supporting online material for -...

www.sciencemag.org/cgi/content/full/314/5800/815/DC1

Supporting Online Material for

FG-Rich Repeats of Nuclear Pore Proteins Form a Three-Dimensional

Meshwork with Hydrogel-Like Properties Steffen Frey, Ralf Peter Richter, Dirk Görlich*

Published 3 November 2006, Science 314, 815 (2006).

DOI: 10.1126/science.1132516

*To whom correspondence should be addressed. E-mail: [email protected] This PDF file includes

Materials and Methods Figs. S1 to S6 Tables S1 to S3 References

Materials and Methods

E.coli expression vectors. The backbone of the indicated plasmids was derived from pQE80

(Qiagen, Hilden, Germany). Plasmids allowed for recominant expression of indicated nucleoporin

fragments in E.coli. Amino acid residues (in parentheses) are numbered according to the

Saccharomyces Genome Database (www.yeastgenome.org). The protein fragments had an N-

terminal His10-tag followed by a TEV-protease recognition site and a unique C-terminal cysteine.

The internal cysteine 144 of the Nup116 GLFG-repeat-domain had been prior removed by site

directed mutagenesis. Plasmid sequences are available on request.

Table S1: E.coli expression vectors

plasmid expressed protein

pSF345 His10-TEV-Nsp1 (1-601)-Cys

pSF361 His10-TEV-Nsp1 FY (1-601)-Cys

pSF362 His10-TEV-Nsp1 FS (1-601)-Cys

pSF350 His10-TEV-Nup116 (1-725)-Cys




Expression, purification, and labelling of nucleoporin FG-repeat-domains. N-terminally His10-

tagged nucleoporin FG-repeat-domains with a unique C-terminal cysteine were expressed in E.coli

and purified on Nickel-Sepharose under denaturing conditions. To obtain fluorescently labelled FG-

repeat-domains, the C-terminal cysteine was reacted with OregonGreen488-maleimide. Labelled

FG-domains were further purified by size exclusion chromatography.

Gelation of Nsp1-repeats. Starting point for gelation had been salt- and solvent-free Nsp1-repeats.

Therefore, purified repeats were dialysed against 6M deionized urea and precipitated with 4

volumes 98% ethanol, 40mM potassium acetate. The precipitate was washed twice with 98%

ethanol, 40mM potassium acetate and once with 100% diethylether and dried under vacuum.

Gelation was initiated by dissolving dried Nsp1 repeat-domains at 500µM in 50mM KOH, 1mM

EDTA yielding a pH of ~11, followed by quick neutralisation with 1/4 volume neutralisation buffer

(250mM K2HPO4/KH2PO4 pH7.5; 50mM KOH; 250mM acetic acid; 1mM EDTA). Resulting gels

were analyzed 20-72h later.

AFM measurements of elasticity. Gels were cast into polypropylene-tubes (2mm inner diameter)

and sealed at both ends with 3% agarose. After 48-72h, tubes with gels were cut into 1mm thick

slices, mounted onto glass-slides and covered with gelation buffer (50mM K2HPO4/KH2PO4 pH7.5;

50mM potassium acetate; 1mM EDTA). Force-displacement curves on hydrogels were acquired

with a NanoWizard AFM (JPK, Berlin, Germany) equipped with oxide-sharpened silicon nitride

cantilevers (Veeco, CA, USA) of 0.06N/m calibrated spring constant. Gel slices were indented in

buffer at approach speeds of 2µm/s. Young’s moduli were derived by fitting the data to the Hertz

cone model (ref. S1). Data and model agreed well for indentations up to 2µm. Multiple force curves

on the same spot did not significantly affect the determined value of Young’s modulus. The

elasticity of the FS mutated Nsp1-repeats was too low to be measured by this method. It was

therefore estimated by macrorheology.

FRAP measurements. A mixture of 1µM of labelled repeat-domain and 400µM of unlabelled

Nsp1 fsFG-domain was allowed to jellify for 20-24h in a sealed microscopy chamber. FRAP

measurements were performed on a Leica SP2 confocal microscope (Leica Microsystems,

Bensheim, Germany), using the 63x oil immersion objective (NA=1.4), the 488nm laser line, and

the FRAP software module. For each time point (t), fluorescence intensities within the bleached

area were integrated using ImageJ and normalised to the fluorescence intensity of an unbleached

control area, yielding the normalised time-dependent fluorescence intensity I(t). With t0 being the

first observed time point after bleaching, the fluorescence recovery (FR) was scaled according to:

!

FR(t) =I(t) " I(t

0)

I(before bleaching) " I(t0)

Mutagenesis. To introduce the FY and FS mutations, the NSP1 gene (Accession number

M37160) was cloned into pALTER®-1, single stranded DNA was prepared, the mutagenic

oligonucleotides (32 each) were annealed, gaps were closed by second strand synthesis and the

resulting DNA was transformed into a mutS E.coli strain. Mutants were selected through an

additional oligonucleotide, which repaired a defective antibiotics resistance. DNA prepared from

singles clones was sequenced and checked for the presence of the desired and the absence of

secondary mutations.

Yeast procedures. Standard yeast procedures used and media preparation are described in ref. S2.

Yeast strains were grown either in YPD (1% yeast extract, 2% peptone, 2% glucose) or synthetic

complete (SC) media lacking indicated amino acids and supplemented with 2% glucose. To select

for cells having lost a functional copy of URA3, cells were plated on medium containing 5-

fluoroorotic acid (FOA). To induce expression from the GAL1 promotor, glucose was replaced in

all media by 2% galactose.

Table S2: yeast plasmids

plasmid description source

pSF234

pADH-nsp1∆fsFG: ADH1 promotor, coding region of Nsp1p rod

domain (amino acids 606-823), NUF2-3´-untranslated region on

ARS/CEN URA3 plasmid

this study

pRS415 empty ARS/CEN LEU2 plasmid ref. S3

pSF423 NSP1 on ARS/CEN LEU2 plasmid this study

pSF424 FY mutated version of NSP1 on ARS/CEN LEU2 plasmid this study

pSF425 FS mutated version of NSP1 on ARS/CEN LEU2 plasmid this study

pSF521 pGAL1-nsp1∆fsFG: GAL1 promotor, coding region of Nsp1p rod

domain (amino acids 606-823), NUF2-3´-untranslated region on

ARS/CEN LEU2 plasmid

this study

pSW1379 NSP1 on ARS/CEN URA3 plasmid ref. S4

Table S3: yeast strains

strain genotype source

SFY119 MATa ade2∆::hisG his3∆200 leu2∆0 lys2∆0 met15∆0 trp1∆63 ura3∆0 this study

SFY244 MATa ade2∆::hisG his3∆200 leu2∆0 lys2∆0 met15∆0 trp1∆63 ura3∆0

nsp1::MET15 + pSF234 (CEN URA3 pADH-nsp1∆fsFG)

this study

SWY2283 MATa ade2-1::ADE2 ura3-1 leu2-3,112 his3-11,15 lys2 can1-100 ref. S4

SWY2284 MATα ade2-1::ADE2 ura3-1 leu2-3,112 trp1-1 his3-11,15 can1-100 ref. S4

SWY3004 MATa his3-11, 15 leu2-3, 112 lys2 ura3-1

flag-LoxP-nsp1∆FG∆FxFG myc-LoxP-nup145∆GLFG

T7-LoxP-nup49∆GLFG + pSW1379 (NSP1 CEN URA3)

ref. S4

SWY2930 MATα his3-11, 15 leu2-3, 112 lys2 ura3-1

T7-LoxP-Nup49∆GLFG flag-LoxP-Nsp1∆FXFG-∆FG

ref. S4


myc-LoxP-Nup57∆GLFG flag-LoxP-Nsp1∆FXFG-∆FG

ref. S4


myc-LoxP-Nup145∆GLFG HA-LoxP-Nup100∆GLFG

flag-LoxP-nsp1∆FG∆FxFG

ref. S4


HTB1-CFP::HIS3 YFP-NSP1

this study


HTB1-CFP::HIS3 YFP-NSP1(FY)

this study


HTB1-CFP::HIS3 YFP-NSP1(FS)

+ pSF521 (CEN LEU2 pGAL1-nsp1∆fsFG)

this study

SFY119 was obtained from a cross of BY4736 and BY4727 (ref. S5). To generate SFY244,

SFY119 was transformed with pSF234 and the genomic copy of NSP1 was replaced with MET15

according to ref. S5. Positive clones were selected on SC-plates lacking methionine and the deletion

of NSP1 was verified by genomic PCR and immunoblots.

Antibodies against the recombinant rod domain of Nsp1p (amino acids 606-823) were raised in

rabbits. Antibodies against Kar2p (ref. S6) were used as described.

NTR

NTR

NTR

Figure S1. Selective phase model for the passage of an nuclear transport receptor (NTR)

through the permeability barrier of nuclear pore complexes.

Inter-repeat contacts between the hydrophobic clusters ( ) of FG-repeat-domains create a sieve-like

barrier which restricts the passage of inert objects larger than the mesh-size. NTRs can overcome

this size-limit, because they possess binding sites ( ) for the hydrophobic clusters. They compete

inter-repeat contacts, thereby open adjacent meshes and dissolve within the barrier. Since the

involved interactions are of low affinity, the NTR can leave the barrier on the other side. For details,

see ref. S7.

Supporting figures

FŸS mutatedNsp1 repeat-domain

wild-type Nsp1 repeat-domain

mutationsPheŸSer

MNFNTPQQNKTPFSFGTANNNSNTTNQNSSTGAGAFGTGQSTFGFNNSAPNNTNNANSSITPAFGSNNTGNTAFGNSNPTSNVFGSNNSTTNTFGSNSAGTSLFGSSSAQQTKSNGTAGGNTFGSSSLFNNSTNSNTTKPAFG GLNFGGGNNTTPSSTGNANTSNNLFGATANANKPAFSFGATTNDDKKTEPDKPAFSFNSSVGNKTDAQAPTTGFSFGSQLGGNKTVNEAAKPSLSFGSGSAGANPAGASQPEPTTNEPAKPALSFGTATSDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGAQKDNEKKTE

Frey, Richter & Görlich, 2006

MNFNTPQQNKTPSSSGTANNNSNTTNQNSSTGAGASGTGQSTSGSNNSAPNNTNNANSSITPASGSNNTGNTASGNSNPTSNVSGSNNSTTNTSGSNSAGTSLSGSSSAQQTKSNGTAGGNTSGSSSLFNNSTNSNTTKPASG GLNSGGGNNTTPSSTGNANTSNNLSGATANANKPASSSGATTNDDKKTEPDKPASSSNSSVGNKTDAQAPTTGSSSGSQLGGNKTVNEAAKPSLSSGSGSAGANPAGASQPEPTTNEPAKPALSSGTATSDNKTTNTTPSSSSGAKSDENKAGATSKPASSSGAKPEEKKDDNSSKPASSSGAKSNEDKQDGTAKPASSSGAKPAEKNNNETSKPASSSGAKSDEKKDGDASKPASSSGAKPDENKASATSKPASSSGAKPEEKKDDNSSKPASSSGAKSNEDKQDGTAKPASSSGAKPAEKNNNETSKPASSSGAKSDEKKDGDASKPASSSGAKSDEKKDSDSSKPASSSGTKSNEKKDSGSSKPASSSGAKPDEKKNDEVSKPASSSGAKANEKKESDESKSASSSGSKPTGKEEGDGAKAAISSGAKPEEQKSSDTSKPASTSGAQKDNEKKTE

Figure S2. Wild-type and FŸS mutated Nsp1 FG-domains

left: Amino acid sequence (residues 1-601 in single letter code) of the wild-type fsFG-repeat-domain of

Nsp1p. right: the FŸS mutated repeat domain of Nsp1p, in which phenylalanines (F) had been mutated to

serines (S).

25°C 37°C

nsp1∆fsFGnup49∆GLFG

nsp1∆fsFGnup57∆GLFG

nsp1∆fsFGnup145∆GLFGnup100∆GLFG

+ CEN LEU2

+ CEN LEU2 NSP1

+ CEN LEU2 nsp1 FŸS

+ CEN LEU2 nsp1 FŸY

+ CEN LEU2

+ CEN LEU2 NSP1



+ CEN LEU2

+ CEN LEU2 NSP1



Figure S3. FŸY mutations within the Nsp1 repeat-domain cause only a partial, but FŸS mutations a

complete loss of function.

The indicated strains are temperature-sensitive, because they combine a deletion of the fsFG-repeats in their

genomic copy of NSP1 with GLFG-repeat-deletions from indicated nucleoporins (S4). Expression of

full-length wild-type Nsp1p from a CEN LEU2 plasmid complemented the temperature-sensitive phenotype

and allowed these strains to grow at 37°C. The FŸY mutated Nsp1p showed a partial complementation in two

of the genetic backgrounds. In contrast, the FŸS mutated Nsp1p did not complement in any strain, and even

suppressed the very slow growth of the nsp1∆fsFG nup57∆GLFG strain at 37°C.

genetic background additional plasmid

Frey, Richter and Görlich, 2006

Figure S4. Nsp1p with FŸS or FŸY mutated repeats is expressed to similar levels as wild-type Nsp1p.

A yeast strain, in which the genomic deletion of NSP1 had been complemented by a CEN URA3 plasmid

encoding the Nsp1 rod domain (amino acids 606-823), was transformed with the indicated plasmids and

cultured in SC-medium lacking leucine for several generations. Cell lysates were analysed by immunoblotting

with antibodies against the Nsp1p rod domain. The anti-Kar2p blot served as a loading control.

The analysis showed that the different full length versions of Nsp1p were expressed to similar levels. The

different mobility on the SDS-gel is due to the different amino acid composition, which apparently affects

SDS-binding.

Yeast cells, which expressed wild-type or FŸY mutated Nsp1p from the CEN LEU2 plasmid, lost the URA3

plasmid encoding the Nsp1 rod domain. In contrast, cells with the empty CEN LEU2 vector or cells expressing

the FŸS mutated Nsp1p could not lose the original plasmid encoding the Nsp1 rod domain.

Frey, Richter and Görlich, 2006

Nsp1p

plasmid-encodedNsp1 rod domain

Kar2p

+ CEN L

EU2 NSP1

+ CEN L

EU2 ns

p1 FŸ

Y

+ CEN L

EU2 ns

p1 FŸ

S

Mr(k

Da)+

CEN LEU2

150

120100

70

85

30

85

70

200


input pulldown via His-tag of Nsp1p

WT

Nsp

1pW

T N

sp1p

-His 10

FŸY

Nsp

1pFŸ

Y N

sp1p

-His 10

FŸS

Nsp

1p

FŸS

Nsp

1p-H

is 10

WT

Nsp

1pW

T N

sp1p

-His 10

FŸY

Nsp

1pFŸ

Y N

sp1p

-His 10

FŸS

Nsp

1p

FŸS

Nsp

1p-H

is 10

M r (k

Da)

150

120

100

60

50

70

85

Nup57p

Nup49p

Nsp1p

Figure S5. FŸY and FŸS mutants of Nsp1p show normal interaction with Nup49p and Nup57p.

Both, Nup49p and Nup57p were co-translated with the indicated versions of Nsp1p in a rabbit reticulocyte

lysate, using 35S-methionine as a label. Complexes containing His-tagged Nsp1p or its mutants were

subsequently recovered with Nickel-Sepharose. Non-tagged Nsp1p versions served as controls for unspecific

binding to the Nickel-matrix. Starting materials and bound fractions were resolved by SDS-PAGE and

analysed with a phosphoimager.

Considering that Nsp1p contains one, while Nup49p and Nup57p contain each six methionines (excluding the

start methionine), the intensity of the invidual bands recovered in the bound fractions suggests that

Nsp1p:Nup49p:Nup57p-complexes with a stoichiometry close to 1:1:1 had been formed. Mutations within

the repeat regions did therefore not impair complex-formation.


Figure S6. FŸY and FŸS mutants of Nsp1p become assembled into NPCs.

In a S.cerevisiae strain expressing CFP-tagged histone 2B (Htb1p-CFP) the genomic copy of NSP1 was

replaced by YFP-tagged wild-type NSP1 or the corresponding FŸY or FŸS mutants. The latter strain was

kept viable by expressing the non-tagged rod-domain of Nsp1p from a galactose-inducible promoter. Cells

were analysed by confocal microscopy. Images show co-localisations of YFP-tagged Nsp1p-derivatives (in

green) and CFP-tagged histone 2B (in red).

Wild-type and FŸY mutated Nsp1p showed the typical NPC-staining pattern. As long as the rod-domain of

Nsp1p had been induced, the FŸS mutated Nsp1p remained mainly cytoplasmic, but a significant

NPC-staining became evident after synthesis of the Nsp1p-rod domain had been repressed by addition of

glucose.

Nsp1p wild-type

FŸY mutated Nsp1p

FŸS mutated Nsp1p + untagged nsp1∆fsFG (induced)

FŸS mutated Nsp1p + untagged nsp1∆fsFG (repressed)

Detection of YFP-tagged full length Nsp1p versions (shown in green)

and CFP-tagged histone H2B (shown in red)

Supporting References

S1. A. J. Engler, L. Richert, J. Y. Wong, C. Picart, D. E. Discher, Surface Science 570, 142

(2004).

S2. M. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics. A Laboratory Course Manual.

(Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York, 1990).

S3. R. S. Sikorski, P. Hieter, Genetics 122, 19 (1989).

S4. L. A. Strawn, T. Shen, N. Shulga, D. S. Goldfarb, S. R. Wente, Nat Cell Biol 6, 197 (2004).

S5. C. B. Brachmann, A. Davies, G. J. Cost, E. Caputo, J. Li, P. Hieter, J. D. Boeke, Yeast 14,

115 (1998).

S6. S. Frey, M. Pool, M. Seedorf, J Biol Chem 276, 15905 (2001).

S7. K. Ribbeck, D. Görlich, EMBO J 20, 1320 (2001).

supporting online material for -...

Documents