Minimal nuclear pore complexes define FG repeat domains essential ...

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Minimal nuclear pore complexes define FG repeat domains essential for transport Lisa A. Strawn1, Tianxiang Shen1,3, Nataliya Shulga2, David S. Goldfarb2 and Susan R. Wente1,4 Translocation through nuclear pore complexes (NPCs) requires interactions between receptor–cargo complexes and phenylalanine-glycine (FG) repeats in multiple FG domain-containing NPC proteins (FG-Nups). We have systematically deleted the FG domains of 11 Saccharomyces cerevisiae FG-Nups in various combinations. All five asymmetrically localized FG domains deleted together were non-essential. However, specific combinations of symmetrically localized FG domains were essential. Over half the total mass of FG domains could be deleted without loss of viability or the NPC’s normal permeability barrier. Significantly, symmetric deletions caused mild reductions in Kap95–Kap60-mediated import rates, but virtually abolished Kap104 import. These results suggest the existence of multiple translocation pathways.

NPCs facilitate the bidirectional translocation of macromolecules across the nuclear envelope1. The mechanism of vectorial translocation remains a major unresolved question. The NPC structure is formed from a symmetrical annulus of eight spokes sandwiched between ring-filament assemblies on either face2. Some of the approximately 30 budding yeast Nups are localized strictly to either the cytoplasmic or nuclear faces3. Others are present on both sides of the NPC

in either symmetrical or biased distributions, and are referred to here collectively as ‘symmetric’. This general NPC architecture is conserved between yeast and vertebrates2–4, and unique Nup localizations may reflect discrete functions at specific nuclear transport steps. For facilitated transport, shuttling transport factors recognize defined nuclear localization sequences (NLSs) or nuclear export sequences (NESs) in cargo5. The karyopherin family of transport factors (also

Table 1 Summary of active transport phenotypes* Strain

Leu-rich NES export†

Histone import†

cNLS import

Nab2NLS import

Pho4-Spo12NLS import†

wild type

+

+

+

+

+

nup100∆GLFG nup145∆GLFG nup57∆GLFG

+

+

+ (↓rate)

– (↓↓rate)

–


+

+

+†

–

nd


+

+

+†

–

nd

nup100∆GLFG nsp1∆FG∆FxFG nup145∆GLFG

+

+

+ (↓rate)

– (↓↓rate)

–


+

+

+†

–

nd


+

+

+†

–

nd

nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF

+

+

+ (↓rate)

+ (↓rate)

+/–

nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG

+

+

+ (↓rate)

+/– (↓↓rate)

–

nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup100∆GLFG

+

+

+†

+/– †

–

*No qualitative defect (+); qualitative defect (–); weak qualitative defect (+/–); reduced import rate relative to wild type: modest = (↓rate), severe = (↓↓rate); nd, not determined. †Determined by qualitative assay only.

1Department

of Cell and Developmental Biology, Vanderbilt University Medical Center, 3120A MRBIII, 465 21st Avenue South, Nashville, TN 37232-8240 USA. of Biology, University of Rochester, Rochester, NY 14627 USA. 3Current address: Department of Pathology, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110, USA. 4Correspondence should be addressed to S.R.W. (e-mail: [email protected]). 2Department

Published online: 22 February 2004, DOI: 10.1038/ncb10097

NATURE CELL BIOLOGY VOLUME 6 | NUMBER 3 | MARCH 2004 ©2004 Nature Publishing Group

©2004 Nature Publishing Group

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b Nup159

Asymmetrically cytoplasmic

Nup42 Nup159-FG Nup42-FG

Nup100 Nup116

Nup100-GLFG Nup116-FG, GLFG Nup49-GLFG Nsp1-FG, FxFG Nup57-GLFG Nup145N-GLFG

Both faces

Asymmetrically nucleoplasmic

Nup49 FG

Nsp1

GLFG

Nup57

FxFG or FxF

Nup145N Nup2

Nup2-FxFG Nup60-FxF Nup1-FxFG

Nup60 Nup1

Asymmetric

c

d

15 °C

23 °C

30 °C

37 °C

Wild type nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF

Wild type

Both faces

nup100∆GLFG nup145∆GLFG nup57∆GLFG nup100∆GLFG nup145∆GLFG nup49∆GLFG nup100∆GLFG nup49∆GLFG nup57∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup49∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup57∆GLFG

Asymmetric & both faces

e Wild type nsp1∆FxFG nup1∆FxFG nup2∆FxFG nup60∆FxF nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup100∆GLFG

Figure 1 Specific symmetric FG domains are essential, asymmetric FG domains are not required. (a) A schematic representation of the NPC with reported steady-state Nup localizations shown (adapted from ref. 19)3,20,22,23,49. (b) A schematic representation of the primary structures for 11 S. cerevisiae FG Nups

(adapted from ref. 11). FG repeat type and position are indicated. Black bars indicate regions deleted. (c–e) Fivefold serial dilutions of yeast homozygous diploid cells were spotted on YPD plates (1.25 × 104 cells in far left spots) and were incubated at 23, 30 and 37 °C for 3 days or at 15 °C for 6 days.

termed importins, exportins and transportins) utilizes the small GTPase Ran to regulate association–disassociation with cargo and the NPC5,6. However, docking of transport factor–cargo complexes at the NPC and translocation are not energy-dependent6. Movement across the NPC requires direct interaction with Nup domains harbouring multiple FG repeats separated by polar spacer sequences7,8. Each karyopherin family member, the mRNA-specific exporter Mex67 (Nxf1) and the Ran import factor Ntf2 bind directly to FG domains9–13. Remarkably, with 8–16 copies of each Nup per NPC3,4, the translocation route is populated by at least 128 FG domains together displaying thousands of individual FG repeats.

Current translocation models are based on transient, low-affinity binding of transport factors to FG repeats3,14–16. Different karyopherin transport pathways may funnel cargoes into a single translocation pathway, or alternatively, multiple distinct or overlapping NPC-mediated pathways may exist. Studies in budding yeast have used in vivo tests to analyse redundancy among FG domains; for example, the FG domains of Nsp1, Nup49, Nup57 have been analysed (ref. 17), and we previously analysed regions in Nup116, Nup100 and Nup145N18. This work suggested partial redundancy between FG domains. It has not been rigorously examined whether FG-binding sites in each NPC substructure are necessary, how many FG domains are required, and which

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Histone H2B1−GFP

b

c

Wild type

a

NLS-NES−GFP2

e

f

g

h

i

j

k

l

RESULTS A genomic strategy to define essential FG domains To dissect FG-domain roles, we used a two-step recombination strategy based on a Cre-LoxP cassette (see Supplementary Information, Fig. S1) to generate precise in-frame chromosomal deletions (∆) of only the sequences encoding respective FG domains. This allowed the parallel production of multiple deletions in different homozygous S. cerevisiae diploids. The aim was to test for phenotypes resulting from all possible double, triple and higher-order FG∆ mutant combinations. We predicted that yeast strains with loss-of-function defects in growth and nuclear transport would pinpoint FG domain requirements. We focused on three specific sub-families of FG domains present in 11 Nups that met the criteria of having multiple clustered FG repeats separated by characteristic spacer sequences19 (Fig. 1a). These FxFG, GLFG or FG repeat regions are collectively referred to here as FG domains. Spacer sequences between FxFG repeats are highly charged and enriched in serine and threonine. GLFG repeats are separated by spacers lacking acidic residues and are rich in serine, threonine, glutamine and asparagine. FG repeats have either spacer type, but lack full FxFG or GLFG tetrapeptides. Nsp1 and Nup116 each contain two discrete domains. Although a mobile Nup20, the Nup2 FxFG domain was included as it met the clustering-spacer criteria. Nup53 and Nup59 were not targeted because their few FG repeats (four and six, respectively) do not cluster. Thus, 13 total FG domains in 11 Nups were analysed. This strategy allowed retention of non-FG domains that are required for NPC assembly and cell viability21. For example, deletions of the FG domains of four essential FG-Nups were each viable (see below), indicating that these mutant proteins were incorporated into NPCs. This allowed us to link mutant defects to specific requirements for FG-regions. FG domains were systematically deleted until additional deletions caused lethality.


nsp1∆FG∆FxFG nup100∆GLFG nup145∆GLFG


d

m 100

Nuclear cells (%)

combinations of FG-binding sites are essential for viability. To address these questions systematically, we have used genomic-based strategies to analyse relationships between all FG domains in the NPC.

80 60 40 20 0

0

2

4

6

8

10

12

14

16

Time (min) Wild type nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG nup100∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup100∆GLFG nup145∆GLFG nup57∆GLFG

Figure 2 Analysis of cNLS–GFP, NLS-NES–GFP2 and histone H2B1–GFP transport in FG∆ mutants. (a–l) GFP reporters were visualized by direct fluorescence microscopy after growth at 23 °C (a–i) or 30 °C (j–l) and shifting to 37 °C for 3 h in homozygous diploids; 37 °C results are shown. Localization at 37 °C was identical in wild-type cells grown initially at 23 °C or 30 °C (data not shown). cNLS–GFP (left), histone H2B1–GFP (middle) and NLS-NES–GFP2 (right) in wild-type (a–c), minimal symmetric FG∆ mutants (d–i), and the asymmetric FG∆ plus nsp1∆FG∆FxFG mutant (j–l). (m) cNLS–GFP import kinetics assayed at 30 °C in duplicate for three independent experiments.

Cells lacking all asymmetric FG domains are viable Five FG-Nups are asymmetrically localized exclusively to either the cytoplasmic (Nup42 and Nup159) or nucleoplasmic (Nup1, Nup2 and Nup60) sides of the NPC3,20,22,23,49 (Fig. 1a). Some have hypothesized that asymmetric Nups are important initial and terminal NPC-binding sites for transport complexes3,14,24,25. First, we generated FG∆ mutants in each asymmetric sub-group (either cytoplasmic or nucleoplasmic). The nup42∆FG nup159∆FG double mutant was viable. However, the homozygous mutant diploid was slightly growth-compromised at 37 °C, compared with wild type (data not shown; haploid mutants had no growth inhibition). The nup42∆FG nup159∆FG diploid showed an increased percentage of binucleate cells (data not shown), a non-lethal phenotype in other nup mutants26. The nup1∆FxFG nup2∆FxFG nup60∆FxF triple mutant was viable and grew identically to wild type at all temperatures tested (data not shown). We then combined these two mutant sets to test removal of all asymmetric FG domains. Surprisingly, the quintuple nup42∆GLFG nup159∆GLFG nup1∆FxFG nup2∆FxFG nup60∆FxF mutant (designated ‘asymmetric FG∆’) was viable at all temperatures tested, but was slightly cold sensitive (Fig. 1c). Thus, all five asymmetric FG domains are dispensable. Symmetric FG domains are essential Six FG-Nups are localized on both NPC sides3 (Fig. 1a). Nup49, Nup57 and Nsp1 are distributed symmetrically3,27. Nup116, Nup100 and Nup145N are also found on both faces, although their localization pat-



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23 °C

37 °C DAPI

Nab2

DAPI

nsp1∆FG∆FxFG nup100∆GLFG nup145∆GLFG


Wild type

Nab2

b

Nuclear cells (%)

100 Wild type nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF

80


60 40


20


0 0

2

4

6

8

10

12

14

16

Time (min)

Figure 3 Nab2 import is severely inhibited in symmetric FG∆ mutants. (a) Nab2 localized by indirect immunofluorescence microscopy in the homozygous diploid yeast cells grown at 23 °C (left) and shifted to 37 °C

(right) for 3 h. DAPI panels indicate nuclei. (b) Nab2-NLS–GFP import kinetics at 30 °C. Data points represent three independent experiments assayed in duplicate.

terns are biased3,28. Nup145N is more predominant on the nucleoplasmic side, whereas Nup116 and Nup100 are cytoplasmically biased. Given that Nup145N, Nup116 and Nup100 are highly similar and most probably result from a gene duplication13,29, the biases effectively result in an overall symmetrical distribution of Nup116–Nup100–Nup145N combined3. Initially, individual deletions of each domain in NUP49, NUP57, NSP1, NUP116, NUP100 and NUP145 were generated. Unexpectedly, removal of the Nup116 FG domain was lethal (data not shown), a phenotype more severe than deletion of the entire NUP116 coding region29. Therefore, all higher-order FG∆ mutants contained the amino-terminal Nup116 FG domain. Other single FG∆ mutants were viable and did not exhibit conditional growth defects (data not shown; see Supplementary Information, Table S1, regarding nup116∆GLFG). Removal of both the FG and FxFG domains of Nsp1 resulted in viability (see Supplementary Information, Table S1), and the combined nsp1∆FG∆FxFG deletion was used in all subsequent studies.

In the next round, mutants were combined in all possible pair-wise combinations. As previously reported18, a double nup116∆GLFG nup100∆GLFG mutant resulted in lethality. However, all other double mutants were viable (see Supplementary Information, Table S1). Finally, a third deletion was added to each double mutant. Only six triple-mutant combinations were viable (Fig. 1d, data not shown). All six viable triple mutants grew more slowly than wild type and were temperature sensitive (Fig. 1d; see Supplementary Information, Table S1, regarding nsp1∆FxFG nup49∆GLFG nup57∆GLFG). Adding another deletion of any other symmetrical FG domain to any of these triple mutants resulted in lethality (data not shown). Although FG domains from three symmetric FG-Nups could be deleted without loss of viability, there were specific requirements and several ‘rules’ emerged. First, the FG-region of Nup116 was essential. Second, the GLFG-region of either Nup116 or Nup100 was necessary because a nup116∆GLFG nup100∆GLFG mutant was

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ARTICLES Pho4-NLS−GFP3 Pho4-NLS−GFP3

30 °C

Spo12-NLS−GFP

c

d

e

f

nup42∆FG nup159∆FG nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup1∆FxFG nup2∆FxFG nup42∆FG nup159∆FG nup60∆FxF nup60∆FxF nup1∆FxFG nup2∆FxFG nup100∆GLFG nsp1∆FG∆FxFG nup60∆FxF

nup100∆GLFG nup145∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup100∆GLFG nup145∆GLFG

s 30 25 Sporulation (%)

37 °C

15 °C

g

h

i

j

k

l

m

n

o

p

q

r

Wild type

b

Wild type

a

20 15 10 5

Wi

ld

typ

e nu nu p100 p1 ∆ nu 45∆ GLF p5 7∆ GLF G G LF G ns G p nu 1∆F p G nu 10 ∆F p1 0∆ xF n 45 GL G nu up42 ∆G FG p1 ∆ L ∆F FG xF nu FG G nu p15 nu p2∆F9∆FG n p6 xF nu nu up42 0∆ G p6 p1 ∆F Fx 0 ∆ ∆F G F Fx xF nu F n G n p1 sp up 59 1∆ 2∆ ∆F nu FG Fx G nu nup p42∆ ∆F FG p6 1∆ F xF 0∆ Fx G n Fx FG up G F n n 15 up up2 9∆ 10 ∆F FG 0∆ xF GL G FG

0

Figure 4 Defects in Pho4-NLS and Spo12-NLS import correlate with defects in sporulation. (a–r) Homozygous diploids expressing Pho4NLS–GFP3 or Spo12-NLS–GFP reporters were grown at 23 °C (a–f) or 30 °C (g–r). Direct fluorescence microscopy localization is shown at permissive

temperatures (g, j, m, p and data not shown) or after a 3-h shift to 37 °C (a-f, h, k, n and q) or 15 °C (i, l, o and r). (s) Sporulation efficiency in FG∆ mutants, each assayed six times.

inviable. However, lethality of the nup49∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup145∆GLFG mutant indicated that together the Nup116 and Nup100 GLFG-regions alone were not sufficient. Third, when the Nup116-GLFG-region was present in a nup100∆GLFG mutant, then any two out of the remaining four domains were required (Nsp1FG-FxFG, Nup49-GLFG, Nup57-GLFG or Nup145GLFG). Fourth, if the Nup100-GLFG region was present in a nup116∆GLFG mutant, then any three of the remaining same Nups were required. This indicates that Nup116-GLFG and Nup100GLFG have overlapping functions that require contributions from either the Nsp1FG-FxFG, Nup49-GLFG and Nup57-GLFG domains, or the Nup145-GLFG domain.

Defining minimal FG-NPCs Starting with the viable asymmetric FG∆ mutant and knowledge of specific requirements for symmetric FG domains, we generated further minimal NPCs. In particular, the sextuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup100∆GLFG mutant was viable, but slightly cold- and temperature-sensitive (Fig. 1e). The only remaining FG domains from Nup116, Nup145N Nup49, Nup57 and Nsp1 were sufficient. Another high-order mutant that removed all asymmetric domains plus those in Nsp1 was both temperature- and cold-sensitive (Fig. 1e). Here, the remaining Nup116, Nup49, Nup57, Nup100 and Nup145N FG domains were also sufficient, but not as effective.



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ARTICLES FG mass remaining (MDa) 0

0.5

1

1.5

2.5

3

3.5

4

4.5

5

5.5

Viable

Wild type nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FxFG nup100∆GLFG nup145∆GLFG nup57∆GLFG nup100∆GLFG nup145∆GLFG nup49∆GLFG nup100∆GLFG nup49∆GLFG nup57∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup49∆GLFG nup100∆GLFG nsp1∆FG∆FxFG nup57∆GLFG nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nup100∆GLFG

2

Inviable

nup116∆GLFG nup100∆GLFG nup116∆GLFG nup145∆GLFG nup57∆GLFG nup116∆GLFG nup145∆GLFG nup49∆GLFG nup116∆GLFG nup49∆GLFG nup57∆GLFG nup145∆GLFG nup49∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup57∆GLFG nup145∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup57∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup145∆GLFG

Figure 5 There is no correlation between viability and the mass of FG domains deleted. Total FG domain mass was calculated for each viable and inviable mutant on the basis of the FG domain boundaries defined in Fig. 1b and the Methods, and the reported abundance of each FG Nup in

the NPC3; 8 copies of Nup42, Nup159, Nup1, Nup60, Nup100 and Nup116; 16 copies of Nup49 and Nup57; 32 copies of Nsp1; an estimated 8 copies of Nup2 and 16 copies of Nup145N.

We also tested for dependence on FG-type. As the nup116∆GLFG nup100∆GLFG mutant was lethal, all GLFG domains cannot be deleted. When all FxFG domains were removed, the quadruple nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FxFG mutant was viable, but both temperature-sensitive and slightly cold-sensitive (Fig. 1e). The Nup116 FG domain was essential, but FG domains from NUP42, NUP159 and NSP1 could be simultaneously deleted (data not shown). Finally, the septuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant was viable although cold- and temperature-sensitive (Fig. 1e). These NPCs contain ‘only’ GLFGNups and were functional at permissive temperatures. Thus, GLFG domains, but not FxFG, domains provide an essential function. It was surprising that so many combinatorial FG∆ mutants were viable. We conducted experiments to measure relative NPC number and levels of remaining FG-Nups. By western blotting, levels of the remaining FG-Nups in the septuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant were similar to wild type and proportional to levels of the non-FG structural NPC protein Nic96 and the spindle pole body protein Spc42 (see Supplementary Information, Fig. S2). Immunofluorescence microscopy analysis of Nic96–Protein A or Nup116 identified distinct peripheral nuclear rim staining identical to wild-type cells (data not shown, and see Supplementary Information, Fig. S2). In addition, biochemical fractionation of representative mutants verified that the remaining FG-Nups behaved as in wild-type cells (Supplementary Information, Fig. S2). There were also no changes in the level or localization of the GTPase Ran (yeast Gsp1; data not shown). Thus, to date, the functional defects in these mutants are most probably caused by loss of functions provided by the deleted FG domains.

representing distinct protein import or export pathways. The mutants were transformed with plasmids expressing green fluorescent protein (GFP) fusions to either the lysine-rich classical (c) NLS from SV40 large T-antigen, the Nab2 NLS, histone H2B1, the Pho4 NLS or the Spo12 NLS, and a double GFP fusion with both the leucine-rich NES from protein kinase inhibitor and SV40 cNLS (NLS–NES–GFP2; see Supplementary Information, Table 1). As summarized30, cNLS–GFP and Nab2-NLS–GFP are imported by Kap60–Kap95 and Kap104, respectively. Pho4 and Spo12 are imported primarily by Kap121 (Pse1)30. Histone H2B1 is imported by Kap114, Kap121, Kap123 or Kap95, with Kap114 predominating30. NLS-NES–GFP2 shuttles through continuous cycles of import by Kap60–Kap95 and export by Xpo1 (Crm1, Kap124)30. The steady-state localization of each reporter was examined by fluorescence microscopy. In wild-type cells, cNLS–GFP, Nab2-NLS–GFP, Pho4-NLS–GFP3, Spo12-NLS–GFP and histone H2B1–GFP each localized to the nucleus, whereas NLSNES–GFP2 was concentrated in the cytoplasm (Figs 2a–c, 4a, b, g–i, Table 1; also see Supplementary Information, Figs S3, S4). At all temperatures tested, the minimal FG∆ mutants did not exhibit defects in the steady-state localizations of cNLS–GFP, histone H2B1–GFP or NLS-NES–GFP2 (Fig. 2d–l and Table 1; also see Supplementary Information, Fig. S3). The steady-state localization of a protein can be influenced by transport efficiency and, for example, by binding to non-diffusible partners (such as histones to DNA). Defects in import rates will also not affect steady-state localizations if the decreased transport rate still exceeds the new cargo synthesis rate. Apparent in vivo import rates are more informative and can be obtained in real-time using published procedures31. Deletion of all five asymmetric FG domains caused an approximately 3–4-fold reduction in the relative import rate of the cNLS–GFP reporter (Fig. 2m). Mutants lacking the asymmetric FG domains plus nsp1∆FG∆FxFG, and those with combinatorial deletions of symmetric FG domains, showed similar reductions in relative cNLS–GFP import rates. Thus, all mutants tested behaved in relatively the same manner,

Specific transport pathways are perturbed in minimal FG∆ mutants To test whether facilitated nuclear transport was perturbed in the FG∆ mutants, we analysed steady-state localizations of reporter cargoes

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ARTICLES despite the combination of FG domains removed. In contrast to the modest effects on cNLS–GFP localization, steadystate nuclear levels of the Nab2-NLS, Pho4-NLS and Spo12-NLS cargoes were significantly perturbed (Fig. 4 and Table 1; also see Supplementary Information, Fig. S4). In all six mutants with minimal subsets of symmetric FG domains, cytoplasmic levels of Nab2 were markedly increased at both 23 °C, and after a shift to 37 °C (Fig. 3a and data not shown). However, there was no qualitative defect in the asymmetric FG∆ mutant (Table 1, also see Supplementary Information, Fig. S4). By analysing the Nab2NLS–GFP import rate, the asymmetric FG∆ mutant did show a mild, reproducible, kinetic defect (Fig. 3b). Strikingly, Nab2-NLS–GFP import was virtually abolished in the symmetric FG∆ mutants (Fig. 3b). The symmetric FG∆ mutants tested also showed clear qualitative defects in nuclear accumulation of both Pho4-NLS–GFP and Spo12NLS–GFP (Fig. 4c–f, j–r and Table 1), and sporulation inefficiently (Fig. 4s). Again, the asymmetric FG∆ mutant was not as severely perturbed with regard to steady-state accumulation. However, sporulation was markedly decreased in the asymmetric FG∆ mutant, suggesting possible transport rate defects. Taken together, different transport pathways were distinctly perturbed in different mutants. Although only kinetic effects were observed for Kap95–Kap60 cNLS–GFP cargo import in the viable symmetric FG mutants, the import pathways for Kap104 and Kap121 cargoes (Nab2 and Pho4–Spo12 NLSs, respectively) were significantly compromised. Transport defects in FG∆ mutants are not caused by increased NPC permeability Several current NPC translocation models predict that the overall mass of FG domains is involved in maintaining the NPC permeability barrier (see Discussion). Thus, transport defects in the FG∆ mutants could reflect disruption or collapse of this permeability barrier. Previous studies in wild-type yeast cells showed that a GFP–NES reporter with a relative molecular mass (Mr) of 51,000 (slightly larger than the NPC sieving limit) is normally excluded from nuclei32. In nup170∆ cells, however, where the passive sieving capacity increases without altering cNLS–GFP import rates32, the 51K GFP–NES diffuses down its concentration gradient across the relatively more permeable NPCs and localizes in the nucleus. The asymmetric FG∆ mutant, and the mutant lacking all five asymmetric FG domains plus the Nsp1 domains, exhibited normal permeabilities. Specifically, the 51K GFP–NES reporter was properly excluded from the mutant cell nuclei (see Supplementary Information Fig. S5). A 31K GFP–NES reporter — small enough to diffuse into wild type nuclei32 — also diffused normally into FG∆ mutant nuclei (data not shown). We conclude that deletion of these FG domains does not measurably alter NPC permeability. The net mass of FG domains is not critical We speculated that a minimum mass of generic FG repeats might be the basis for determining mutant viability and NPC function. The number of repeats and mass remaining in each mutant was calculated (see Methods) and the mass values are shown in Fig. 5. Wild-type NPCs contain ~3,500 repeats with a combined mass of 5.27 MDa. Over 50% of the FG repeats and FG-domain mass could be deleted without lethality. For example, the viable septuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant contains only 2.47 MDa of FG domains. We also found no correlation between viability and total mass or number of deleted FG repeats. In some mutants, a remarkably large mass was eliminated with minimal effect (2.80 MDa removed in nup42∆FG nup159∆FG nup1∆FxFG

nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG). In others, relatively little FG mass was deleted, resulting in lethality (0.86 MDa removed in nup116∆GLFG nup100∆GLFG). There was also no correlation between the amount of FG mass deleted and the severity of the transport defect. The nup100∆GLFG nup145∆GLFG nup57∆GLFG with 4.16 MDa of FG mass had a more severe inhibitory effect on the Nab2NLS–GFP import rate than did the nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF mutant with 3.84 MDa FG mass (Figs. 3b and 5). DISCUSSION We have pinpointed the minimal, functionally important, FG domains in the yeast NPC. Our key findings revolve around the fact that all FG domains are not equivalent: first, the asymmetric FG domains do not perform any essential functions, whereas specific combinations of symmetric FG domains localized in discrete sub-complexes are strictly required; second, there is no simple threshold of required FG-mass and there is no overall correlation between FG-mass and transport efficiency. Finally, different karyopherin transport pathways show requirements for different FG-Nup subsets, suggesting distinct transport pathways exist in the NPC. Our finding that asymmetric FG domains are not required for viability is in agreement with previous studies33 and goes further to eliminate all currently known asymmetric FG domains. Asymmetric FG domains may be required for some non-essential transport pathways not yet assayed (for example, Kap123), or for only the most highly efficient transport. Interestingly, we found specific requirements for symmetric FG domains. FG domains in Nup116, Nup145, Nsp1, Nup57 and Nup49 are collectively sufficient and found in at least two biochemically defined Nup sub-complexes (Fig. 6). The exact FG domains may be less important than their association with distinct NPC sub-complexes. Recent studies suggest that FG domains lack secondary structure and form extended random coils34. Thus, a single FG domain could topologically span a broad area within the NPC and its localization may not

Nup42-FG Nup159-FG Nsp1 Nup116 Nup82 Nsp1 Nup49 Nup57 Nic96

Nup116-FG, GLFG

Nup100-GLFG

and two of Nup49-GLFG Nsp1-FG, FxFG Nup57-GLFG Nup145N-GLFG

and three of or

Nup49-GLFG Nsp1-FG, FxFG Nup57-GLFG Nup145N-GLFG

Nup116 Nup2-FxFG Nup60-FxF Nup1-FxFG

Figure 6 Minimal NPCs require FG domains in two specific Nup subcomplexes. Sub-complexes with eight-fold NPC rotational symmetry are present in multiple copies. Nsp1–Nup49–Nup57 form one complex with Nic96 on both faces (purple; ref. 50). A second complex consists of Nup116 and Nsp1 with Nup82 on the cytoplasmic face (blue-green; summarized in ref. 21). Nup116 may also associate with an unidentified nucleoplasmic sub-complex, or nucleoplasmic localization could reflect a dynamic population not stably associated with the NPC. ‘Rules’ for required FG domains are also illustrated (right).



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ARTICLES be defined precisely. Regarding the FG∆ mutants, remaining FG domains may redistribute and compensate for losses. We can now test these hypotheses further using the Cre-loxP system to move and anchor specific FG domains in discrete NPC sub-structures. Until now, the evidence for most translocation models have been based on in vitro binding studies between FG domains and karyopherins11–13, the predicted high density of FG repeats in all NPC substructures3,8 and rapid translocation rates16. As discussed below, the FG∆ mutant phenotypes change the perceptions upon which these NPC translocation models are based. Affinity gradient mechanisms postulate that transport factors bind sequentially to different FG domains with progressively increasing affinity from one NPC side to the other, with highest affinity sites at respective exit points14,24. Asymmetric sites may also contribute to transport directionality and coordinate cargo release and transport factor recycling. Others have suggested that the Nup42 and Nup100 FG domains provide respective low- and medium-affinity binding sites for Kap95 translocation from the cytoplasm to a high-affinity non-FG site on Nup1 (ref. 24). Here, NPCs lacking all five asymmetric FG domains are only mildly defective in cNLS–GFP import rates. The additional deletion of the Nsp1-FG-FxFG-regions has no further impact, and we predict the same for the asymmetric FG∆ mutant also lacking the Nup100-GLFG region. Taken together, these results are inconsistent with a major role for FG domains in an affinity gradient mechanism. If affinity gradients exist, they may use either non-FG-binding sites in regions not deleted here, or compensation by remaining symmetric FG domains. Given that the affinities of importin-β for three different FG domains are almost identical14, there is no evidence for an affinity gradient amongst only symmetric FG domains. There is recent documentation of Kap95-binding sites in the non-FG domains of Nup60 and Nup1 (ref. 24, 35). With the minimal FG∆ mutants, we will be able to assay and define requirements for such non-FG docking sites. Other models postulate that transport is mediated by FG domains which provide multiple independent low-affinity binding sites. In selective phase partitioning, an interacting meshwork of FG repeats forms a hydrophobic sieve blocking translocation of molecules not bound by transport factors16,36. Binding to FG repeats locally disrupts the hydrophobic meshwork, or permeability barrier, allowing partitioning into the NPC. The ‘oily-spaghetti’ model also invokes interactions between hydrophobic FG repeats and a step-wise transit of karyopherin–cargo complexes along the NPC channel1. The Brownian affinity (virtual) gating model is based on the hypothesis that FG domains are involved in establishing an entropic barrier for diffusion through the confined NPC central channel3,15. Binding to FG domains allows macromolecules to overcome the entropic barrier and traverse the NPC. The selective phase partitioning and oily spaghetti mechanisms depend on large numbers of FG repeats to establish the permeability barrier1,16. If true, we predicted that deleting FG domains should alter diffusive permeability and active transport rates. However, a remarkably large proportion of the FG domain mass can be eliminated without increasing the permeability barrier (Fig. 5; also see Supplementary Information, Fig. S5). NPC permeability is, therefore, not especially sensitive to loss of FG domains1,16,36. Interestingly, in vivo studies have implicated non-FG-Nups in control of permeability37. Of similar significance, there is no direct correlation between the FG mass removed and viability or transport efficiency. If such physical or energetic barriers exist, they can withstand significant FG repeat reductions. In addition, the location of a hydrophobic sieve, oily spaghetti network, or entropic FG ‘bristles’ barrier must be limited to discrete subsets of FG domains (Fig. 6). We favour a model in which specific FG domains in

defined locations are key determinants for NPC translocation. The hypothesis that multiple karyopherin-mediated targeting pathways funnel cargoes into a single translocation pathway is inconsistent with our results. The minimal symmetric FG∆ mutants resulted in mild reductions in Kap95–Kap60-mediated import rates, but virtually abolished Kap104 and Kap121-mediated import. This argues for the existence of at least two distinct translocation pathways across the NPC. It is of note that the virtual gating model15 does account for alternative transport pathways. These conclusions correlate with the preferential binding of different karyopherins to various FGNups11,12,38–46. Why Kap104 and Kap121 pathways are more greatly affected in the symmetric FG∆ mutants is not understood. All of the karyopherins bind to the symmetric FG-Nups in vitro11,13; however, Kap104 and Kap121 show less binding to the asymmetric Nup1–Nup2–Nup60, compared with Kap95–Kap60 (ref. 11). Some FG domains may be more strongly required by subsets of transport factors. Thus, the NPC may not be governed by a single universal translocation mechanism. We speculate that distinct mechanisms for specific karyopherin pathways may exist and that NPC translocation as a whole is based on a ‘hybrid’ of current models. Kap95–Kap60 may be more dependent on high affinity non-FG-binding sites24, or a modified affinity gradient mechanism. Kap104 and Kap121 may utilize symmetric FG domains in a non-discriminatory manner in a modified selective phase partitioning or oily spaghetti mechanism. These FG∆ mutants will serve as a powerful platform for further refinement of NPC transport mechanism(s).

METHODS Plasmids, yeast strains, and biochemical analysis. Plasmids, yeast strains and biochemical methods used in this study are detailed in Supplementary Information Table S1. Yeast strains were grown in either YPD (1% yeast extract, 2% peptone and 2% glucose) or synthetic complete (SC) media lacking appropriate amino acids and supplemented with 2% glucose, unless otherwise indicated. Generation of FG∆ mutants and calculation of FG mass. The FG domain coding sequences were deleted by loxP-his5+-loxP disruption cassettes followed by rescue of the his5+ marker for use in multiple rounds of deletions (also see Supplementary Information, Fig. S1). All FG regions were deleted from the sequence encoding the first amino acid of the first designated repeat (FG, FxFG, GLFG) to the last amino acid of the last repeat (FG, FxFG, GLFG; Fig. 1b). Most deletions removed all FG repeats in the given domain. Some FG repeats were not included as they fell outside the clustering-spacer criteria (Nup100, Nup49, Nup145N and Nup2). Deletions were also designed in part to correlate with domains used in previous biochemical studies11,12. The previously described two-step gene integration method was used47. For each repeat domain, PCR was used to generate a loxP-his5+-loxP deletion cassette flanked on each side by 60 basepairs (bp) immediately outside the region to be deleted. These DNA fragments were introduced into diploid yeast and colonies were selected on SC medium lacking histidine. Correct integration was confirmed by PCR. The his5+ marker was rescued through expression of Cre recombinase from pSH47, as described48. Excision of his5+ resulted in the replacement of sequence for the FG region with the sequence encoding the epitope tag (24–33 bp), one loxP site (34 bp), and 38 bp of vector sequence. his5+ excision was confirmed by PCR, and mutant protein expression was confirmed by immunoblotting with antibodies to the epitope tag (mAb HA.11 16B12; Covance, Princeton, NJ) to the HA tag, mAb 9E10 (Covance) to the c-Myc tag, mAb to the T7 tag (Novagen, Madison, WI), or rabbit polyclonal anti-Flag (Affinity BioReagents, Golden, CO). Using the criteria stated in the Results section, the following FG domain boundaries were defined: Nup42-FG as 4–364, Nup159-FG as 464–876, Nup49GLFG as 2–236, Nup57-GLFG as 2–223, Nup145-GLFG as 10–209, Nup100GLFG as 2–570, Nup116FG as 2–95, Nup116-GLFG as 205–715, Nsp1-FG as 13–169, Nsp1-FxFG as 179–591, Nsp1-FG-FxFG as 13–591, Nup60-FxF as

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ARTICLES 397–512, Nup1-FxFG as 384–888, and Nup2-FxFG as 189–527.

COMPETING FINANCIAL INTERESTS The authors declare that they have no competing financial interests.

Microscopic analysis of steady-state GFP cargoes. Strains containing pGAD–GFP (cNLS–GFP), pNS167 (Nab2-NLS–GFP), pJON280 (histone H2B1–GFP), pKW430 (NLS-NES–GFP2), pEB0836 (Pho4-NLS–GFP3), or pSpo1276–130–GFP (Spo12-NLS–GFP) (see Supplementary Information, Table 1) were grown to early or mid-log phase at 23 °C or 30 °C in SC media lacking appropriate nutrients and containing 2% glucose (or 3% raffinose for pJON280). Portions of each culture were shifted to 15 °C or 37 °C for 3 h. For strains containing pJON280, histone H2B1–GFP expression was induced at the time the culture was divided by adding galactose to 3%. Images were collected with an Olympus BX50 microscope attached to an UPlanF1 100x/1.30 oil immersion objective and a Photometrics Coolsnap HQ camera. Images within an experiment were prepared identically and compiled using MetaVue v4.6 and Adobe Photoshop 7.0 software. In vivo transport rate analysis and diffusive permeability assays. Assays to assess the relative import kinetics of cNLS–GFP and Nab2-NLS–GFP reporters were performed basically as reported31,32. Briefly, approximately two A600 units of logarithmically growing cells were pelleted and re-suspended in 1 ml glucosecontaining synthetic medium (SC-Glu) containing 20 mM deoxyglucose and 20 mM sodium azide to induce equilibration of the GFP reporters. After 20 min rotation at 30 °C, the cells were washed with ice-cold water and kept on ice before beginning the import assays. Cold cells were pelleted and resuspended in 35 µl SC-Glu at 30 °C to initiate import. At various times, 2 µl of cells were removed, mounted under coverslips on glass slides and scored at room temperature using an Olympus BH-2 microscope with an Olympus SPlan 100 oil immersion objective. Re-import kinetics were quantified as described31. The steady-state localization of sized GFP–NES reporters in wild-type and mutant cells was monitored as in ref. 32 using a Leica TCS NT confocal microscope equipped with UV, Ar, Kr-Ar, and He-Ne lasers and a Nikon fluorescence microscope (SPlan 100 objective (NA 1.25)). Light and confocal images were processed using Adobe Photoshop. Generation of Nab2 antibodies and indirect immunofluorescence microscopy. A polyclonal antiserum was raised in rabbits against recombinant, bacterially expressed, Nab2 (Cocalico Biologicals, Inc., Reamstown, PA). Nab2 antibodies were affinity purified against bacterially expressed Nab2 immobilized on nitrocellulose. Yeast strains were grown to early log phase in YPD at the indicated temperature. Aliquots were shifted to 15 °C or 37 °C for 3 h, as indicated. Cells were fixed (10 min) and processed for indirect immunofluorescence microscopy as previously described29,46. Cells adhered to slides were incubated overnight with either affinity purified Nab2 antibodies (1:200), affinity-purified rabbit anti-mouse IgG to detect protein A (ICN Pharmaceuticals, Inc., 1:1,000), or Ran mAbs (Transduction Laboratories, 1:2,000). Bound antibodies were detected with Texas Red-conjugated goat anti-rabbit IgG (ICN Pharmaceuticals, Inc.; 1:200), fluorescein-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:200), or fluorescein-conjugated goat anti-mouse IgG (ICN Pharmaceuticals, Inc., Aurora, OH ; 1:200). DNA was stained with 0.1 µg/ml 4′6-diamidino-2-phenylindole (DAPI). Images were viewed, captured and compiled as above. Determination of sporulation efficiency. Diploids were incubated on YPD plates for approximately 16 h at 30 °C before transfer to sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose, 0.002 N sodium hydroxide and 2% agar). After 5 days at 23 °C, cells were scored for ascus formation by microscopic examination. At least 200 cells were counted. Note: Supplementary Information is available on the Nature Cell Biology website. ACKNOWLEDGEMENTS We thank the following for generously sharing yeast strains, plasmids, and antibodies: J. Aitchison, J. Aris, G. Blobel, C. Cole, C. Hardy, J. Hegemann, E. Hurt, J. Loeb, E. O’Shea, M. Rout and K. Weis. We also thank M. Suntharalingam for purification of Nab2 for antibody production and initial characterization of the antibodies. We appreciate discussion and comments from C. Cole. M. Rexach, R. Wozniak, and Wente lab members. This work was supported by funds from the National Institutes of Health R01 GM51219 to S.R.W. and R01 GM67838 to D.S.G.

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Floer, M. & Blobel, G. Putative reaction intermediates in Crm1-mediated nuclear protein export. J. Biol. Chem. 274, 16279–16286 (1999). 26. Bogerd, A. M., Hoffman, J. A., Amberg, D. C., Fink, G. R. & Davis, L. I. nup1 mutants exhibit pleiotropic defects in nuclear pore complex function. J. Cell Biol. 127, 319–332 (1994). 27. Fahrenkrog, B., Hurt, E. C., Aebi, U. & Pante, N. Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes. J. Cell Biol. 143, 577–588 (1998). 28. Ho, A. K. et al. Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p. Mol. Cell Biol. 20, 5736–5748 (2000). 29. Wente, S. R., Rout, M. P. & Blobel, G. A new family of yeast nuclear pore complex proteins. J. Cell Biol. 119, 705–723 (1992). 30. Chook, Y. M. & Blobel, G. Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11, 703–715 (2001). 31. Shulga, N. et al. In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation. J. Cell Biol. 135, 329–339 (1996). 32. Shulga, N., Mosammaparast, N., Wozniak, R. & Goldfarb, D. S. Yeast nucleoporins involved in passive nuclear envelope permeability. J. Cell Biol. 149, 1027–1038 (2000). 33. Walther, T. C. et al. The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import. J. Cell Biol. 158, 63–77 (2002).



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SUPPLEMENTARY INFORMATION:

METHODS

Plasmid Construction

Plasmids used in this study are detailed in Table S1. Plasmids were constructed using standard molecular biology techniques and maintained in DH5a. The loxP-his5+loxP disruption cassette (pSW1307) was constructed by replacing kanMX in pUG6 with S. pombe his5+. A DNA fragment containing his5+ with XhoI or BglII restriction sites at either end was amplified by PCR using pGFP-HIS5 (gift from J. Aitchison) as template. The cleaved fragment was ligated into the XhoI/BglII sites of pUG6, resulting in pSW1307. To facilitate the simultaneous deletion of a region of a gene and in frame insertion of sequence encoding an epitope tag, the respective sequences encoding small epitope tags were added upstream of one loxP site in pSW1307. The epitope tags included the DNA sequences coding for YPYDVPDYA from the influenza virus hemagglutinin (HA) protein, the sequence CEQKLISEEDL from c-myc, the sequence MASMTGGQQMG from the T7 gene 10 protein, and the flag epitope DYKDDDDK. Complementary oligonucleotides were generated and annealed resulting in a fragment with the sequence coding for the epitope tag flanked by HinDIII and SalI sites at either end. The oligonucleotide duplexes were ligated into pSW1307 cleaved with HinDIII and SalI, resulting in pSW1308 (HA-loxP- his5+-loxP), pSW1309 (myc-loxP- his5+-loxP), pSW1311 (T7-loxP- his5+-loxP), and pSW1312 (flag-loxP- his5+-loxP).

1 ©2004 Nature Publishing Group

Yeast strains

Parental strains used in this study are derivatives of W303 and detailed in Table S1. Strain SWY2285 was generated by crossing haploid progeny (SWY2283 and SWY2284) from the cross and dissection of SWY518 and YCH128. All other strains were derived from SWY2285.

Cre-loxP in vivo recombination strategy for making internal inframe deletions of genomic sequences encoding FG domains.

For this study a disruption cassette with the S. pombe his5+ gene flanked on both sides with loxP sites was constructed (see above and Fig. S1). DNA fragments consisting of the disruption cassette flanked by sequence homologous to the FG repeat regions to be deleted were generated by PCR and transformed into diploid yeast cells as in Baudin et al1. Homologous recombination resulted in the replacement of the sequence encoding the FG region from one copy of the NUP with the disruption cassette. The his5+ gene was then removed by introduction of Cre recombinase. This left an inframe epitope tag and one loxP site in place of the FG repeat region. Correct integration and excision of his5+ was confirmed by PCR. Expression of each mutant Nup protein, with the exception of Nup42∆FG and Nup60∆FxF, was confirmed by immunoblot with antibodies against the epitope tag. Haploid cells containing the deleted FG region were obtained by sporulation and tetrad analysis. Haploids of opposite mating type were then crossed to create a


homozygous diploid, which was used as the starting strain for deletion of the next repeat domain. Alternatively, haploids containing deletions in different FG domains were crossed to obtain a heterozygous diploid, which was sporulated to obtain double (or triple, quadruple, etc.) deletion mutants. Deletions were added in this way until all FG domains of interest were deleted or until the cells were inviable. For inviable combinations a wild type copy of one NUP on a URA3 plasmid was introduced into the heterozygous diploid before sporulation. Then, haploid progeny covered by the plasmid were tested for growth on media containing 5-fluoroorotic acid, lethal to cells that cannot survive without the plasmid-borne wild type Nup. As an example of the overall strategy, Fig. S1 contains a flow-chart illustrating the construction of strains containing a minimal number of asymmetric FG domains. Table S1 contains a summary of all strains constructed.

Biochemical analysis of ∆FG mutants.

For analysis of relative Nup and NPC levels in wild type versus the nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant, sequence encoding IgG domains from Protein A was integrated at the 3’ end of chromosomal SPC42 and NIC96 by a previously described method1. This allowed detection by the same antibody on a single blot with the proteins were both epitope tagged at their carboxy-terminus with IgG binding domains from Staphylococcus aureus Protein A in the same strain. The Protein A tags did not interfere with protein function since non-FG structural NPC protein Nic962 with the spindle pole body protein Spc423 are each


essential and no changes in growth were detected in tagged strains compared to the untagged parents. Crude lysates were made from equal masses of cell pellets, and serial dilutions were analyzed by western blotting with rabbit polyclonal anti-Nup116GLFG antibodies4 (1:1000) and developed as above. For biochemical analysis of soluble and nuclear-associated pools of Nups, spheroplasts were generated from 25 A600 units of each strain, osmotically lysed, and fractionated as previously described5. Immunoblotting was conducted with rabbit polyclonal anti-Nup116GLFG antibodies4 (1:1000), rabbit polyclonal antibodies raised against Nsp16 (1:2500), guinea pig polyclonal antibodies against Nup1597 (1:10,000) with a rabbit anti-guinea pig secondary (ICN Pharmaceuticals, Inc.; 1:250), rabbit polyclonal antibodies against Snl18 (1:1000), rabbit polyclonal antibodies against Nup145C9 (1:100), mouse mAbs against Pgk1 (Molecular Probes; 1:1000), and mouse mAb D77 against Nop110 (1:20). Bound antibodies were detected with alkaline phosphatase-conjugated anti-rabbit or anti-mouse IgG (Promega; 1:7500). Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate (Promega). To examine Ran levels the total lysate fractions were immunoblotted with mAbs against Ran (Transduction Laboratories; 1:1000).

SUPPLEMENTARY REFERENCES 1.

Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. & Cullin, C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21, 3329-30 (1993).


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Grandi, P., Doye, V. & Hurt, E. C. Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96. EMBO J 12, 3061-71 (1993).

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Donaldson, A. D. & Kilmartin, J. V. Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication. J Cell Biol 132, 887-901 (1996).

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Bucci, M. & Wente, S. R. A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective for nuclear pore complex assembly. Mol Biol Cell 9, 2439-61 (1998).

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Bogerd, A. M., Hoffman, J. A., Amberg, D. C., Fink, G. R. & Davis, L. I. nup1 mutants exhibit pleiotropic defects in nuclear pore complex function. J Cell Biol 127, 319-32 (1994).

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Nehrbass, U. et al. NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell 61, 979-89 (1990).

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Gorsch, L. C., Dockendorff, T. C. & Cole, C. N. A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes. J Cell Biol 129, 939-55 (1995).

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Ho, A. K., Raczniak, G. A., Ives, E. B. & Wente, S. R. The integral membrane protein Snl1p is genetically linked to yeast nuclear pore complex function. Mol Biol Cell 9, 355-73 (1998).


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Emtage, J. L., Bucci, M., Watkins, J. L. & Wente, S. R. Defining the essential functional regions of the nucleoporin Nup145p. J Cell Sci 110, 911-25 (1997).

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Aris, J. P. & Blobel, G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J Cell Biol 107, 17-31 (1988).

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Dilworth, D. J. et al. Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex. J Cell Biol 153, 1465-78 (2001).

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Kaffman, A., Rank, N. M. & O'Shea, E. K. Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes Dev 12, 2673-83 (1998).

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Shulga, N. et al. In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation. J Cell Biol 135, 329-39 (1996).

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Schlenstedt, G., Saavedra, C., Loeb, J. D., Cole, C. N. & Silver, P. A. The GTPbound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. Proc Natl Acad Sci U S A 92, 225-9 (1995).

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Stade, K., Ford, C. S., Guthrie, C. & Weis, K. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90, 1041-50 (1997).

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Shulga, N., Mosammaparast, N., Wozniak, R. & Goldfarb, D. S. Yeast nucleoporins involved in passive nuclear envelope permeability. J Cell Biol 149, 1027-38 (2000).


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Guldener, U., Heck, S., Fielder, T., Beinhauer, J. & Hegemann, J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24, 2519-24 (1996).

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Chaves, S. R. & Blobel, G. Nuclear import of Spo12p, a protein essential for meiosis. J Biol Chem 276, 17712-7 (2001).

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Hardy, C. F. Characterization of an essential Orc2p-associated factor that plays a role in DNA replication. Mol Cell Biol 16, 1832-41 (1996).

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Bucci, M. & Wente, S. R. In vivo dynamics of nuclear pore complexes in yeast. J Cell Biol 136, 1185-99 (1997).

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Fabre, E., Schlaich, N. L. & Hurt, E. C. Nucleocytoplasmic trafficking: what role for repeated motifs in nucleoporins? Cold Spring Harb Symp Quant Biol 60, 67785 (1995).

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Iovine, M. K., Watkins, J. L. & Wente, S. R. The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor. J Cell Biol 131, 1699-713 (1995).


FIGURE LEGENDS

Figure S1: A: loxP-his5+-loxP cassette and strategy for making genomic disruptions. B: Flow-chart for construction of strains with a minimal number of FG domains in the asymmetric Nups. Lower left panels: To detect expression of the ∆FG mutant proteins, western blots from representative strains were probed with antibodies to the epitope tags.

Figure S2: A: There is no detectable change in the relative number of NPCs and levels of GLFG Nups in the septuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant. Left panel: Spc42-Protein A and Nic96-Protein A were localized properly by indirect immunofluorescence microscopy with rabbit anti-mouse IgG in the indicated haploid strains grown at 30°C. Right panel: Lysates were prepared from equivalent cell amounts and five-fold serial dilutions were analyzed by western blotting with anti-Nup116GLFG antibodies. The antibodies recognized Nup116, Nup100, Nup57, Nup49, and the Protein A tags on Spc42 and Nic96. The relative protein levels were unchanged compared to wild type. The identity of each protein was determined by comparing lysates of individual ∆FG mutants or Protein A-tagged strains with wild type (data not shown). Many of the unlabelled bands were identified as proteolytic products of Nup116 (data not shown). The ratio of Nic96Protein A to Spc42-Protein A remained the same in the septuple nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup60∆FxF nsp1∆FG∆FxFG mutant compared to wild type.


Thus, no gross changes were detected in the relative number of NPCs or levels of remaining FG Nups.

Figure S2: B: The remaining FG Nups in the minimal ∆FG mutants biochemically fractionate as in wild type cells. Homozygous diploid yeast strains with deletions of symmetric FG-domains NPC were grown at 23˚C (a) and strains with deletions of all asymmetric FG-domains and selected symmetric FG-domains were grown at 30˚C (b). Spheroplasts were prepared, lysed (total, T), and separated into soluble (S) and insoluble pellet (P) fractions. The levels of FG Nups and control proteins in the total (T), S, and P fractions were analyzed by western blotting. Fractions were blotted with antibodies to the cytoplasmic protein Pgk1, the nucleolar protein Nop1, the non-FG NPC protein Nup145C, the endoplasmic reticulum and nuclear envelope protein Snl1, and the FG Nups Nup116, Nsp1, and Nup159. The Nup116GLFG antibodies recognize Nup116, Nup100, Nup57, and Nup49. The Nsp1 antibody recognizes Nup2 in addition to Nsp1. As controls, the cytoplasmic protein Pgk1 was found predominantly in the S fractions whereas the nucleolar protein Nop1, the non-FG NPC protein Nup145C, and the nuclear envelope and endoplasmic reticulum integral membrane protein Snl1 were predominantly in the P fractions. The distribution of each of the control proteins between the S and P fractions was identical in wild type and ∆FG mutant cells. The fractionation of the remaining FG Nups was monitored with anti-Nup116GLFG, Nsp1, or Nup159 antibodies. Blotting with the Nup116GLFG antibody indicated Nup116, Nup100, Nup57, and Nup49 fractionated almost exclusively in the P fractions in both wild type and each of the ∆FG mutants tested. Similarly, Nsp1 and Nup159 also were found


predominantly in the P fractions. Consistent with Nup2 being a mobile, shuttling Nup11, it was found in both the S and P fractions. The ratio of Nup2 between the S and P fractions appeared identical in wild type and ∆FG mutant cells. Thus, the remaining FG Nups tested in the ∆FG mutants biochemically fractionated as in wild type cells.

Figure S3: cNLS-GFP and histone H2B1-GFP are imported and NLS-NES-GFP2 is exported in ∆FG mutants. The GFP reporter was visualized in the indicated homozygous diploids grown at 30˚C and shifted to 37˚C (a) or 15˚C (b) for three hours. Import of histone H2B1-GFP at 15˚C could not be assayed because expression of histone H2B1-GFP could not be detected in cells induced at 15˚C for 3 hours (data not shown).

Figure S4: Strains containing deletions of FG domains on both NPC faces have a diminished capacity to import Nab2NLS-GFP. Nab2NLS-GFP was localized in homozygous diploid yeast cells with the indicated deletions. Nab2NLS-GFP in wild type cells (a,h), strains containing deletions of FG domains localized on both faces of the NPC (b-g), a strain containing deletions of all asymmetric FG domains (i), a strain containing deletions of all asymmetric FG domains and the FG and FxFG domains of Nsp1 (j), and a strain containing deletions of all asymmetric FG domains and Nup100GLFG (k). The cells were visualized by direct fluorescence after growth at 23˚C or after shift to 15 or 37˚C for three hours. The asterisk (row b, 37˚C) indicates an exposure time equivalent to twice that used for all other panels.


Figure S5: The NPC permeability barrier is maintained. The 51 kDa GFP-NES reporter was localized by confocal microscopy after incubation on ice for one hour. The corresponding DIC panels are shown on the right. Arrows indicate the positions of nuclei.


Table S1: Plasmids and yeast strains Plasmid/Strain pEB0836 pGAD-GFP pGFP-HIS5 pJON280 pKW430 pNS101 pNS167 pProtA-HIS3-URA3 pProtA-HIS5 pSH47 pSpo1276-130-GFP pSW1308 pSW1309 pSW1311 pSW1312 pUG6 YCH128 SWY518 SWY2283 SWY2284 SWY2285

∆FG Strainsa SWY2831-2833; SWY2834 SWY2807-2809; SWY2809

Description PHO4 NLS fused to three GFP under PHO4 promoter (URA3 CEN) SV40 cNLS fused to GFP under ADH promoter (LEU2 2m) GFP-his5+ cassette Histone H2B1 fused to GFP under GAL1-10 promoter (URA3 CEN) SV40 cNLS and protein kinase inhibitor NES fused to two GFP under ADH promoter (URA3 2m) 51 kDa GFP-NES reporter under MET25 promoter (URA3 CEN) NAB2 NLS fused to GFP under MET25 promoter (URA3 CEN) Protein A-HIS3-URA3 cassette Protein A-his5+ cassette Cre recombinase under GAL1 promoter (URA3 2m) SPO12 NLS fused to GFP under TPI promoter (LEU2 2µ) HA-loxP- his5+-loxP cassette myc-loxP- his5+-loxP cassette T7-loxP- his5+-loxP cassette flag-loxP- his5+-loxP cassette loxP-kanMX-loxP cassette

Source 12 13 J. Aitchison 14 15

MATa ade2-1 ade3::HISG ura3-1 leu2-3,112 his3-11,15 lys2 can1-100 MATa ade2-1::ADE2 ura3-1 leu2-3,112 trp1-1 his3-11,15 can1-100 MATa ade2-1::ADE2 ura3-1 leu2-3,112 his3-11,15 lys2 can1-100 MATa ade2-1::ADE2 ura3-1 leu2-3,112 trp1-1 his3-11,15 can1-100 MATa/a ade2-1::ADE2/ade2-1::ADE2 ura3-1/ura3-1 leu2-3,112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15 lys2/LYS2 can1-100/can1-100

19 20 This study This study This study

∆FG Genotype nup42∆FG nup159∆FG 12 ©2004 Nature Publishing Group

16 16 J. Aitchison J. Aitchison 17 18 This study This study This study This study 17

Phenotypeb viable viable

SWY2801-2802; SWY2803 SWY2729-2731; SWY2737 SWY2771-2774; SWY2775 SWY2825-2827; SWY2828 SWY2751-2754; SWY2757 SWY2867-2869; SWY2870 SWY2762-2765; SWY2766 SWY2789-2792; SWY2793 SWY2878-2880; SWY2881c SWY2811-2813; SWY2814 SWY3029-3030; na SWY2919-2922; SWY2923 SWY2991-2994c; na SWY2844-2846; SWY2847 SWY2892-2894; SWY2895 SWY2856-2857, 3069; SWY2858 SWY2985, 2995c; na SWY2915-2916; na SWY2819-2821; SWY2822 SWY2839-2842; SWY2843 SWY2972-2973, 2982; SWY2974 SWY2783-2785; SWY2786 SWY2835-2837; SWY2838 SWY2924-2926; SWY2927 SWY2963-2965; SWY2966 SWY2882-2884; SWY2885 SWY2975-2977; SWY2978 SWY2958-2961; SWY2962 SWY2933-2935; SWY2936 SWY2928-2931; SWY2932 SWY3027-3028; SWY3031 SWY2796-2799; SWY2800 SWY2815-2817; SWY2818

nup1∆FxFG nup2∆FxFG nup60∆FxF nup49∆GLFG nup57∆GLFG nup145∆GLFG nup100∆GLFG nup116∆GLFG nup116∆FG nsp1∆FG nsp1∆FxFG nsp1∆FG∆FxFG nup116∆1 nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nup2∆FxFG nup60∆FxF nup116∆GLFG nup100∆GLFG nup116∆GLFG nup145∆GLFG nup116∆GLFG nup57∆GLFG nup116∆GLFG nup49∆GLFG nup100∆GLFG nup145∆GLFG nup100∆GLFG nup57∆GLFG nup100∆GLFG nup49∆GLFG nup145∆GLFG nup57∆GLFG nup145∆GLFG nup49∆GLFG nup57∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nsp1∆FG∆FxFG nup57∆GLFG nsp1∆FG∆FxFG nup49∆GLFG nsp1∆FxFG nup49∆GLFG nsp1∆FxFG nup2∆FxFG nup159∆FG nsp1∆FG


viable viable viable viable viable viable viable viableg lethal viable viable viable lethal viable viable viable lethal viable (ts) viable (ts) viable (ts) viable viable viable viable (ts) viable (ts) viable (ts) viable (ts) viable (ts) viable (ts) viable (ts) viabled viable viable

SWY2848-2849; na SWY2908-2910; SWY2955 SWY2911-2912; SWY2956 SWY2913-2914; SWY2957 SWY2896-2897; SWY2898 SWY3023-3025c; na na SWY2997-2999c; na SWY2950-2953; SWY2954 SWY2967-2969; SWY2970 SWY2871-2873; SWY2874 SWY3020-3021e; na SWY3037-3040c; na SWY3033-3035c; na SWY3017-3018c; na SWY2980-2981, 2983; SWY3005 SWY3012-3014; SWY3015 SWY3007-3009; SWY3010 SWY2987-2989f; na SWY3003-3004f; na SWY3001f; na na SWY2861-2863; SWY2864 SWY2899-2902; SWY2904 SWY2850-2853; SWY2854 SWY2937-2939; SWY2940 SWY2941-2943; SWY2944 SWY2945-2948; SWY2949 SWY2904-2906; SWY2907 SWY2971, 3041; SWY3045 SWY3042-3043; SWY3044 SWY3062-3064, 3066; SWY3065

nup42∆FG nsp1∆FG nup42∆FG nup159∆FG nup1∆FxFG nup42∆FG nup159∆FG nup2∆FxFG nup42∆FG nup159∆FG nup60∆FxF nup1∆FxFG nup2∆FxFG nup60∆FxF nup116∆GLFG nup145∆GLFG nup57∆GLFG nup116∆GLFG nup145∆GLFG nup49∆GLFG nup116∆GLFG nup57∆GLFG nup49∆GLFG nup100∆GLFG nup145∆GLFG nup57∆GLFG nup100∆GLFG nup145∆GLFG nup49∆GLFG nup100∆GLFG nup57∆GLFG nup49∆GLFG nup145∆GLFG nup57∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup145∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup116∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup100∆GLFG nup145∆GLFG nsp1∆FG∆FxFG nup100∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup100∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup57∆GLFG nsp1∆FG∆FxFG nup145∆GLFG nup49∆GLFG nsp1∆FG∆FxFG nup57∆GLFG nup49∆GLFG nsp1∆FxFG nup57∆GLFG nup49∆GLFG nsp1∆FxFG nup2∆FxFG nup1∆FxFG nsp1∆FxFG nup2∆FxFG nup60∆FxF nup42∆FG nup159∆FG nsp1∆FG nup42∆FG nup159∆FG nup2∆FxFG nup60∆FxF nup42∆FG nup159∆FG nup1∆FxFG nup60∆FxF nup42∆FG nup159∆FG nup1∆FxFG nup2∆FxFG nsp1∆FxFG nup2∆FxFG nup60∆FxF nup1∆FxFG nup42∆FG nup159∆FG nup60∆FxF nup1∆FxFG nup2∆FxFG nup42∆FG nup159∆FG nup60∆FxF nup1∆FxFG nup2∆FxFG nup100∆GLFG nup42∆FG nup159∆FG nup60∆FxF nup1∆FxFG nup2∆FxFG nsp1∆FG∆FxFG


viable viable viable viable viable lethal lethal lethal viable (ts) viable (ts) viable (ts) lethal lethal lethal lethal viable (ts) viable (ts) viable (ts) lethal lethal lethal lethalg viable viable viable viable viable viable viable (ts) viable viable viable (ts,cs)

a

Where multiple strains are listed haploid and homozygous diploid strains are listed before and after semicolon, respectively. na, not available b ts, temperature sensitive at 37˚C; cs, cold sensitive at 15˚C c Contains pSW131 (NUP116 URA3 CEN) d Not tested for conditional growth e Contains pSW125 (NUP49 URA3 CEN) f Contains pSW1379 (NSP1 URA3 CEN) g Any differences between the phenotypes generated here and previous reports are addressed below. The most notable methodological difference is that in this work all deletions followed internally consistent criteria for construction and were exclusively chromosomal deletions. Two strain comparisons of note are detailed here. 1) It has been previously reported that a triple nsp1∆FxFG nup49∆GLFG nup57∆GLFG mutant is viable21. However, our mutant was inviable (data not shown). There are several differences in the two strains. First, the exact sequences deleted in the two studies differed. Second, our strain contained precise chromosomal deletions of the repeat domains and all genes were under the control of their endogenous promoters. In the study by Fabre et al., Nup49∆GLFG under its own promoter and Nup57∆GLFG under the NOP1 promoter were expressed from a plasmid21. Third, different yeast strain backgrounds were used. Therefore, the phenotype difference could be due to a difference in the sequence deleted, protein expression levels, or yeast strain background. 2) We had previously divided Nup116 into three regions and analyzed the role of each region in cells22. Region 2 of Nup116 (amino acids 186 to 725) contains the GLFG repeats flanked by short regions on either side, and a nup116∆2 mutant exhibited a temperature sensitive growth defect22. In our present study, a precise deletion of the NUP116 GLFG domain (amino acids 205 to 715) did not result in a conditional phenotype (data not shown). In each case Nup116∆2 or Nup116∆GLFG was expressed from the chromosome under control of the endogenous promoter, so the difference in growth between nup116∆2 and nup116∆GLFG is likely attributable to the difference in the exact sequence deleted.





