Supplementary methods
Plant transformation and selection. Each PIPline construct was transformed into C58 GV3101
Agrobacterium strain and selected on YEB media (5g/L beef extract; 1g/L yeast extract; 5g/L
peptone; 5g/L sucrose; 15g/L bactoagar; pH 7.2) supplemented with antibiotics (Spectynomicin,
Gentamycin). After two days of growth at 28°C, bacteria were collected using a single-use cell scraper,
re-suspended in about 200mL of transformation buffer (10mM MgCl2; 5% sucrose; 0.25% silweet)
and plants were transformed by dipping.
Primary transformants (T1) were selected in vitro on the appropriate antibiotic/herbicide (glufosinate
for CITRINE-tagged PIPline, hygromycin for CHERRY-tagged PIPline and kanamycin for CyPETtagged PIPline). 24 independent T1s were selected for each PIPline. In the T2 generation at least 3
independent transgenic lines were selected using the following criteria when possible: i) good
expression level in the root for detection by confocal microscopy, ii) uniform expression pattern, iii)
single insertion line (1 sensitive to 3 resistant segregation ratio) and, iv) line with no obvious abnormal
developmental phenotypes. PIPlines for which we could not find fluorescence out of 24 independent
lines or that did not associated with any membrane compartments (including the PM) were not further
analysed. The remaining PIPlines were rescreened in T3 using similar criteria as in T2 with the
exception that we selected homozygous lines (100% resistant). At this step, we selected one transgenic
line for each PIPline that was used for further analysis and crosses and that will be distributed to the
Arabidopsis stock centres.
Quantitative image analysis. The quantification of low and high affinity phosphoinositide biosensor
distribution was done using ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij).
For the comparison between 1xPHFAPP1 vs 2xPHFAPP1 and 1xPHPLC vs 1xPHPLC, cell edges were
delimited manually and the fluorescence intensities were quantified within the cell, i.e. without the PM,
and in the entire cell using the “Integrated Density” measurement tool of ImageJ. Quantitative results
were expressed as the ratio of PM signal intensity to the intracellular signal intensity. This ratiometric
measurement allowed to minimized variation in signal intensities between different cells or roots. For
the comparison between 1xFYVEHRS and 2xFYVEHRS we used FM4-64 (3µM, 3 min) to counterstain
the PM and delimitate cell edges. First, the fluorescence was quantified in the entire cell as described
above. Next, we selected endomembrane compartments using the “Threshold” tool of ImageJ, in
order to select and then quantify the fluorescence of objects (i.e. endosomes) with higher signal than
the cytoplasm. Quantitative results were expressed as the ratio of endosomal signal intensity to the
signal intensity of the cytosol only.
For quantitative co-localisation, we used an object-based analysis (OBA). OBA is used to determine
the centroid of each spot (intracellular compartment) and to compare their respective localisation. Colocalisation between the two structures is determined if the distance between the two centroids is
below the optical resolution (Bolte & Cordelieres, 2006). The OBA was performed as followed; first
the intracellular compartments were automatically detected in each channel using the spot detector
function of the Icy software (http://icy.bioimageanalysis.org/). Next, using Fiji software
(http://fiji.sc/) we removed the remaining PM signal and performed the OBA using the Jacop plugin
of Fiji (Bolte & Cordelieres, 2006). Co-localisation was quantified in 30 cells per crosses.
Protein extraction and protein-lipid overlay assay: 1mg of fresh material (7 days-old seedlings) was
grinded in liquid nitrogen, resuspended in 200μL of ice-cold extraction buffer (500mM sucrose; 50mM
Hepes; 5mM EDTA; 5mM EGTA; pH 7.4, complete protease inhibitor cocktail, Roche) and
centrifuged twice at 13,000g (15 min, 4°C) or until the supernatant was cleared. 20µL of cleared
protein extracts were analysed by western blot (1:1500 anti-GFP, Roche) and 100µL were used for
protein-lipid overlay assay. Membrane strips pre-spotted with lipids (PIP strip and PIP array, tebu-bio)
were blocked 1h in blocking solution (10mM Tris-HCl; pH8.0; 150mM NaCl; tween-20 0,1%; 3%
BSA) and then incubated 1h with 100μL of protein extracts in 8mL of blocking solution. The
immuno-detection was performed according to the manufacturer’s instruction using an anti-GFP
antibody (1:1500, Roche).
32
P-phospholipid labelling, extraction and analysis: A protocol recently described in detail by
Munnik and Zarza (2013) was used. In brief, five-days-old seedlings grown on 0.5 MS 0.8 % agar
plates supplemented with 1% sucrose, were transferred to 2 ml Safe-lock Eppendorf tubes, containing
200 µL of labelling buffer (2.5 mM MES (2-[N-Morpholino]ethane sulfonic acid, pH 5.7 with KOH,
plus 1 mM KCl); each tube holding three seedlings. To label their phospholipids, seedlings were
incubated overnight for ~16 hrs with 1 µL (2-10 µCi) carrier-free 32P-PO43- (32Pi; PerkinElmer, The
Netherlands). Next day, seedlings were treated by adding 200 µL of buffer at room temperature (22 °C
= control), or buffer supplemented with NaCl (250 mM final, at RT) or buffer at 40 °C (heat stress).
Reactions were stopped by adding 50 µL of 50% (w/v) perchloric acid, followed by 10 min of shaking.
The total solvent was then removed and 400 μl of CHCl3/MeOH/HCl (50:100:1) added to start
extracting their lipids. After 10 min of shaking, two phases were induced by adding 400 μl CHCl3 and
200 μl 0.9% (w/v) NaCl. The organic lower phase was then transferred to a tube containing 400 μl
CHCl3/MeOH/1M HCl (3:48:47), which was again shaken for 5 min, afterwhich the upper phase was
removed and the organic lower phase dried down in a vacuum centrifuge at 54 °C. The residue was
resuspended in 100 μl CHCl3 and sampled for lipid analysis.
Phospholipids were analysed by thin-layer chromatography (TLC) on silica gel 60 plates (20x20
cm; Merck) that were K-oxalate impregnated and heat activated (Munnik and Zarza, 2013). An alkaline
solvent, composed of CHCl3/MeOH/NH4OH(25%)/H2O (90:70:4:16), was used to separate the
different phospholipid species. Radiolabelled phospholipids were visualized by autoradiography and
quantified by phosphoimaging. Each sample was composed of three seedlings and each experiment
was performed in triplicate, which was repeated independently twice.
Cloning of the PIP-line constructs: 1xFYVEHRS and 2xFYVEHRS were PCR amplified using
FYVE-B2R / FYVE-B3wSTOP primers (see Table S2) and myc-2xFYVE plasmid as template (gift
from H. Stenmark, The Norwegian Radium Hospital, Oslo, Norway) (Gillooly et al., 2000). The PCR
products corresponding to 1xFYVE and 2xFYVE amplicons were then recombined into the
pDONRP2R-P3 (Invitrogen) by BP reaction (Gateway, Invitrogen) to obtain 1xFYVEp3’ and
2xFYVEp3’ respectively. 1xAtPH1 was PCR amplified using AtPH1-B2R-Xho / AtPH1-B3wSTOP
primers and arabidopsis seedling cDNA as template. The PCR product was then recombined into
pDONRP2R-P3 to obtain 1xAtPH1p3’. AtPH1 was then PCR-amplified using AtPH1-Xho-F / AtPH1Xho-R primers and cloned into 1xAtPH1p3’ using XhoI restriction enzyme to obtain 2xAtPH1p3’.
1xPXp40 was PCR amplified using p40phox-B1 / p40phox-B2GAGAnoSTOP primers and p40PX-EGFP
(addgene plasmid # 19010 (http://www.addgene.org/), gift of Michael Yaffe, MIT Cambridge, USA)
(Kanai et al., 2001). The corresponding PCR product was recombined into pDONR221 (Invitrogen) to
obtain 1xp40p221. 1xPHOSH2 and 2xPHOSH2 were PCR amplified using OSH2-B2R / OSH2-B3 primers
and pTL511[(GFP-PH(osh2)-linker-PH(osh2)-GFP)] plasmid as template (gift from Timothy Levine
UCL, London, UK) (Roy & Levine, 2004). The PCR products corresponding to 1xPHOSH2 and
2xPHOSH2 amplicons were then recombined into the pDONRP2R-P3 to obtain 1xOSH2p3’ and
2xOSH2p3’ respectively. 1xPHFAPP1 was PCR amplified using FAPP1-B2R-XhoI / FAPP1-B3-wSTOP
primers and pTL334[GFP-PH(FAPP1)] plasmid as template (gift from Timothy Levine UCL, London,
UK)
(Levine & Munro, 2002). The corresponding PCR product was then recombined into
pDONRP2R-P3 to obtain 1xFAPP1p3’. 1xPHFAPP1 was then PCR-amplified using FAPP1-XhoI-F /
FAPP1-XhoI-R primers and cloned into 1xFAPP1p3’ using XhoI restriction enzyme to obtain
2xFAPP1p3’. 1xGRAMAtg26 was PCR amplified using GRAM-B2R-XhoI / GRAM-B3wSTOP and
pJCF392[GFP-Atg26] (gift from Jean Luc Farré, UCSD, San Diego, USA). The corresponding PCR
product was then recombined into pDONRP2R-P3 to obtain 1xGRAMp3’. 1xGRAMAtg26 was then
PCR-amplified using GRAM-Xho-F / GRAM-Xho-R primers and cloned into 1xGRAMp3’ using XhoI
restriction enzyme to obtain 2xGRAMp3’. 1xPHOSBP was PCR amplified using OSBP-B2R-Bgl2 / OSBPB3wSTOP primers and pTL332[GFP-PH(OSBP)] plasmid as template (gift from Timothy Levine UCL,
London, UK) (Levine & Munro, 2002). The corresponding PCR product was then recombined into
pDONRP2R-P3 to obtain 1xOSBPp3’. 1xPHOSBP was then PCR-amplified using OSBP-Bgl2-F / OSBPBgl2-R primers and cloned into 1xOSBPp3’ using BglII restriction enzyme to obtain 2xOSBPp3’.
3xPHDING2 was PCR amplified using PHD-B2R / PHD-B3wSTOP primers and GFP-PHDx3 plasmid
as template (gift of Or Gozzani, Stanford, USA) (Gozani et al., 2003). The corresponding PCR
product was then recombined into pDONRP2R-P3 to obtain 3xING2p3’. 1xPXp47 was PCR amplified
using p47phox-B1 / p47phox-gaga-B2noSTOP primers and pGEX-p47PX plasmid as template (addgene
plasmid # 19007, gift of Michael Yaffe, MIT Cambridge, USA) (Kanai et al., 2001). The corresponding
PCR product was then recombined into pDONR221 to obtain 1xp47p221. 1xPHTAPP1 was PCR
amplified using TAPP1-B2R-gaga / TAPP1-B3wSTOP primers and TAPP1/pEGFP-C1 plasmid as
template (gift from Aaron Marshall, University of Manitoba, Canada) (Marshall, Krahn, Ma, Duronio,
& Hou, 2002). The corresponding PCR product was then recombined into pDONRP2R-P3 to obtain
1xTAPP1p3’. 1xPHTAPP2 was PCR amplified using TAPP2-B2R-gaga / TAPP2-B3wSTOP primers and
TAPP2/pEGFP-C1 plasmid as template (gift from Aaron Marshall, University of Manitoba, Canada)
(Marshall et al., 2002). The corresponding PCR product was then recombined into pDONRP2R-P3 to
obtain 1xTAPP2p3’. 1xENTHEnt3p was PCR amplified using Ent3-B1 / Ent3-B2noSTOP primers and
GST-ent3p(ENTHdomain) plasmid as template (gift from Silvie Friant, GMGM, Strasbourg, France)
(Friant et al., 2003). The corresponding PCR product was then recombined into pDONR221 to obtain
1xEnt3p221. 1xENTHEnt5p was PCR amplified using Ent5-B1 / Ent5-B2noSTOP primers and GSTent5p(ENTHdomain) plasmid as template (gift from Silvie Friant, GMGM, Strasbourg, France) (Eugster
et al., 2004). The corresponding PCR product was then recombined into pDONR221 to obtain
1xEnt5p221. 1xPHPLCδ1 and 2xPHPLCδ1 were PCR amplified using PLC-B2R / PLC-B3wSTOP primers
and pTL336[GFP-PH(PLCd1)dimer)] plasmid as template (gift from Timothy Levine UCL, London,
UK) (Levine & Munro, 2002). The PCR amplicons corresponding to 1xPHPLCδ1 and 2xPHPLCδ1 were
then recombined into pDONRP2R-P3 to obtain 1xPLCp3’ and 2xPLCp3’ respectively. 1xTUBBYCTUBBY was PCR amplified using Tubby-B2R / Tubby-B3wSTOP primers and pGFPtub-fl/pEGFP-C1
plasmid as template (gift from Lawrence Shapiro, NYU, New York, USA) (Santagata et al., 2001). The
corresponding PCR product was then recombined into pDONRP2R-P3 to obtain 1xTUBBYp3’.
1xTUBBY-CTUBBY was then PCR-amplified using TUBBY-Xho-F / TUBBY-linkerBsrgI-R primers and
cloned into 1xTUBBYp3’ using XhoI and BsrgI restriction enzymes to obtain 2xTUBBYp3’. 1xPHAKT
was PCR amplified using AKT-B1 / AKT-B2noSTOP primers and pcDNA3-AKT-PH-GFP plasmid as
template (addgene plasmid # 18836, gift from Craig Montell, John Hopkin university, Baltimore, USA)
(Kwon, Hofmann, & Montell, 2007). The corresponding PCR product was then recombined into
pDONR221 to obtain 1xAKTp221. 1xPHBTK was PCR amplified using BTK-B1 / BTK-B2noSTOP
primers and pDONR223-BTK plasmid as template (addgene plasmid # 23918, gift from William Hahn
and David Root) (Johannessen et al., 2010). The corresponding PCR product was then recombined
into pDONR221 to obtain 1xBTKp221. 2xCHERRY and 2xCyPET were PCR amplified from
2xCHERRY-6xmyc/pBJ36 and 2xCyPet-3xFlag-6xHis/pBJ36
(both vectors are gift from J. Long,
UCLA, Los Angeles, USA) and recombined into both pDONR221 and pDONRP2R-P3 to obtain
2xCHERRY-1xmyc_noSTOP/pDONR221, 2xCyPet-3xFlag6xHis_noSTOP/pDONR221, 2xCHERRY4xMyc/pDONRP2R-P3, 2xCyPet-3xFlag6xHis_noSTOP/pDONRP2R-P3 (See Table S3 for a list of the
primers
used).
UBIQUITIN10prom/pDONRP4-P1R,
mCITRINEnoSTOP/pDONR221,
mCITRINE/pDONRP2R-P3 have been described before (Jaillais et al., 2011). Final destination vectors
were obtained by using three fragments recombination system (Invitrogen) using the pB7m34GW,
pH7m34GW, pK7m34GW destination vectors (Table S4) (Karimi, Bleys, Vanderhaeghen, & Hilson,
2007). The final constructs are named PnY for the CITRINE constructs in pB7m34GW, PnR for the
CHERRY constructs in pH7m34GW and PnC for the CyPet constructs in pK7m34GW (Table S4).
Construction of yeast and human expression vectors. Each LBD (with the exception of 1xPXp40)
was cloned with a C-terminal STOP codon into the pDONR221 to give 1xFYVEwSTOPp221,
1xFAPP1wSTOPp221, 1xOSBPwSTOPp221, 1xPLCwSTOPp221, 1xTUBBYwSTOPp221 (See table S2
for a name and full sequence of the primers used). These entry vectors were then recombined into
pAG425GPD-EGFP-ccdb (addgene clone #14322, gift of Susan Lindquist) and pcDNA-DEST53
(Invitrogen) for N-terminal GFP fusion and expression in yeast and human cells respectively.
1xp40p221 was recombined into pAG425GPD-ccdb-EGFP (addgene clone #14202, gift of Susan
Lindquist) and pcDNA-DEST47 (Invitrogen) for C-terminal GFP fusion and expression in yeast and
human cells respectively.
Reporter expression combined with immunofluorescence analysis in human cell line. 2x105
Huh-7 cells (Dreux et al., 2012) were plated on coverglass (Thermo Fisher Scientific). Next day, cells
were transfected with 1.5 μg of purified plasmid by using Xtreme-GENE HP DNA Transfection
Reagent (Roche) and following the manufacturer’s instructions. Twenty-four later, cells were washed
with PBS (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3mMNa2HPO4, and 1.4mMKH2PO4) and fixed 20
min at room temperature in 4% paraformaldehyde. Immunofluorescence detections were performed
as previously described (Dreux, Gastaminza, Wieland, & Chisari, 2009) using antibodies against EEA1 0.5 μg/ml (BD Biosciences) and GM130 (BD Biosciences) at 2.5 μg/ml, respectively, and Alexa555-conjugated anti-mouse antibodies (Molecular Probes) diluted in PBS supplemented with 3% BSA
and 0.1% Triton X-100. Nuclei were labeled with Hoechst dye (Molecular Probes) and coverglass were
mounted on microscope slide by using SlowFade antifade Kit (Molecular Probes).
Supplementary method references:
Bolte, S., & Cordelieres, F. P. (2006). A guided tour into subcellular colocalization analysis in light
microscopy. Journal of Microscopy-Oxford, 224(Pt 3), 213–232. doi:10.1111/j.1365-2818.2006.01706.x
Burd, C. G., & Emr, S. D. (1998). Phosphatidylinositol(3)-phosphate signaling mediated by specific
binding to RING FYVE domains. Molecular cell, 2(1), 157–162.
Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P., & Alessi, D. R. (2000).
Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositidebinding specificities. Biochemical Journal, 351(Pt 1), 19–31.
Dreux, M., Garaigorta, U., Boyd, B., Décembre, E., Chung, J., Whitten-Bauer, C., et al. (2012). ShortRange Exosomal Transfer of Viral RNA from Infected Cells to Plasmacytoid Dendritic Cells
Triggers Innate Immunity. Cell host & microbe, 12(4), 558–570. doi:10.1016/j.chom.2012.08.010
Dreux, M., Gastaminza, P., Wieland, S. F., & Chisari, F. V. (2009). The autophagy machinery is
required to initiate hepatitis C virus replication. Proceedings of the National Academy of Sciences of the
United States of America, 106(33), 14046–14051. doi:10.1073/pnas.0907344106
Eugster, A., Pécheur, E.-I., Michel, F., Winsor, B., Letourneur, F., & Friant, S. (2004). Ent5p is
required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular
body. Molecular biology of the cell, 15(7), 3031–3041. doi:10.1091/mbc.E03-11-0793
Farré, J.-C., Vidal, J., & Subramani, S. (2007). A cytoplasm to vacuole targeting pathway in P. pastoris.
Autophagy, 3(3), 230–234.
Franke, T. F., Kaplan, D. R., Cantley, L. C., & Toker, A. (1997). Direct Regulation of the Akt ProtoOncogene Product by Phosphatidylinositol-3,4-bisphosphate. Science, 275(5300), 665–668.
doi:10.1126/science.275.5300.665
Friant, S., Pécheur, E. I., Eugster, A., Michel, F., Lefkir, Y., Nourrisson, D., & Letourneur, F. (2003).
Ent3p Is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body.
Developmental Cell, 5(3), 499–511.
Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Gaullier, J. M., et al. (2000).
Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. The EMBO Journal,
19(17), 4577–4588. doi:10.1093/emboj/19.17.4577
Godi, A., Campli, A. D., Konstantakopoulos, A., Tullio, G. D., Alessi, D. R., Kular, G. S., et al. (2004).
FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nature
Cell Biology, 6(5), 393–404. doi:10.1038/ncb1119
Gozani, O., Karuman, P., Jones, D. R., Ivanov, D., Cha, J., Lugovskoy, A. A., et al. (2003). The PHD
finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide
receptor. Cell, 114(1), 99–111.
Jaillais, Y., Hothorn, M., Belkhadir, Y., Dabi, T., Nimchuk, Z. L., Meyerowitz, E. M., & Chory, J.
(2011). Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering
membrane release of its kinase inhibitor. Genes and development, 25(3), 232–237.
doi:10.1101/gad.2001911
Johannessen, C. M., Boehm, J. S., Kim, S. Y., Thomas, S. R., Wardwell, L., Johnson, L. A., et al.
(2010). COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.
Nature, 468(7326), 968–972. doi:10.1038/nature09627
Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., et al. (2001). The PX domains of
p47phox and p40phox bind to lipid products of PI(3)K. Nature Cell Biology, 3(7), 675–678.
doi:10.1038/35083070
Karimi, M., Bleys, A., Vanderhaeghen, R., & Hilson, P. (2007). Building blocks for plant gene
assembly. Plant Phys, 145(4), 1183–1191. doi:10.1104/pp.107.110411
Kwon, Y., Hofmann, T., & Montell, C. (2007). Integration of phosphoinositide- and calmodulinmediated regulation of TRPC6. Molecular cell, 25(4), 491–503. doi:10.1016/j.molcel.2007.01.021
Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., & Schlessinger, J. (1995). Specific and
high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proceedings
of the National Academy of Sciences of the United States of America, 92(23), 10472–10476.
Levine, T. P., & Munro, S. (2002). Targeting of Golgi-specific pleckstrin homology domains involves
both PtdIns 4-kinase-dependent and -independent components. Current Biology, 12(9), 695–704.
Marshall, A. J., Krahn, A. K., Ma, K., Duronio, V., & Hou, S. (2002). TAPP1 and TAPP2 are targets
of phosphatidylinositol 3-kinase signaling in B cells: sustained plasma membrane recruitment
triggered by the B-cell antigen receptor. Molecular and cellular biology, 22(15), 5479–5491.
Munnik T. & Zarza X. (2013) Analyzing plant signaling phospholipids through 32Pi-labeling and TLC.
Methods Mol. Biol. 1009: 3-16.
Rameh, L. E., Arvidsson, A.-K., Carraway, K. L., Couvillon, A. D., Rathbun, G., Crompton, A., et al.
(1997). A comparative analysis of the phosphoinositide binding specificity of pleckstrin
homology domains. The Journal of biological chemistry, 272(35), 22059–22066.
Roy, A., & Levine, T. P. (2004). Multiple Pools of Phosphatidylinositol 4-Phosphate Detected Using
the Pleckstrin Homology Domain of Osh2p. The Journal of biological chemistry, 279(43), 44683–
44689. doi:10.1074/jbc.M401583200
Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., et al. (1996). Distinct
specificity in the recognition of phosphoinositides by the pleckstrin homology domains of
dynamin and Bruton's tyrosine kinase. The EMBO Journal, 15(22), 6241.
Santagata, S., Boggon, T. J., Baird, C. L., Gomez, C. A., Zhao, J., Shan, W. S., et al. (2001). G-protein
signaling through tubby proteins. Science (New York, NY), 292(5524), 2041–2050.
doi:10.1126/science.1061233
Simonsen, A., Lippé, R., Christoforidis, S., Gaullier, J. M., Brech, A., Callaghan, J., et al. (1998). EEA1
links PI(3)K function to Rab5 regulation of endosome fusion. Nature, 394(6692), 494–498.
doi:10.1038/28879
Yamashita, S. I., Oku, M., Wasada, Y., Ano, Y., & Sakai, Y. (2006). PI4P-signaling pathway for the
synthesis of a nascent membrane structure in selective autophagy. The Journal of Cell Biology, 173(5),
709–717. doi:10.1083/jcb.200512142
Yu, J. W., Mendrola, J. M., Audhya, A., Singh, S., Keleti, D., Dewald, D. B., et al. (2004). Genomewide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Molecular cell,
13(5), 677–688.