350 cutoff ln(growth-ratio) Rescuer candidates that localize to the plasma membrane. 1

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Olivia Owen
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1 DOI: 1.138/nc2444 Δdnf2 35 cutoff 3 25 # of strins ln(growth-rtio) Rescuer cndidtes tht loclize to the plsm memrne. LEM3 THI73 PUN1 FUS1 ROD1 SSO1 SSO2 YPS1 PSR2 PMP3 YPS3 SNC2 LDB19 SUR7 TPN1 Memrne protein of the plsm memrne nd ER, intercts specificlly in vivo with the phospholipid trnslocse (flippse) Dnf1p; involved in trnsloction of phospholipids nd lkylphosphocholine drugs cross the plsm memrne. Puttive plsm memrne permese proposed to e involved in croxylic cid uptke nd repressed y thimine; sustrte of Df2p/Mo1p kinse; trnscription is ltered if mitochondril dysfunction occurs Plsm memrne protein with role in cell wll integrity; co-loclizes with Sur7p in punctte memrne ptches; null mutnt displys decresed thermotolernce; trnscription induced upon cell wll dmge nd metl ion stress Memrne protein loclized to the shmoo tip, required for cell fusion; expression regulted y mting pheromone; proposed to coordinte signling, fusion, nd polriztion events required for fusion; potentil Cdc28p sustrte Memrne protein tht inds the uiquitin ligse Rsp5p vi its 2 PY motifs; overexpression confers resistnce to the GST sustrte o-dinitroenzene,zinc, nd clcium; proposed to regulte the endocytosis of plsm memrne proteins Plsm memrne t-snare involved in fusion of secretory vesicles t the plsm memrne nd in vesicle fusion during sporultion; forms complex with Sec9p tht inds v-snare Snc2p; syntxin homolog; functionlly redundnt with Sso2p Plsm memrne t-snare involved in fusion of secretory vesicles t the plsm memrne; syntxin homolog tht is functionlly redundnt with Sso1p Asprtic protese, memer of the ypsin fmily of proteses involved in cell wll growth nd mintennce; ttched to the plsm memrne vi glycosylphosphtidylinositol (GPI) nchor Functionlly redundnt Psr1p homolog, plsm memrne phosphtse involved in the generl stress response; required with Psr1p nd Whi2p for full ctivtion of STRE-medited gene expression, possily through dephosphoryltion of Msn2p Smll plsm memrne protein relted to fmily of plnt polypeptides tht re overexpressed under high slt concentrtion or low temperture, not essentil for viility, deletion cuses hyperpolriztion of the plsm memrne potentil Asprtic protese, memer of the ypsin fmily of proteses involved in cell wll growth nd mintennce; ttched to the plsm memrne vi glycosylphosphtidylinositol (GPI) nchor Vesicle memrne receptor protein (v-snare) involved in the fusion etween Golgi-derived secretory vesicles with the plsm memrne; memer of the synptorevin/vamp fmily of R-type v-snare proteins Protein involved in regulting the endocytosis of plsm memrne proteins y recruiting the uiquitin ligse Rsp5p to its trget; locliztion chnges in response to nutrient levels; null mutnt hs reduced ffinity for lcin lue dye Plsm memrne protein tht loclizes to furrow-like invgintions (MCC ptches); component of eisosomes; ssocited with endocytosis, long with Pil1p nd Lsp1p; sporultion nd plsm memrne sphingolipid content re ltered in mutnts Plsm memrne pyridoxine (vitmin B6) trnsporter; memer of the purine-cytosine permese sufmily within the mjor fcilittor superfmily; proton symporter with similrity to Fcy21p, Fcy2p, nd Fcy22p Supplementry Figure 1 Figure S1 Genome-wide screen for rescuers of Rdi1 over-expression growth defect. () Distriution of ln(growth-rtio) over the entire yest hploid nonessentil deletion lirry fter 98 hrs of growth t 23C (see Supplementry Methods). Growth rtio is defined s the rtio of growth of given strin with to without Rdi1 overexpression. Green line represents the vlue nd the cyn ox represents the cutoff rnge of the ln(growth-rtio) for rescuer cndidtes, including lem3 (the pink line). dnf2 (lue line) hd slightly lower ln(growth-rtio) thn the cutoff nd ws therefore not in the initil rescuer list. () A list of the rescuer cndidtes tht loclize to the plsm memrne, with SGD description ( 1

2 Lem3-GFP Dnf1-GFP c Dnf2-GFP 4 p = 1.3E-5 Intensity rtio 3 2 p = 4.3E-6 p = 2.3E-7 1 Lem3-GFP Dnf1-GFP Dnf2-GFP d Men fluorescence intensity (.u.) p =.67 p = Gl induction (min) Men fluorescence intensity (.u.) p = p = Gl induction (min) e ; Bni1-GFP f Intensity t perimeter (.u.) Perimeter (Pixels) g Bni1 distriution (2*FWHM)/perimeter h i Normlized intensity (.u.) Perimeter (Pixels) Figure S2 The Lem3 flippse complex is polrized to the polr cortex nd the effect of on the shpe of Cdc42 distriution. Shown re confocl imges of unudded polrized cells expressing Lem3-GFP (), Dnf1-GFP () nd Dnf2-GFP (c), nd ox plots for quntifiction of the fluorescence intensity rtios of the hlf of the plsm memrne contining the polr cp over the other hlf s descried in Figure 3g legend. P-vlue is mesured using one-tiled one smple t-test to compre whether the intensity rtios re greter thn 1. which corresponds to uniform distriution. Smll Squre is the men; ox rnge shows SEM; whiskers re stndrd devition (SD); line is medin. (d) Quntifiction of the men fluorescence intensity over the entire cell for (n = 1, 1, 11, from left to right) nd lem3 (n = 11, 9, 11, from left to right) cells t different Rdi1 induction time points s indicted. Error rs represent stndrd error of men (SEM). (e) The delivery window size in lem3 ws mesured y the distriution of GFP tgged formin Bni1 s descried previously 1. Confocl imges represent unudded polrized cells expressing Bni1-GFP. (f) The fluorescence distriution long the perimeter ws fitted to Gussin model. (g) The window width is pproximted s 2 times the full width hlf mx (FWHM) of the Gussin distriution. Shown is the men nd SEM (n=16). (h) Representtive imges of GFP-Cdc42 t the polr cp in unudded nd lem3 cells. (i) Fluorescence intensity trces long the perimeter re normlized nd ligned with respect to pek intensity nd shown djcent to ech cell type. Scle r: 2 μm. 2

() Confocl imges of unudded polrized cells in cycling popultion expressing Cdc42 tgged with mcherry t the

3 GFP-Lct-C2 mcherry-cdc42 Colocliztion GFP-Lct-C2 Figure S3 PS polriztion overlps with Cdc42 nd PS distriution in udded cells. () Confocl imges of unudded polrized cells in cycling popultion expressing Cdc42 tgged with mcherry t the N-terminus nd GFP-Lct-C2. Scle r: 2 μm. () Imges of udded or lem3 cells expressing GFP-Lct-C2. Arrow points to enriched GFP signl in the smll uds of lem3 cells compred to cells. Scle r: 4 μm. 3

TC-Cdc42 Rdi1-6XHis * 181.8 115.5 82.2 64.2 48.8 37.1 25 15 1 75 5 37 25.9 19.

) 7 6 5 4 3 2 1 Prenylted Cdc42 Nonprenylted Cdc42 Figure S4 SDS-PAGE

Prenyl group is required for TC-Cdc42 ssocition with the SLB.

the yest plsm memrne (See Supplementry method).

cteri cell lyste. Asterisks mrk the respective protein nd of interest.

prenylted Cdc42 (purified from yest plsm memrne).

non-prenylted Cdc42 (purified from cteri), with the rest of the conditions nd

4 TC-Cdc42 Rdi1-6XHis * * c Prenylted Cdc42 d Nonprenylted Cdc42 e 2.3E-1 c Men fluorescence intensity (.u.) Prenylted Cdc42 Nonprenylted Cdc42 Figure S4 SDS-PAGE followed y Coomssie stining of the purified proteins. Prenyl group is required for TC-Cdc42 ssocition with the SLB. () Prenylted TC-Cdc42 (GST-HA-TC-Cdc42) protein ws expressed nd purified from the yest plsm memrne (See Supplementry method). () C-terminlly hexhistdine-tgged Rdi1 protein ws expressed nd purified from cteri cell lyste. Asterisks mrk the respective protein nd of interest. (c) TIRF imge of the SLB with 4% PS, 15% PE loded with 126 nm FlAsH leled prenylted Cdc42 (purified from yest plsm memrne). (d) SLB with 4% PS, 15% PE similrly leled s ove with 126 nm FlAsSH leled non-prenylted Cdc42 (purified from cteri), with the rest of the conditions nd imge cquisition identicl to c. Imges were presented with identicl contrst. Scle r: 1 µm. (e) Men fluorescence intensity smpled over different opticl fields on SLBs loded with FlAsH-leled prenylted or nonprenylted Cdc42 s explined ove. Box plots re shown s descried for Figure S2c. 4

SGl-LEU, 23 C, 4 dys SD-LEU, 23 C, 4 dys ; Vector ; Vector ;

pgal-rga1 (colony2) ; Vector ; Vector ; pgal-rga1 (colony2).3 p =.

does not rescue Cdc42 dissocition rte t lem3 polr cortex.

were spotted on SGl-Leu nd SD-Leu pltes.

Notice tht Rg1 overexpression cuses growth defect, which is

5 SGl-LEU, 23 C, 4 dys SD-LEU, 23 C, 4 dys ; Vector ; Vector ; pgal-rga1 (colony1) ; pgal-rga1 (colony2) ; pgal-rga1 (colony1) ; pgal-rga1 (colony2) ; Vector ; Vector ; pgal-rga1 (colony1) ; pgal-rga1 (colony2) ; pgal-rga1 (colony1) ; pgal-rga1 (colony2).3 p =.62 ifrap rte (1/s).2.1. ; Vector ; pgal-rga1 Figure S5 Over-expression of Rg1 (Cdc42 GAP) does not rescue Cdc42 dissocition rte t lem3 polr cortex. () Seril tenfold dilutions of cultures for the indicted strins were spotted on SGl-Leu nd SD-Leu pltes. Pltes were scnned fter 4 dys incution t 23 C. Notice tht Rg1 overexpression cuses growth defect, which is excerted y Dlem3. () ifrap rtes (1/s) of GFP-Cdc42 in lem3 cells ering vector control or pgal1-rga1. Box plots re shown s descried for Figure S2c. 5

6 References 1 Slughter, B. D., Ds, A., Schwrtz, J. W., Ruinstein, B. & Li, R. Dul modes of Cdc42 recycling fine-tune polrized morphogenesis. Dev. Cell 17, (29). 6

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 1.138/nc2975 GM13 / DNA F-ctin α shrna CLASP1 shrna shrna shrna CLASP1 shrna shrna e Tuulin CLASP1 CLASP1 shrna #32 shrna #33 #55 #58 Tuulin c Tuulin ppmlc E-Cdherin shrna CLASP1 shrna shrna f Golgi