Supporting Online Material Materials and Methods...Jun 08, 2004 · Loewen et al., Supporting...

Loewen et al., Supporting Material

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Supporting Online Material

Materials and Methods

Yeast strains Yeast genotypes and strain names were as follows: wild-type –

RS453B [ref S1], prp20-1 (RAN-GEF ts) – PSY713 [ref S2], pis1 mutant –

D278-2A [ref S3], leptomycin B sensitive (Crm1 T539C) – AAY1312 [ref S4],

pik1-83ts – YES102 [ref S1], stt4-4 ts – AAY102 [ref S1], mss4-2 ts – SD102 [ref

S1], ∆vps34 – PHY102 [ref S1], galactose inducible Scs2p – TLY251 [ref S5].

Other strains carrying gene deletions were obtained from Euroscarf (Frankfurt,

Germany): ∆opi1– BY4742 YHL020c::kanMX4, ∆lsb6 – FMAY003-02D(AL),

∆fab1– BY4742 YFR019w::kanMX4, and ∆spo14 – BY4742 YKR031C::kanMX4.

For reporter assays, ∆opi1 cells were mated with RS453B and sporulated to

produce TLY501, a ∆opi1 spore that allowed integration at LEU2 and URA3.

Plasmids

Uniform expression of GFP-Opi1p was achieved using the integrating plasmid

pTL211 [ref S5]. Other plasmids including Opi1p preceded by GFP were:

pTL214 – CEN URA3, based on pRS416; and pTL215 – a CMV immediate early

gene promoter plasmid, based on pCL384 [ref S5]. Variants of the CEN URA3

plasmid carried either mutations in the basic domain (K136A K137A R138A –

pTL216), or mutations in the FFAT motif (D203A – pTL217; EFFD200-203ALLA

– pTL218), or 8 mutations in the basic domain (L124A Y127A L129A M131A

I133A K136A K137A R138A – pTL229). Integrating plasmids coding for the

promoter (277 bp), open reading frame (1215 bp) and terminator (307 bp) of


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OPI1 contained either all wild-type sequence (pTL221), or mutations in the

FFAT motif (D203A – pTL227; EFFD200-203ALLA – pTL228). Opi1p sequences

(wild-type and EFFD200-203ALLA ) were cloned as a MBP fusion proteins in

pMAL-c2e (New England Biolabs), and expressed as described [ref S6]. Other

Opi1p sequences described in the text were made in the same vector

backbones using the following portions of Opi1p:Opi1N: 1-199; Opi1C: 211-

404 Q1: 1-102 Q2: 103-189 + variations as described in Fig. S4F; Q3: 203-

286; Q4: 311-404. For expression of Scs2 in bacteria, residues 1-225 were

cloned following the sequence MHHHHHH, and the expressed protein purified

on nickel agarose. For reporter assays of INO1 transcription, the INO1

promoter was cloned upstream of the lacZ open-reading frame in pRS405 for

integration at LEU2 (pTL5ILZ).

Yeast were grown in minimal synthetic medium without inositol, unless

otherwise stated, and assayed at mid-log phase (approx. OD600 = 1).

Confocal microscopy was carried out as described [ref S1]. COS-7 cells were

microinjected with PBS containing rhodamine dextran (0.5 mg/ml) and

bacterial phospholipase D (Streptomyces sp., Sigma; 5 U/µl).

Quantitation of INO1 transcripts Immediately prior to, and 5, 10, 15, 20,

and 30 mins following addition of 75 µM inositol, cells were harvested by

filtration and immediately frozen on dry ice. Total RNA was isolated by

SDS/acid phenol extraction [ref S7] and 10 µg RNA was analyzed by slot

blotting, probing for INO1 or ACT1, and quantitation by PhosphorImager.

Values were expressed as a ratio of INO1 transcripts to ACT1 transcripts, and


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scaled to an initial value of 1.

Metabolic labelling of glycerophospholipids 100µCi of [32P]H3PO4/mL was

added to wild type cells grown without inositol. Samples were taken at 2, 5,

10, 12, 15 and 20 min of labelling, with the addition of inositol (final 75 µM) at

10 minutes. The cells were harvested, suspended in 5% trichloroacetic acid,

and placed on ice for 30 minutes Lipids were extracted [ref S8], individual

phospholipid species were resolved by two-dimensional paper chromatography

[ref S9], and quantified by PhosphorImager analysis.

Liposome binding assays Dioleoyl lipids were obtained from Avanti Polar

Lipids (Alabaster, AL) or Sigma, and made up in 95% chloroform/5%

methanol. PC and mixtures of PC and single other lipids in 1:1 molar ratio were

made into suspensions of small unilamellar liposomes in Binding Buffer (BB)

(125 mM KCl, 25 mM HEPES, 1 mM DTT, 25 µM CaCl2, 1 mM AEBSF, 1 mg/ml

soybean trypsin inhibitor, pH 7.4) as described [ref S1]. MBP-tagged to Opi1p

and to Opi1 segments was purified to ~90% homogeneity as described [ref

S6], and eluted with 20 mM maltose in BB. Binding reactions were carried out

in 50 µl of BB, incubated for 30 min at 22 °C before sedimentation at 30,000

rpm for 5 min (S100AT3, Sorvall). Liposome pellets were rinsed once with BB

and resuspended in sample buffer for SDS-PAGE analysis. Phospholipids were

assumed to be equally distributed between the two halves of the bilayer.

Assay for ß-galactosidase activity pTL5ILZ was integrated into TLY501 to

make pTLY502, which was further transformed with Opi1p expression plasmids

pTL214 (wild-type) or pTL216 (mutated in PA binding). Cells were grown to


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mid-log phase in normal medium ± 100 µM inositol. Cells (1.5 OD600 units)

were collected by centrifugation, resuspended in 500 µl of 100 mM phosphate

buffer (pH 7.2) containing 10 mM potassium chloride, 1 mM magnesium

sulphate, 0.05% (w/w) SDS, and 3% (w/w) chloroform, mixed with glass

beads (425-600 µm diameter) and shaken vigorously for 20 seconds. Lysate

was mixed with O-nitrophenyl galactopyranoside solution (final concentration

0.67 mg/ml) and incubated for 12 hours at 25˚C, prior to stopping the reaction

with sodium carbonate (final concentration 300 mM). Enzyme activity was

measured by the absorbance at 420 nm, and normalized to the number of cells

added, as assessed by the optical density at 600 nm of the initial cell

suspensions.

Legends for Supporting Figures:

Fig. S1. Analysis of the nuclear translocation of Opi1p.

(A) Line scan (green line) across a single cell (top) 1 min after addition of

inositol showed peaks on the nuclear envelope (filled arrow) and lower peaks

at the cell periphery (open arrow).

(B) Line scan (green line)across the same cell (top) 6 mins after addition of

inositol – with greatly enhanced nuclear localization. Note scale 2x that of (A),

and loss of peripheral fluorescence. This increase was not due to altered

protein levels, as determined by Western blotting of cells with/without inositol.


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(C) Line scans at different time points (1 and 5 mins) for cells incubated

without addition. Data for each time point represent averaged, smoothed scans

from 10 nuclei, with adjustment for bleaching measured for the entire field.

The fluorescence profile across nuclei showed no significant change.

(D) Line scans across for time points 1 to 5 mins after addition of inositol,

quantified as in C. The first detectable effect was seen at 2 minutes with slight

accumulation in the centre of the nucleus. At 3 minutes accumulation started

at the NE, though still to a lesser extent than at the centre. Redistribution of

GFP-Opi1p ceased by 4 minutes.

(E) Role of nuclear export signals in localization of GFP-Opi1p. GFP-Opi1p was

expressed in strain AAY1312 that carries a leptomycin B-sensitive allele of the

nuclear export signal receptor Crm1p [ref S4]. The localization of GFP-Opi1p

grown in the absence of inositol was not altered by addition of leptomycin B

(LMB) at 100 ng/ml, a concentration that inhibited Crm1p function (as

confirmed by the inhibition of further cell growth in this strain but not wild-type

yeast). Note that punctae in the NE were seen in cells with the highest levels of

GFP-Opi1p., as noted previously [ref S5]. Deleting or inactivating other genes

with recognized functions in nuclear export (CSE1, MSN5 and LOS1) also did

not affect localization of GFP-Opi1p.

Fig. S2. Rapid transmission of an inositol-based signal to the nucleus by

Opi1p does not require PI/PIP kinases but affects newly synthesized PA.


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(A) GFP-Opi1p was expressed in yeast carrying mutations/deletions in each of

the 6 PI/PIP kinases involved in production of PI(4)P, PI(4,5)P2, PI(3)P and

PI(3,5)P2: stt4-4ts, pik1-83ts, mss4-2ts, ∆lsb6, ∆vps34, ∆fab1. Cells were grown

without inositol, and imaged at mid-log phase and 15 minutes after addition of

100 µM inositol. Strains with temperature sensitive (ts) alleles were shifted

from 25˚C (permissive) to 37˚C (non-permissive) 30 minutes earlier to

compromise PIP production [ref S1]. In each case, conditions of kinase

inactivation had no discernible effect on translocation of GFP-Opi1p.

(B) Phospholipid labelling without inositol. Glycerophospholipids were extracted

2, 5 and 10 minutes following the addition of 32P to cells growing in log-phase

in the absence of inositol. Lipids were separated by 2-D thin layer

chromatography and quantified as described in Methods. Label first appeared

in PA, followed by CDP-DAG, with some labelling (in decreasing order) of PS, PI

and PE.

(C) Phospholipid labelling with inositol. As in A, except inositol (75 µM) was

added simultaneously with 32P label. PI was almost exclusively the sole lipid

species labelled.

Fig. S3. Interactions of Opi1p with phospholipids

(A) Interaction of Opi1p with membranes treated with phospholipase D. GFP-

Opi1p was extracted from COS cells (in which it is mostly intra-nuclear, see

Fig. 4E) using a high salt buffer (400 mM KCl, 40 mM HEPES pH 7.8, 10%v/v


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glycerol, 0.5 mM EDTA). Extract was diluted to 14 µg protein/ml in 144 µl

buffer (200 mM sucrose, 25 mM KCl, 25 mM HEPES pH7.2, 2.5 mM MgSO4

1mM DTT, 1mM EGTA) containing cytosol of untransformed COS cells (final 0.4

mg protein/ml) and CHO-K1 “light” membranes (from a 0.8-1.2 M sucrose

interface [ref S10], final 66 µg protein /ml), together with (in lane 3 only)

bacterial phospholipase D (bPLD, Streptomyces sp., Sigma, 2.5 units). After

incubation on ice for 30 minutes, vesicles were recovered by centrifugation

(13,000xg for 10 min at 4˚C), and bound GFP-Opi1p assayed by

immunoblotting. Lane 1: 5% of extract used in binding assays. Lane 2:

vesicles (ves.) in incubation without bPLD; Lane 3: vesicles incubated with

bPLD. In the absence of phospholipase D approximately 5% of input pelleted;

however, binding of GFP-Opi1 increased to approximately 50% following PLD

treatment of the membranes, which converts PC into PA.

(B) Interaction of Opi1p extracted from yeast with liposomes treated with

phospholipase D. GFP-Opi1p was extracted from wild-type yeast using a high

salt buffer (400 mM KCl, 40 mM HEPES pH 7.8, 0.5 mM EDTA). Extract was

incubated on ice for 30 minutes with PC liposomes together with (in lane 2

only) bacterial phospholipase D (bPLD, Streptomyces sp., Sigma, 2.5 units).

Liposomes were recovered by centrifugation (30,000xg for 5 min at 4˚C), and

bound GFP-Opi1p assayed by immunoblotting. Lane 1: PC liposomes (lipos.) in

incubation without bPLD; Lane 2: liposomes incubated with bPLD. In the

presence of phospholipase D to produce PA, binding of GFP-Opi1 increased

approximately 5-fold.


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(C) Interaction of Opi1p with selected phospholipids. MBP-Opi1p fusion protein

was incubated with different concentrations of small unilamellar liposomes,

containing PA (lanes 2-4), DGPP (lanes 5-7), PG (lanes 8-10), and PI(4)P

(lanes 11-13). Binding was analysed as described for Fig. 3B. Concentrations

of lipid available for binding were: lanes 2, 5, 8, 11 – 25 µM; lanes 3, 6, 9, 12

– 50 µM, and lanes 4, 7, 10, 13 – 100 µM. For comparison, lane 1 contains

100% of input protein.

Fig S4. Role of binding to Scs2p in the function of Opi1p.

(A) Effect of mutating FFAT motif on binding of Opi1p to Scs2p. MBP-Opi1p

fusions, either wild-type (lane 1) or carrying four mutations in the FFAT motif

(lane 2, EFFD200-203ALLA), were expressed in bacteria, purified and

incubated with nickel-agarose beads pre-loaded with his6-tagged Scs2p

(truncated to lose its hydrophobic transmembrane domain [ref S5]) in 250 mM

NaCl, 25 mM HEPES, 1 mM DTT, 1 mM AEBSF, pH 7.4. Beads were washed

twice with buffer and bound protein analyzed by SDS-PAGE. Wild-type Opi1p

bound five times more efficiently than mutant Opi1p.

(B) Translocation of Opi1p mutant that binds Scs2p poorly. Opi1p carrying the

quadruple mutation of its FFAT motif tested in (A) above (GFP-FFATmut4) was

expressed from plasmid pTL219 [ref S5] in wild-type cells. Left: in cells grown

in the absence of inositol (left), the construct was diffuse in the cytoplasm,

enriched in the nucleoplasm, and weakly targeted the NE, although cell growth


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was minimal (in keeping with an inositol auxotrophic phenotype – see (E)

below). Right: 8 mins after re-addition of inositol (100 µM), the construct had

translocated into the nucleoplasm, with no discernible targeting of the NE,

unlike wild-type Opi1p (see Fig. 1).

(C) Role of Scs2 levels on the translocation of Opi1p. GFP-Opi1p was

expressed in TLY251, in which expression of Scs2p is inducible/repressible [ref

S5], and its response to inositol (100 µM) observed after 10 minutes. Top:

when expression of Scs2p was repressed, GFP-Opi1p was poorly localized to

membranes. Nevertheless, in response to inositol, there was complete and

rapid translocation from the cytoplasm to the nucleus, though with no

accumulation on the NE (as in (B) above, where Opi1p was mutated to reduce

its affinity for Scs2p). Bottom: in the same strain grown to induce high levels

of Scs2p, GFP-Opi1p was more tightly membrane localized in the absence of

inositol, and upon addition of inositol, translocation to the NE was prominent.

(D) Activity of Opi1p mutated in the FFAT motif. Opi1p mutants were tested in

a reporter assay using a beta-galactosidase reporter containing the INO1

promoter (pTL5ILZ) in a ∆opi1 strain (TLY501) carrying either no other

plasmid, or integrated OPI1 plasmids expressing either wild-type (pTL221) or

mutant OPI1 (pTL227, pTL228). Cells were grown either without inositol (black

bars) or with 100 µM inositol (grey bars). The single or quadruple mutations

are in the FFAT motif, and reduce binding to Scs2p ([ref S5], and (A) above).

Results were expressed in arbitrary units (A.U.) after normalization for the

number of cells (measured by OD600). In the absence of Opi1p, there was


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high expression from the INO1 promoter that was not repressed by inositol.

Re-introduction of wild-type Opi1p reduced expression without inositol, and

allowed inositol to induce further repression. Both the single and quadruple

FFAT mutations reduced expression 2-fold further, indicating strong gain of

Opi1p function. Similar results were obtained using CEN vectors in which the

Opi1p was tagged with GFP (pTL211, pTL217 and pTL218). Thus, reduction in

binding to Scs2p was associated with activation of Opi1p.

(E) Inositol auxotrophy induced by mutation of the FFAT motif in Opi1p. A

∆opi1 strain (TLY501) was transformed with plasmids expressing Opi1p (wild-

type – pTL221, and mutants – pTL227,pTL228) (as in (D) above),

transformants were spotted onto medium containing 100 µM or no inositol in

serial dilutions (2000, 100 and 5 cells per spot) and grown for 2 days at 37˚C.

Increased activity of Opi1p was associated with inositol auxotrophy, in that

cells containing either mutant form of Opi1p failed to grow without added

inositol. As with mutations of SCS2 [ref S11], there was no inositol

requirement for growth at 30˚C. Similar results were obtained for four colonies

of each genotype. As in (D), reduced binding to Scs2p was associated with

activation of Opi1p, indicating that one function of Scs2p is to inhibit Opi1p.

Fig S5. Location and function of PA binding sites in Opi1p

(A) Binding of Opi1 segments to PA beads. Constructs tagged with GFP were

expressed in COS cells to test for binding to agarose coated with PA, as in Fig.


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3A. Lanes contained: 1/2 –Opi1N; 3/4 – Opi1C. Lanes 1/3 contain 5% of input

material, lanes 2/4 contain bound material. Binding is weaker than with full-

length Opi1p (see Fig. 3A).

(B) Binding to PA liposomes of Opi1 segments. MBP-Q1 (lanes 1-4) , MBP-Q3

(lanes 5-8) and MBP-Q4 (lanes 9-12) fusion proteins were incubated with

different concentrations of small unilamellar liposomes containing PA as in Fig

3B, with concentrations of lipid available for binding of 25 µM, 50 µM and 100

µM, with 100% of input protein included for comparison. Expression of MBP-Q1

was extremely poor and inclusion of Q1 had a similar effect when present in

longer constructs, as expression of Opi1N was poor compared to that of Q2

(see Fig 4B). Binding of all three main bands (arrows) was not significant.

(C) Binding to PA liposomes of variants of Q2. In one experiment (top row),

binding to PA liposomes was tested for MBP-Q2 (wild-type sequence) in

comparison to MBP-Q2A3 and MBP-Q2B5 (with alanine substitutions as indicated

in F below). In a second experiment (bottom row, left and center), MBP-Q2

was compared to MBP-Q2HC8 (with alanine substitutions as indicated in F

below). In a third experiment, the smallest truncation variant that localized in

vivo (MBP-113-168, see F below), was shown to bind PA, although with

reduced affinity compared to MBP-Q2. Binding and analysis as in Fig 3B, with

concentrations of lipid available for binding of 50µM, 100 µM, 200 µM and 400

µM. Indicated lanes contain 100% of input protein. Breakdown products

(asterisk) did not interact, acting as internal negative controls. The affinity of

binding relative to that of MBP-Q2 is indicated for each construct.


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(D) Effect of inositol on localization of GFP-Q2 in yeast. GFP-Q2 expressed in

yeast growing in medium containing 0 µM or 100 µM inositol, showing

increased peripheral targeting with high levels of inositol.

(E) Translocation of Opi1p in the absence of yeast PLD1. GFP-Opi1p expressed

in ∆spo14 yeast that were grown without inositol and imaged before and 10

minutes after addition of 100 µM inositol. Translocation was indistinguishable

from wild-type cells studied in parallel.

(F) Mapping of the PA binding site and NLS in Q2. Q2 truncation variants and

basic residue mutants were expressed as GFP-fusion proteins in yeast, and

compared against wild-type GFP-Q2 for targeting to the plasma membrane and

strength nuclear localization signal. Truncations (top row of images): loss of

10 residues at the N-terminus and up to 23 residues at the C-terminus

individually still allowed plasma membrane targeting, as did the combined

truncation (113-168, i.e. 56 residues). The NLS was largely lost on truncation

of residues 103-112. Mutations (bottom row of images): mutation of group A

(K109A R110A K112A) accounted for the reduced plasma membrane targeting

and loss of NLS seen in N-terminal truncations; mutation of group B (R115A

K119A K121A K125A K128A) had no effect on targeting; mutation of group C

(K136A K137A R138A) reduced plasma membrane targeting without affecting

the NLS; mutation of group C together with 5 hydrophobic residues (L124A

Y127A L129A M131A I133A, indicated by arrowheads) to make Opi1HC8m

abolished plasma membrane targeting without affecting the NLS, even though

in vitro PA binding was not affected more than in Opi1C3m (see Fig. S5C).


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(G) Localization of GFP-Opi1HC8m in wild-type cells, grown without inositol. The

eight-fold mutant was constitutively translocated to the nucleus.

(H) Inositol auxotrophy induced by reduction of PA binding in Opi1HC8m. A

∆opi1 strain (TLY501) was transformed with an empty plasmid or plasmids

expressing Opi1p (wild-type – pTL221, and Opi1HC8m mutant – pTL229)

transformants were spotted onto medium containing 100 µM or no inositol

(100 cells per spot) and grown for 2 days at 37˚C (as in Fig S4E). Increased

activity of Opi1p was associated with inositol auxotrophy, in that cells

containing the mutant form of Opi1p grew poorly in the absence of inositol.


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Supporting References:

S1. T.P. Levine and S. Munro, Curr. Biol. 12 695 (2002).

S2. T. Beilharz, B. Egan, P. A. Silver, K. Hofmann, T. Lithgow, J. Biol. Chem.

278, 8219 (2003).

S3. J. Nikawa and S. Yamashita, Eur. J. Biochem. 143, 251 (1984).

S4. A. Audhya and S. D. Emr, EMBO J. 22, 4223 (2003).

S5. C. J. Loewen, A. Roy, T. P. Levine, EMBO J. 22, 2025 (2003).

S6. A. Sreenivas, M. J. Villa-Garcia, S. A. Henry, G. M. Carman, J. Biol. Chem.

276, 29915 (2001).

S7. P. T. Spellman et al., Mol. Biol. Cell 9 3273 (1998).

S8. J.L. Patton-Vogt et al., J. Biol. Chem. 272, 20873 (1997)

S9. M.R. Steiner and R.L. Lester Biochim. Biophys. Acta 260 222 (1972).

S10. N.T. Ktistakis, H.A. Brown, P.C. Sternweis and M.G. Roth. Proc. Natl.

Acad. Sci. U. S. A. 92 4952 (1995).

S11. S. Kagiwada and R. Zen, J. Biochem. (Tokyo) 133, 515 (2003).

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