120XL light source, Plan Apochromat 63X oil objective (NA \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 17\n1.4), and an AxioCam CCD camera. Quinacrine was visualized using a 38 HE EGFP shift-free \nfilter set and FM4-64 was visualized with a 42 HE CY 3 shift-free filter set.  Exposure times were \nset using WT vacuoles for each fluorescence channel and scripts acquired non-specific images \nfollowed by specific reporters. This ensures that bleaching is consistent to negate it as a factor \nin calculating intensity ratios. Exposure times were held constant within an experiment. \n \nImages were analyzed using ImageJ software (NIH). Vertex enrichment was determined by first \nmeasuring maximum fluorescence intensity in each channel at each contact point between \nmembranes, i.e., vertex domain within a cluster. Next, fluorescence intensity was measured in \neach channel at outer membrane domains where vacuoles are not in contact with other \nmembranes. The ratio of specific ( e.g., GFP) to non-specific ( e.g., FM4-64) was determined for \nvertices and outer membrane domains and compared for relative enrichment. Measurements for \neach condition were taken of 15-20 clusters to yield 100-300 vertices for each condition/strain \nper experiment. Data from multiple experiments are combined in column plots showing \nindividual values as well as the geometric means and geometric standard deviation for each \ncondition.  \n \nWestern blotting \nVacuoles were solubilized with 95 °C 1-5X Laemmli buffer for 5 min. Extracts were resolved \nusing 10% SDS-PAGE and transferred to nitrocellulose for immunoblotting. Rabbit antibodies \nagainst Actin, Nyv1, Vam3, Vps18, Vps33 and Ypt7 were prepared as described (Eitzen et al, \n2002; Nichols et al , 1997; Seals et al , 2000; Haas et al , 1995). Goat anti-rabbit IgG (H+L) \nantibody DyLight 650 conjugate was used as a secondary antibody. Fluorescence was \nmeasured with an Azure 400. \n \nBio-Layer Interferometry (BLI) \nVam7 binding to lipids was measured by BLI as described (Calderin et al, 2025). Biotinylated \nPI3P and PI(3,4,5)P3 were resuspended in PS buffer to a final stock concentration of 0.1 mM. \nLipids were diluted to 500 nM with BLI running buffer (PBS with 0.002% Tween-20 (v/v) and 190 \nµL was added to wells in a 96-well microplate. GST-Vam7, and GST-Vam7\nY42A was diluted to \n100, 200, 400, and 800 nM with BLI running buffer and 190 µL of each dilution, for each analyte, \nwere loaded to corresponding wells.  \n \nStatistical analysis \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 18\nFusion results were expressed as the mean ± SEM, mean ± 95% confidence interval (CI), or \ngeometric mean ± SD as needed. Experimental replicates (n) are defined as the number of \nseparate experiments. For comparison of vertex enrichment all the ratio data was log-\ntransformed to yield near-normal distribution with comparable variances. Non-parametric \nanalysis gave indistinguishable results. Statistical analysis was performed by unpaired two-\ntailed t-test or One-Way ANOVA for multiple comparisons using Prism 10 (GraphPad, San \nDiego, CA). Statistical significance is represented as follows: * p<0.05, ** p<0.01, *** p <0.001, \n**** p<0.0001. Tukey, Dunnett, and Šidák post hoc analysis was used for multiple comparisons \nand individual p-values.  \n \n \nData availability \nAll data generated or analyzed during this study are available upon request. Addition data \nsharing information is not applicable to this study. \n \nAuthor contributions \nConceptualization: C.Z., J.D.C. and R.A.F.; Data curation: C.Z., J.D.C. and R.A.F.; Formal \nanalysis: C.Z., J.D.C. and R.A.F.; Investigation: C.Z., J.D.C., A.T., V.S., J.M.K., C.K., R.A., D.G., \nE.W. and R.A.F.; Methodology: C.Z., J.D.C. and R.A.F.; Visualization: R.A.F.; Writing-original \ndraft: C.Z., J.D.C. and R.A.F.; Writing-review and editing: All authors; Resources: R.A.F.; \nSupervision: R.A.F.; Project administration: R.A.F.; Funding acquisition: R.A.F. \n \nAcknowledgements \nThe authors wish to thank Dr. William Wickner for the generous gifts of antibodies and Dr. \nNicholas Leslie for gifted plasmids. This research was supported by a grant from the National \nScience Foundation (MCB 2216742) to RAF. JDC was partially supported by an NIGMS-NIH \nChemistry-Biology Interface Training Grant (5T32-GM070421). \n \nConflict of interest \nThe authors declare that they have no conflict of interest. \n \nABBREVIATIONS \nBLI, bio-layer interferometry; C8, dioctanoyl; GDI, GDP dissociation inhibitor; NEM, N-\nethylmaleimide; NSF, NEM sensitive factor; PC, phosphatidylcholine; PE, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 26\nUetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, \nSrinivasan M, Pochart P, et al (2000) A comprehensive analysis of protein-protein \ninteractions in Saccharomyces cerevisiae. Nature 403: 623–627 \nUngermann C, Sato K & Wickner W (1998) Defining the functions of trans-SNARE pairs. Nature \n396: 543–548 \nWang L, Merz AJ, Collins KM & Wickner W (2003) Hierarchy of protein assembly at the vertex \nring domain for yeast vacuole docking and fusion. J Cell Biol 160: 365–374 \nWang L, Seeley ES, Wickner W & Merz AJ (2002) Vacuole fusion at a ring of vertex docking \nsites leaves membrane fragments within the organelle. 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It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 27\nFigures and Legends \n \n \n \nFigure 1. C8-PI(3,4,5)P\n3 potently inhibits fusion after vacuole tethering.  \nFusion reactions were treated with buffer alone or dosage curves of: C8-PI3P, C8-PI(3,5)P 2 and \nC8-PI(3,4,5)P3 (A); C8-PC, C8-PS and C8-PE (B); Ins(1,3,5)P3, Ins(1,3,4)P3 and Ins(1,3,4,5)P4 \n(C) and incubated for 90 min at 27ºC. Fusion was normalized to the maximum fusion (buffer \nalone) set to 1 for each curve. Data points show the average of multiple experiments (n ≥3) and \nSE. Each set was fit to one-phase decay curves. IC 50 values were determined using Graphpad \nPrism 10. (D) Gain of resistance fusion reactions were performed with buffer alone, 1 mM NEM, \n2 µM GDI or 150 µM C8-PI(3,4,5)P 3. Individual reactions were treated with reagents or buffer at \nthe indicated time points. A second set of buffer-treated reactions were placed on ice at the \nindicated times. Fusion reactions were incubated for a total of 120 min. The amount of fusion for \neach reaction was normalized to the untreated control for the indicated time point at 27°C set to \n1. Data points show the average of multiple experiments (n ≥3) and SE. Each set was fit to one-\nphase decay curves. (E) Calculated half-times of resistance from assays in (C). Error bars \nrepresent SEM (n=3) \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 28\n \n \n \n \nFigure 2. PIP3 specific reagents inhibit vacuole fusion. \nFusion reactions were treated with buffer alone or dose response curves of: GST-Grp1-PH and \nGST-Grp1-PHK237A (A); GST-PTEN (B); PIT-1 and 3,5-dimethyl PIT-1 (DMPIT) (C) and \nincubated for 90 min at 27ºC. Each curve was normalized to the maximum fusion (no treatment) \nset to 1. Data points shown are the averages of multiple experiments (n ≥3) and SE. Each data \nset was fit to one-phase decay curves and IC 50 values were determined using Graphpad Prism \n10. (D) Fusion inhibition by PTEN (200 nM) was rescued with 200 µM PIT-1. Fusion efficiency \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 29\nwas normalized to PTEN alone set to 1. (E) Fusion inhibition by Grp1-PH (2.3 µM) was rescued \nwith 200 µM PIT-1. Fusion efficiency was normalized to Grp1-PH alone set to 1 (F) Fusion \ninhibition by wild type Grp1-PH and Grp1-PH K237A (2.3 µM) was rescued with 200 µM DMPIT. \nFusion efficiency was normalized to Grp1-PH or Grp1-PH K237A alone set to 1.  (G) Fusion \ninhibition by PTEN (200 nM) was rescued with 200 µM DMPIT. Fusion efficiency was \nnormalized to PTEN alone set to 1. (H)  Gain of resistance fusion reactions were performed with \nbuffer alone, 1 mM NEM, 2 µM GDI 2 µM GST-Grp1-PH, or 200 nM PTEN. Reactions were \ntreated with reagents or buffer at each time point. A second set of untreated reactions was \nplaced on ice at each timepoint. Fusion reactions were incubated for a total of 120 min and \nfusion for each reaction was normalized to the untreated control for the each timepoint at 27°C \nset to 1. Data sets show the average of multiple experiments (n ≥3) and SE. Each set was fit to \none-phase decay curves. (H)  Calculated half-times of resistance from assays in (G) . Error bars \nrepresent mean ± SE (n=3). In panels D-G, significance was determined unpaired two-tailed t-\ntest. * p<0.05, ** p < 0.01. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 30\n \n \n \nFigure 3. PI(3,4,5)P3 is made by Vps34 and localizes at the vertices of docked vacuoles.   \nDocking reactions were incubated for 30 min at 27ºC with 150 nM GST-Grp1-PH to mark \nPI(3,4,5)P3. Grp1-PH was visualized by adding CF488-anti GST polyclonal antibody (1:500) at \nthe end of the reaction and 5 µM FM4-64 to label entire vacuoles. Vacuoles were pelleted \n(5,000 g, 5 min, 4 °C) to remove excess antibody fluorescence and resuspended in PS buffer. \nDocking reactions were treated with: 200 µM SAR405 or DMSO (A); 50 nM PTEN or PS buffer \nalone (C). Reactions were mixed 1:1 with 0.6% low melt agarose and prepared for fluorescence \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 31\nmicroscopy. (B) Quantitation of ratiometric fluorescence intensities of vertices (V) and outer \nedge (O) in panel A. Data points were pooled from multiple experiments and each experiment \ncontained ratios from 10 or more fields with 10-15 clusters made of ≥  5 vacuoles (n > 500 \nvertices and > 200 outer edges for each condition). Error bars represent geometric means ± \ngeometric SD (n=3). Significance was determined using one-way ANOVA for multiple \ncomparisons. Tukey’s post-hoc test of multiple comparison was used for individual p-values. **** \np < 0.0001. Scale bars: 5 µm. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 32\n \n \n \nFigure 4. PI(3,4,5)P3 competes Grp1-PH binding to vertices \n(A) Docking reactions were incubated for 30 min at 27ºC with 150 nM GST-Grp1-PH and \nvisualized by adding CF488-anti GST antibody as described in Figure 3. Docking reactions were \ntreated with 300 nM C8-PI(4,5)\n2, C8-PI(3,4,5)P 3, or buffer alone. At the end of the incubation \nperiod, reactions were placed on ice, labeled FM4-64 and prepared for fluorescence microscopy \nas described. (B) Quantitation of ratiometric fluorescence intensities of vertices (V) and outer \nedge (O) in panel A. Data points were pooled from multiple experiments as described in Figure \n3. Error bars represent geometric means ± geometric SD (n=3). Significance was determined \nusing one-way ANOVA for multiple comparisons. Tukey’s post-hoc test of multiple comparison \nwas used for individual p-values. **** p < 0.0001. NS, not significant. Scale bars: 5 µm. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 33\n \n \n \nFigure 5. Grp1-PH blocks GFP-Ypt7 enrichment at vertices \n(A) Docking reactions with vacuoles harboring GFP-Ypt7 were treated with unlabeled 2 µM \nGrp1-PH to inhibit fusion or buffer and incubated for 30 min at 27°C. At the end of the incubation \nperiod, reactions were prepared for fluorescence microscopy. (B) Quantitation of ratiometric \nfluorescence intensities of vertices (V) and outer edge (O) in panel A. (C) Docking reactions with \nGFP-Ypt7 vacuoles were treated with 250 µM C8-PI(3,4,5)P 3 to inhibit fusion or buffer and \nincubated for 30 min at 27ºC. (D) Quantitation of ratiometric fluorescence intensities of vertices \n(V) and outer edge (O) in panel A. Data points were pooled from multiple experiments as \ndescribed. Error bars represent geometric means ± geometric SD (n=3). Significance was \ndetermined using one-way ANOVA for multiple comparisons and Tukey’s post-hoc test was \nused for individual p-values. **** p < 0.0001. Scale bars: 5 µm. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 34\n \n \n \nFigure 6. Free PI(3,4,5)P3 is required for Vps33 localization to vertex microdomains. \n(A) Docking reactions were performed with vacuoles harboring Vps33-GFP in the presence \nbuffer (control), 250 µM C8-PI(3,4,5)P3 or 2 µM Grp1-PH At the end of the incubation period, \nreactions were processed for fluorescence microscopy as described above. (B-C) Quantitation \nof ratiometric fluorescence intensities of vertices (V) and outer edge (O) in panel (A). Error bars \nrepresent geometric means ± geometric SD (n=3). Significance was determined using one-way \nANOVA for multiple comparisons and Tukey’s post-hoc test for individual p-values. **** p < \n0.0001; Scale bars: 5 µm. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 35\n \n \n \nFigure 7. PTEN inhibits trans-SNARE pairing.  \n(A) Fusion reactions were treated with buffer alone (Control), 2 mM NEM, or GST-PTEN at the \nindicated concentrations. Reactions were incubated for 60 min at 27\n°C. Protein complexes \ncontaining CBP-Vam3 were isolated with calmodulin beads. Proteins were resolved by SDS-\nPAGE, transferred to nitrocellulose and immunoblotted for Vam3, Nyv1, Vam7, Vti1, Vps33 and \nVps18. (B) Quantitation of proteins bound to CBP-Vam3 in the presence of NEM. Data \nrepresents mean and SEM (n=3). Significance was determined using one-way ANOVA for \nmultiple comparisons and Dunnett’s post-hoc of multiple comparison was used for individual p-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 36\nvalues. ** p < 0.01. (C) Quantitation of proteins bound to CBP-Vam3 in the presence of PTEN. \nData represents mean and SEM (n=3). Significance was determined using one-way ANOVA for \nmultiple comparisons and Šidák’s post-hoc test for pairwise comparisons between CBP-Vam3 \nand specified proteins in the presence of PTEN for individual p-values. * p < 0.05, ** p < 0.01, \n*** p < 0.001. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 37\n \n \n \nFigure 8. Sequestering PI(3,4,5)P3 with GST-Grp1-PH releases Vam7 from membranes.  \n(A) Fusion reactions were treated with buffer alone (Control) or GST-Grp1-PH at the indicated \nconcentrations. Reactions were incubated for 60 min at 27 °C. Membrane-bound and unbound \nproteins were separated by centrifugation (16,000, 10 min, 4 °C) into supernatant (unbound) and \npellet (bound) fractions. Pellets were resuspended in PS buffer to match the starting volumes. \nBound and unbound fractions were mixed with SDS-loading buffer, separated by SDS-PAGE, \ntransferred to nitrocellulose and probed for with antibodies against Actin, Nyv1, Vam7, Ypt7, \nVps33 and Vps18. (B) Quantitation of proteins released from vacuoles in the presence of Grp1-\nPH. Data represents mean and SEM (n=4). Significance was determined using one-way \nANOVA for multiple comparisons and Šidák’s post-hoc test for pairwise comparisons between \neach protein released absence or presence of GST-Grp1-PH for individual p-values. **** p < \n0.0001. L, ladder; W, whole reaction. (C-D) BLI binding curves of response units versus protein \nconcentrations of wild type Vam7 and Vam7\nY42A.   \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint \n\nVacuole Fusion Regulation by PI(3,4,5)P3 \n 38\n \n \n \n \n \n \nTable 1. Strains used in this study \n \nTable 1. Strains used in this study. \nStrain Genotype Source \nBJ3505 MATa pep4::HIS3 prb1-∆ 1.6R his3-200 lys2-801 trp1∆ 101 \n(gal3) ura3-52 gal2 can1 \n(Jones et al., \n1982)  \nDKY6281 MATα leu2-3,112 ura3-52 his3-∆ 200 trp1-∆ 901 lys2-801  \nsuc2-Δ9 pho8Δ::TRP1 \n(Klionsky and Emr, \n1989) \nSEY6210 MAT\nα leu2-3,112 ura3-52 his3-∆ 200 trp1-∆ 901 lys2-801  \nsuc2-Δ9 \n(Klionsky and Emr, \n1989) \nGFP-Ypt7 SEY6210, ypt7::HIS3 pRS304:GFP-Ypt7 (TRP1) (Wang et al., \n2002) \nVps33-GFP DKY6281, VPS33-GFP (Wang et al., \n2002) \nGFP-Vps39 SEY6210, vps39::HIS3 pYlPlac211-GFP-VPS33 (TRP) (Wang et al., \n2002) \nCBP-Vam3 \nnyv1Δ \nBJ3505, CBP-VAM3::Kan\nr nyv1::natr (Collins & \nWickner, 2007) \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 2, 2025. ; https://doi.org/10.1101/2025.08.01.668199doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}