Download Defining the essential functional regions of the nucleoporin Nup145p

911
Journal of Cell Science 110, 911-925 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS8112
Defining the essential functional regions of the nucleoporin Nup145p
Jennifer L. T. Emtage, Mirella Bucci, Janis L. Watkins and Susan R. Wente*
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO
63110, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
Studies of the essential nucleoporin Nup145p have shown
that its depletion is coincident with a block in RNA export
and that deletion of its amino-terminal domain results in
clustering of nuclear pore complexes. To further define the
functional domains of Nup145p, we have characterized a
panel of nup145 mutants. Deletions from both the amino
terminus and the carboxy terminus resulted in temperature
sensitive mutants that accumulated polyadenylated RNA in
the nucleus at the non-permissive temperature. In addition,
these mutants also displayed constitutive clustering of
nuclear pore complexes in localized patches of the nuclear
envelope. These results suggested that an internal region of
Nup145p consisting of amino acids 593-893 is essential for
function. Accordingly, when this region was deleted,
growth was not supported at any temperature, whereas the
region alone was able to complement a null mutation when
INTRODUCTION
The nucleus of a eukaryotic cell is separated from the
cytoplasm by two membranes, and pores formed through the
bilayers are necessary for all nucleocytoplasmic communication. The nuclear pore complex (NPC) allows the passive
diffusion of ions and small molecules and mediates the active
transport of large macromolecules with a diameter of up to 26
nm (Feldherr et al., 1984). High resolution electron microscopy
images of vertebrate NPCs reveal an intricate structure with a
molecular mass of 125 MDa (Reichelt et al., 1990). It has a
distinct eightfold symmetry, and is comprised of multiple rings,
a spoke network, a basket-like structure on the nuclear face,
cytoplasmic filaments, and connections to the nuclear lamina
and lattice (for reviews see Akey, 1995; Panté and Aebi, 1996).
Functionally, yeast and vertebrate NPCs are highly homologous: they recognize the same import and export signals
(Osborne and Silver, 1993; Gerace, 1995), proteins in both
share antibody epitopes and characteristic sequence motifs
(Rout and Wente, 1994), and in one example to date, a gene
encoding a vertebrate NPC protein complements a yeast NPC
mutant background (Aitchison et al., 1995b).
Based on biochemical analysis of purified NPCs (Rout and
Blobel, 1993) and on calculations from their total size, it is
estimated that the NPCs may contain as many as a hundred
different proteins (called nucleoporins or NUPs). In the yeast
Saccharomyces cerevisiae, genetic and biochemical strategies
expressed on a high copy plasmid. Previous studies have
suggested that Nup145p is cleaved into two polypeptides of
approximately 65 and 80 kDa. Interestingly, our experiments suggest that cleavage occurs in vivo. However, a
small internal deletion of 17 amino acid residues that
abolished cleavage had no effect on cell growth. Therefore,
cleavage is not necessary for Nup145p function. When a
sequence harboring the Nup145p cleavage site required for
Nup145p cleavage was inserted in a chimeric protein, it was
not sufficient for mediating cleavage. Cleavage likely
requires a second region from amino acid residues 247-524
in addition to the cleavage site.
Key words: Nuclear pore complex, Nucleocytoplasmic transport,
Trafficking, Clustering, Cleavage
to date have identified the genes encoding 21 nucleoporins (for
review see Rout and Wente, 1994; Doye et al., 1994; Grandi et
al., 1995a; Hurwitz and Blobel, 1995; Grandi et al., 1995b;
Pemberton et al., 1995; Li et al., 1995; Aitchison et al.,
1995a,b; Gorsch et al., 1995; Kraemer et al., 1995; Heath et
al., 1995; Siniossoglou et al., 1996). Only eight of these genes
are essential. One of them, NUP145, encodes an essential
polypeptide with a predicted molecular mass of 145 kDa
(Fabre et al., 1994). NUP145 was independently isolated by its
cross-reactivity with an antibody that recognizes other yeast
nucleoporins (Wente and Blobel, 1994) and by its synthetic
lethality with another nucleoporin, Nsp1p (Fabre et al., 1994).
Nup145p can be divided into three structural regions. First, its
amino-terminal 220 amino acids contain 12 repeats of the
tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG), a
repeating sequence element common to four other yeast nucleoporins: Nup49p, Nup57p, Nup100p, and Nup116p. Together,
these five proteins comprise the GLFG family (Wente et al.,
1992; Wimmer et al., 1992; Grandi et al., 1995b). Second, the
middle 370 amino acids of Nup145p share homology with the
carboxy-terminal regions of Nup116p and Nup100p. The first
two regions are referred to together as the amino-terminal (Nterminal) region of Nup145p. Finally, the carboxy-terminal (Cterminal) region of Nup145p, which spans 727 amino acids, is
unique.
Two possibly distinct NPC functions have been attributed to
Nup145p: maintaining proper NPC distribution and mediating
912
J. L. T. Emtage and others
RNA export. When Nup145p was depleted by placing NUP145
under control of an inducible promoter, 30% of the cells had
accumulated polyadenylated RNA in their nuclei after 3 hours;
by 12 hours, almost all of the cells showed nuclear accumulation (Fabre et al., 1994). Inhibition of protein import into the
nucleus lagged behind this RNA export defect. These results
suggest that Nup145p has a primary role in RNA export.
However, because the experimental strategy requiring
Nup145p depletion was dependent on the turnover of the wildtype protein, it is not possible to directly link the inability of
the cells to export RNA to the loss of functional Nup145p.
Cells harboring a nup145∆N mutation, wherein the sequence
encoding the N-terminal region of Nup145p was deleted and
the LEU2 or URA3 gene inserted, were viable at all growth
temperatures but contained clusters of NPC-like structures in
‘aggregated’ patches of the nuclear envelope (Wente and
Blobel, 1994). This is in sharp contrast to the NPCs of wildtype cells which are distributed over the entire nuclear surface
at a density of ~15 NPC/µm2, except for exclusion from areas
where the nuclear and vacuolar membranes abut (Severs et al.,
1976). The presence of NPC/nuclear envelope perturbations
suggests that Nup145p may play a role in the assembly of
NPCs. Because of the manner in which the nup145∆N
mutation was generated, it was not clear whether the constitutive clustering of nup145∆N NPCs was due to the absence of
the N-terminal region of Nup145p or to a lower expression
level of the remaining C-terminal portion, which has no
obvious promoter.
Interestingly, Nup145p in yeast cell extracts is proteolyzed
into two polypeptide fragments with apparent molecular
masses of ~80 kDa and ~65 kDa, corresponding to the Cterminal and N-terminal regions, respectively (Wente and
Blobel, 1994). However, it is not known whether this reflects
specific Nup145p cleavage in vivo or if it is a consequence of
cell breakage. If cleavage occurs in intact cells, the physiological consequences of producing exclusively full length protein
could prove insightful. The goal of the experiments in this
paper was to pinpoint the structural region(s) of Nup145p
whose absence or mutation results in NPC clustering, RNA
export, and/or non-cleavage phenotypes.
MATERIALS AND METHODS
Standard techniques were used for growth and transformation of
yeast, sporulation of diploids, dissection of tetrads, and extraction of
DNA from yeast (Kaiser et al., 1994). Strains were grown in YEPD
(1% yeast extract, 2% bacto-peptone, 2% dextrose) unless plasmid
selection required an appropriate selective minimal medium. Standard
methods were used for restriction digests, alkaline phosphatase
treatment of vectors, ligations, and all handling of bacterial strains,
including growth, transformation with and extraction of DNA
(Sambrook et al., 1989).
Yeast strains
The yeast strains used in this study are listed in Table 1. The
nup145::LEU2 null allele was produced by transformation of PstIdigested pSW168 into the diploid strain W303. pSW168 was constructed by ligating the 300 bp SacI/BamHI fragment of pSW165, the
2 kbp PstI/SacI LEU2-bearing fragment of pJJ282 (Jones and Prakash,
1990), and the 5.2 kbp BamHI/partial NsiI vector fragment of pSW102
(Wente and Blobel, 1994). This construct removes amino acids 67
through 1,290 of Nup145p and replaces them with the LEU2 gene
oriented in the opposite direction to NUP145. Leu+ transformants
were screened by Southern analysis to identify a heterozygous null
strain (SWY203). Carboxy-terminal in-frame fusions of the five IgG
binding domains of Protein A to N-terminal regions of Nup145p were
constructed using plasmid pProtA/HU and the strategy described by
Aitchison et al. (1995a,b).
Plasmid construction
The 3′ end of NUP145 was amplified by PCR from Z1 λ DNA (Wente
and Blobel, 1994) using oligonucleotides 145-X (5′-TCGGGATCCCCTTTGGCGGGACTTGGACTTTC-3′) and 145-Y2 (5′-GCTGGATCCATTCAAGGCTACCACAGGTGGAGG-3′),
and
the
resulting fragment was cut with AatII and BamHI and inserted into
the corresponding sites of pSW69 (Wente and Blobel, 1994) to form
the full length NUP145 gene in pBSKS (pSW181). The 6.2 kbp
BamHI/SalI fragment bearing NUP145 was excised from pSW181
and inserted into the corresponding sites of pRS316 and pRS313
(Sikorski and Hieter, 1989) to form pSW190 (URA3) and pSW191
(HIS3), respectively.
Deletions of NUP145 were made as follows (unless otherwise
noted pSW181 was used as the template for PCR). For
pSW303(nup145-58/HIS3), pSW191 was digested with XbaI to
remove a 2.7 kbp fragment and the resulting vector religated. The first
100 kDa (nup145-100) was placed in HIS3-CEN and URA3-2µ
vectors by inserting the 4.8 kbp EcoRI fragment from pSW69 into
pRS313 and pRS426 (Sikorski and Hieter, 1989) to form pSW360 and
pSW249, respectively. pSW363, (nup145∆GLFG/HIS3), was made in
a three-step process. First, a 2.2 kbp BamHI/SalI fragment amplified
by PCR using oligonucleotides T3 (5′-ATTAACCCTCACTAAAG-3′)
and 145-P (5′-CCAGGATCCTATTAAACATAAGGTGGCTAC-3′),
was inserted into the corresponding sites of pRS306 (Sikorski and
Hieter, 1989) to form pSW261. Next, the 3.7 kbp BamHI-digested
PCR product of oligonucleotides T7 (5′-AATACGACTCACTATAG3′) and #161 (5′-TCGGGATCCCATTCCCAAGATCCGGT-3′) was
inserted into the BamHI site of pSW261 to form pSW280. Finally, the
2.8 kbp SnaBI/MluI fragment from pSW280 replaced the corresponding fragment of pSW191 to form pSW363. pSW388
(nup145∆NS/HIS3) was constructed by replacing the BamHI fragment
of pSW363 with the 2 kbp BamHI-digested PCR product made with
oligonucleotides T7 and 145-X. pSW459 (nup145∆NL/HIS3) was
constructed as pSW388, except that a 2.7 kbp BamHI-digested PCR
product made with oligonucleotides T7 and 145-CS (5′GGGGGATCCCGATGAAAGATACGACG-3′) was used. pSW540,
(nup145∆524/592/HIS3), was constructed by ligating the
BamHI/SalI-digested PCR product of oligonucleotides 145-D4 (5′CTAGGATCCTAGAGGAACGAAATATTACAATTTTACC-3′) and
T3 and the BamHI/XbaI-digested PCR product of oligonucleotides
145-Y3 (5′-TGCTCTAGATTCAAGGCTACCACAGGTGGAGGTG3′) and 145-X into XbaI/SalI-digested pRS313. pSW542,
(nup145∆592/893/HIS3), was constructed in two steps as follows.
First, pSW538 was constructed by inserting the BamHI/XbaI-digested
PCR product of oligonucleotides 145-D2 (5′-GCAGGATCCCTTCTAACGAAATAGAACAAATATTTC-3′) and 145-Y3 into pRS313.
Next, the BamHI/SalI-digested PCR product of oligonucleotides 145D1 (5′-AGGGGATCCTATAGGATATATAGTTCATTTCCC-3′) and
T3 was inserted into the corresponding sites of pSW538 to form
pSW542. pSW543, (nup145∆592/608/HIS3), was constructed in two
analogous steps. First, pSW539 was constructed by inserting the
BamHI/XbaI-digested PCR product of oligonucleotides 145-D3 (5′GCAGGATCCGGGGGTTAGTCAATGAAGAAGATGCGG-3′) and
145-Y3 in pRS313. Next, the BamHI/SalI-digested PCR product of
oligonucleotides 145-D1 and T3 was inserted into the corresponding
sites of pSW539 to form pSW543. pSW610, encoding amino acids
593 through 893 on a high copy vector, was constructed by ligating
the 3.0 kbp EcoRI fragment from pSW388 into pRS423 (Sikorski and
Hieter, 1989).
Mapping the functional regions of Nup145p
913
Table 1. Strains and relevant genotypes
Strain
W303
SWY203
SWY211
SWY476
SWY556
SWY647
Relevant genotype
Description
MATa/MATα leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/his3-11,15 ade2-1/ade2-1
trp1-1/trp1-1 can1-100/can1-100
MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/
his3-11,15
MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/
his3-11,15 pSW191 (NUP145/HIS3/CEN)
MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/
his3-11,15 pSW249 (nup145-100/URA3/2µ)
MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/
his3-11,15 pSW363 (nup145∆GLFG/HIS3/CEN)
MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/
his3-11,15 pSW388 (nup145∆NS/HIS3/CEN)
W303 with one copy of NUP145 deleted and
replaced by LEU2
SWY203 + pSW191
SWY203 + pSW249
SWY203 + pSW363
SWY203 + pSW388
SWY122
MATa nup145∆N::LEU2 leu2-3,112 his3-11,15
W303 derivative with the N-terminus of NUP145
replaced by LEU2 (Wente and Blobel, 1994)
SWY294
SWY389
SWY513
SWY656
SWY690
SWY849
SWY390
SWY535
SWY391
SWY536
SWY392
SWY537
SWY393
SWY538
SWY394
SWY540
SWY395
SWY541
SWY396
SWY539
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW191 (NUP145/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW249 (nup145-100/URA3/2µ)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW363 (nup145∆GLFG/HIS3/CEN)
MATa nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW388 (nup145∆NS/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW459 (nup145∆NL/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW295 (nup145-A5/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW369 (nup145-A5/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW296 (nup145-E6/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW370 (nup145-E6/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW297 (nup145-L2/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW371 (nup145-L2/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW298 (nup145-O1/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW372 (nup145-O1/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW300 (nup145-R4/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW374 (nup145-R4/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW302 (nup145-V8/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW375 (nup145-V8/URA3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW299 (nup145-R2/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW373 (nup145-R2/URA3/CEN)
Segregant of SWY211
SWY294 with pSW191 replaced by pSW190
Segregant of SWY476
Segregant of SWY556
Segregant of SWY647
SWY389 with pSW190 replaced by pSW459
SWY389 with pSW190 replaced by pSW295
SWY390 with pSW295 replaced by pSW369
SWY389 with pSW190 replaced by pSW296
SWY391 with pSW296 replaced by pSW370
SWY389 with pSW190 replaced by pSW297
SWY392 with pSW297 replaced by pSW371
SWY389 with pSW190 replaced by pSW298
SWY393 with pSW298 replaced by pSW372
SWY389 with pSW190 replaced by pSW300
SWY394 with pSW300 replaced by pSW374
SWY389 with pSW190 replaced by pSW302
SWY395 with pSW302 replaced by pSW375
SWY389 with pSW190 replaced by pSW299
SWY396 with pSW299 replaced by pSW373
SWY1333
SWY389 + pRS313
SWY1349
SWY1350
SWY1351
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pRS313 (HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pSW191 (NUP145/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pSW540 (nup145∆524/592/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pSW542 (nup145∆592/893/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pSW543 (nup145∆592/608/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN)
pSW610 (nup145-33/HIS3/2µ)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW191 (NUP145/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW543 (nup145∆592/608/HIS3/CEN)
MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW540 (nup145∆524/592/HIS3/CEN)
SWY1360
MATa leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 pSW545 (GAL4BD-GFPS65T/TRP1)
SWY1361
MATa leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 pSW604 (GAL4BD-nup145GFPS65T/TRP1)
MATa/MATα NUP145/nup145 (1-594) – Protein A:HIS3:URA3 ura3-1/ura3-1
his3-11,15/his3-11,15
SWY1334
SWY1335
SWY1336
SWY1337
SWY1364
SWY1396
SWY1397
MATa/MATα NUP145/nup145 (1-626) – Protein A:HIS3:URA3 ura3-1/ura3-1
his3-11,15/his3-11,15
HF7c
MATa ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3,112 gal4-542 gal80-538
LYS2::GAL1-HIS3 URA3::(GAL4 17mers)3-CYC1-lacZ
pSW545, which encodes a fusion of the green fluorescent protein
(GFP) carboxy-terminal to the DNA binding domain of Gal4p was
constructed by inserting a BamHI/EcoRI GFP-encoding fragment
from pRSETB-S65T (Heim et al., 1995) into pGBT8 (Bartel and
Fields, 1995). pSW604 was constructed by inserting the
SWY389 + pSW191
SWY389 + pSW540
SWY389 + pSW542
SWY389 + pSW543
SWY389 + pSW610
SWY1334 without pSW190
SWY1337 without pSW190
SWY1335 without pSW190
ADE2 W303a (M.B. and S.R.W., in press)
+ pSW545
ADE2 W303a (M.B. and S.R.W., in press)
+ pSW604
W303 with one copy of NUP145 replaced by the
first 594 amino acids of Nup145p fused to
Protein A
W303 with one copy of NUP145 replacedby the
first 626 amino acids of Nup145p fused to
Protein A
Gift from H. Feilotter, Cold Spring Harbor, NY
EcoRI-digested
PCR
product
of
oligonucleotides
CA
(5′-GCCGAATTCGAGCTCTTCGGTAAAATTGTAATATTTCG-3′)
and CB (5′-GCCGAATTCTTGTTTACTCAAATCGTCTTCATC-3′)
into the EcoRI site of pSW545.
For pSW259, the N-terminal region of Nup145p (encoding residues
914
J. L. T. Emtage and others
2-664) was fused to the DNA binding region of Gal4p by subcloning
a BamHI/SacI-digested PCR product (generated with oligonucleotides
#124 (5′-TCGGGATCCTTAATAAAAGTGTAAATAGTGGT-3′) and
#112 (5′-CGAGAGCTCTTACCTGATAGACTTCC-3′)) into pGBT8
(Bartel and Fields, 1995). For pSW253, the C-terminal region of
Nup145p (encoding residues 593-1317) was fused to the activation
domain of Gal4p by subcloning the BamHI-digested PCR product
generated from 145-X and 145-Y2 into pACTII (gift of S. Elledge).
Generation of temperature-sensitive nup145 alleles
pSW191 (NUP145 HIS3) was mutagenized in vitro with hydroxylamine for 16 hours at 37°C (Kaiser et al., 1994) and transformed into
SWY389. His+ transformants were selected at ambient temperature,
replica-plated to medium containing 5-fluoroorotic acid (5-FOA), and
then tested for growth at 37°C on YEPD (Sikorski and Boeke, 1991).
Temperature-sensitive colonies were tested for the ability to grow at
37°C in the presence of pSW190 to identify mutations in other genes
and on 5-FOA medium at 23°C to identify NUP145 null mutations.
Plasmids were extracted from appropriate strains and retransformed
into SWY389 for further testing. These strains were allowed to lose
pSW190 and tested for growth at 37°C. From screening a total of
12,000 transformants, seven strains (referred to as SWY390SWY396) were unable to grow at 37°C and were designated temperature-sensitive nup145 alleles.
Gap repair and sequencing of the new temperaturesensitive nup145 alleles
pSW182, a fusion of the C-terminal region of Nup145p to glutathione
S-transferase (GST) behind the GAL1 promoter, was constructed by
ligating the BamHI-digested PCR product of oligonucleotides 145-X
and 145-Y2 from Z1 λ DNA into the BamHI site of pBJ382 (a
GAL1/GST fusion in pRS424 provided by C. Hug). The temperaturesensitivity of strains SWY390-SWY396 was complemented by transformation with pSW182, indicating that the mutations were within the
C-terminal region. pSW190 was cut with restriction enzymes delineating different regions of the C-terminal region of Nup145p and
transformed into strains SWY390-SWY396. Ura+ transformants were
selected at 23°C and then tested for growth at 37°C. SWY390,
SWY394, and SWY396 were unable to grow at 37°C when pSW190
was digested with MluI and AatII. The mutated area was further
narrowed to the region between the MluI and AflII sites. SWY391SWY393 and SWY395 were unable to grow when pSW190 was
digested with MluI and AvrII, or with HindIII alone. The Ura+ temperature-sensitive strains were allowed to lose the HIS3-plasmidborne nup145 alleles to form strains SWY535-SWY541. The relevant
regions of the nup145/HIS3 plasmids were sequenced. nup145-L2,
nup145-O1, nup145-V8, and nup145-A5 changed C to T to make glutamines #1193, 1147, 1091, and 1061, respectively, into stop codons.
nup145-E6 changed C to A to make serine #1140 a stop codon.
nup145-R4 deleted a T between amino acids #1012-1013; the
frameshift results in a stop codon at #1013. nup145-R2 deleted the C
in amino acid #1017, which alters the amino acid sequence from
#1017 and terminates 34 codons later.
Microscopy
Cells were processed for immunofluorescence as described by Wente
et al. (1992) and for in situ hybridization with a digoxigenin-labeled
oligonucleotide poly-(dT) probe as described (Wente and Blobel,
1993). mAb414 (Davis and Blobel, 1986) was used for pore complex
staining, mAb D77 for Nop1p staining (Aris and Blobel, 1988), and
5× diluted mAb B512 for tubulin staining (gift of J. Kilmartin).
Fixation times were 15 minutes for staining with mAb414 and 1 hour
for all other antibodies. Samples were examined with an Olympus
microscope through a ×100 objective. Photographs were taken using
an attached camera with Kodak T-MAX 400 film.
After fixation overnight in 2% glutaraldehyde and 2% formaldehyde, cells were prepared for electron microscopy as described by
Wente and Blobel (1993) using the method preserving both protein
and membrane structures. Samples were viewed with a Zeiss-902
electron microscope and photographs taken on Kodak electron
microscopy film.
Immunoblotting and production of antiserum to the Cterminal region
The C-terminal region of Nup145p was fused in frame behind the
maltose binding protein (MBP) to make pSW184 by inserting the
BamHI-digested PCR product of the 145-X2 (5′-TCGGGATCCAATCCCTTTGGCGGGACTTGGACT-3′) and 145-Y oligonucleotides from Z1 λ DNA into the BamHI site of pMAL-cRI (Maina
et al., 1988). pSW184 was transformed into DH5α, and fusion protein
was expressed and purified using amylose resin (New England
Biolabs) according to the manufacturer’s directions and sent to
Cocalico Biologicals, Inc. (Reamstown, PA) for production of rabbit
antiserum WU599. For affinity purification of the antiserum, 10 mg
of MBP and 7 mg of the fusion between MBP and the C-terminal
region of Nup145p were purified as described above and each was
coupled to 2 g CNBr-activated Sepharose 4B (Pharmacia Biotech,
Uppsala, Sweden) according to the manufacturer’s directions. WU599
was dialyzed into PBS (4°C) and centrifuged for 10 minutes at 9,000
g. The supernatant was incubated with MBP-Sepharose 4B for 2
hours, cleared of beads, and incubated with beads coupled to the
fusion protein overnight. The beads were washed with 0.5 mM NaCl,
20 mM sodium phosphate, pH 7.5, packed into a column, and eluted
with 0.1 M glycine-HCl, pH 2.8. The fractions were immediately
adjusted to pH 7.5-8.0 with 1 M Tris base, dialyzed into PBS, and
titered.
Total yeast cell extracts and immunoblotting were conducted as
described (Iovine et al., 1995). Protein A blots were incubated with a
rabbit anti-mouse antibody (1:500 dilution; Cappel), and GFP blots
were incubated with a rabbit polyclonal antibody (1:500; Clonetech).
Bands were visualized by the ECL system (Amersham Corp.)
according to the manufacturer’s directions or by incubating the blots
with alkaline phosphatase-conjugated anti-rabbit antibody (Promega)
and developing with nitro blue tetrazolium and 5-bromo-4-chloro-3indolyl-1-phosphate.
RESULTS
Analysis of N-terminal Nup145p deletions
In the previously reported nup145∆N::LEU2 mutation, the
LEU2 gene replaced the sequence encoding amino acids 65
through 549 of Nup145p (Wente and Blobel, 1994). Expression
of the C-terminal region was inferred from the fact that the
nup145∆N::LEU2 cells are viable whereas a complete nup145
null mutant is inviable (Fabre et al., 1994). Because the Cterminal region in the nup145∆N::LEU2 mutant has no
obvious promoter, the NPC clustering phenotype could be due
to the absence of the N-terminal region, or due to a lowered
expression level (or stability) of the C-terminal region. To
directly test whether the N-terminal region of Nup145p is
required for maintaining proper NPC distribution, three
different deletions from the N terminus were constructed and
placed under the control of the endogenous NUP145 promoter
(Fig. 1A). The nup145∆GLFG allele deleted the first 214
amino acids containing all the GLFG repeats. The nup145∆NL
allele deleted the first 562 amino acids, removing all of the
GLFG repeat region and most of the middle region. Finally,
the nup145∆NS allele deleted the first 592 amino acids,
removing the entire N-terminal region. Strains carrying the Nterminal deletion alleles as their sole source of Nup145p were
Mapping the functional regions of Nup145p
A
CLUSTERS
TS
TS RNA
EXPORT
NUP145
-
-
-
nup145∆GLFG
-
-
-
ALLELE
915
591
220
1317
215
563
nup145∆NL
-
-
ND
593
LEU2
nup145∆NS
+
+
+
nup145∆N::LEU2
+
-
ND
nup145-L2
-
+
ND
1146
nup145-O1
-
+
ND
1139
nup145-E6
-
+
ND
nup145-V8
+
+
+
nup145-A5
+
+
+
1016
nup145-R2
+
+
ND
1012
nup145-R4
+
+
+
nup145-100 (2µ)
+
+
+
nup145-58 (2µ)
NA
NC
NA
AAAAAAAAAA
AAAAAAAAAA
64
550
B
1192
1090
1060
893
551
C
8
NUP145
7
nup145-V8
relative A600
6
nup145-A5
5
nup145-R4
4
nup145-100
3
nup145 ∆ NS
2
1
time at 37°C (hours)
14
12
10
8
6
4
2
0
-2
0
Fig. 1. Mutant alleles of NUP145. (A and B) Diagrams of the
polypeptides encoded by the nup145 deletion mutations. The
deletion positions are denoted by amino acid number. The GLFG
repeats are represented by boxes, the middle region is gray, and the
C-terminal region is white. Clusters +, NPCs in clusters; clusters −,
NPCs were evenly distributed. Temperature-sensitive (ts) −, growth
at 37°C; ts +, no growth at 37°C. RNA export +, has a poly(A+)
RNA export defect at 37°C; −, no export defect. ND, not done; NC,
non-complementing; NA, not applicable. (C) Growth curves of the
nup145 mutants at 37°C. NUP145 (SWY389), nup145-V8
(SWY541), nup145-A5 (SWY535), nup145-R4 (SWY540),
nup145-100 (SWY513), and nup145∆NS (SWY690) strains were
grown in YEPD at 30°C and shifted to 37°C at time 0. Strains were
maintained in logarithmic phase throughout the course of the
experiment by diluting as needed to keep the A600 under 0.8. Thus,
the relative A600 is the actual A600 times the dilution factor(s).
assayed for growth at 37°C. The nup145∆NS cells showed
impaired growth at the high temperature (see Fig. 1C). The
strains expressing the two milder N-terminal deletions,
nup145∆GLFG and nup145∆NL (data not shown), were indistinguishable from wild type.
Indirect immunofluorescence was conducted to assess
whether the NPCs were clustering (Fig. 2A). Wild-type cells
showed punctate nuclear rim staining, whereas the signal in
nup145∆N::LEU2 cells was concentrated in discrete foci (NPC
clusters). Removal of the GLFG region had no detectable effect
on the surface distribution of the pore complexes (not shown).
This is in agreement with the published characterization of a
Protein A-Nup145p fusion which removed the GLFG region
(Fabre et al., 1994). The nup145∆NL cells also appeared
similar to wild type. The NPC staining in the nup145∆NS cells
was overall very weak, and therefore a definitive evaluation of
the presence or absence of NPC clusters was determined by
thin-section electron microscopy. Pore complexes in wild-type
and mutant cells are represented by electron dense patches that
span the double nuclear membranes. In wild-type cells (see
Fig. 5A), the NPCs were generally found singly distributed
around the entire circumference of the nuclear envelope
section. When thin sections of nup145∆NS cells were
examined (Fig. 2B), clusters of NPC-like structures were
observed (large arrowheads). These NPCs were grouped in
localized patches of the nuclear envelope and closely resemble
structures reported for nup145∆N::LEU2 cells. The clusters
were characterized by multiple NPC-like structures assembled
in grape-like aggregates (both micrographs, at ~11:00).
Besides the NPC clusters, another distinct morphological perturbation was observed (small arrowheads, left micrograph). In
apparently NPC-free areas, the nuclear envelope sometimes
appeared discontinuous and as a lace-like meshwork. This was
in contrast to the clearly continuous bilayer observed in wildtype cells.
NPC clustering was not observed in the nup145∆NL cells,
916
J. L. T. Emtage and others
B
and therefore the absence of the N-terminal region alone was
probably not responsible for the NPC clustering phenotypes.
To test whether the protein levels of the C-terminal region were
altered in the nup145∆N::LEU2 and nup145∆NS cells, proteins
were extracted from equivalent cell numbers and immunoblot-
Fig. 2. Characterization of the N-terminal Nup145p deletions.
(A) Examination of NPC distribution. NUP145 (SWY294),
nup145∆NL (SWY849), nup145∆NS (SWY690), and
nup145∆N::LEU2 (SWY122) strains were grown at 30°C,
fixed, permeabilized, and stained with antibodies against the
NPC and fluorescein-linked secondary antibodies. DNA was
stained with DAPI. Bar, 2 µm. (B) Examination by electron
microscopy of NPC clusters in nup145∆NS cells. The
nup145∆NS strain (SWY690) was
grown at 30°C and processed for
thin section electron microscopy.
Large arrowheads point to
clusters of NPCs. Small
arrowheads point to unusual
membrane structures in A. n,
nucleus; c, cytoplasm. Bar, 0.25
µm. (C) Poly(A+) RNA export
phenotype of the nup145∆NS
strain. NUP145 (SWY294) and
nup145∆NS (SWY690) cells were
grown to early logarithmic phase
at 30°C and shifted to 37°C for 5
hours. Cells were fixed,
permeabilized, and stained with a
digoxigenin-linked
oligonucleotide poly-(dT) probe
plus fluorescein-labeled antidigoxigenin antibodies. DNA was
stained with DAPI. Bar, 5 µm.
ted with a polyclonal Nup145p C-terminal specific antibody.
The C-terminal region from wild-type cells and from both of
the mutants migrated as an ~90 kDa fragment (Fig. 3A). An
unrelated yeast protein of greater molecular mass was also recognized by the antibody. Both of the mutants contained sig-
Mapping the functional regions of Nup145p
203
118
-
86 -
non-specific
C-term
51.6 -
203
-
118
-
86 -
51.6 -
nup145-R4 37°C
nup145-R4 23°C
NUP145 37°C
NUP145 23°C
nup145∆NS 37°C
nup145∆NS 23°C
nup145∆N::LEU2
B
NUP145
A
917
non-specific
C-term
C-term*
Fig. 3. Expression levels of Nup145p C-terminal region in deletion mutants. nup145 strains were grown at 23°C and shifted to 37°C for 1 hour
before harvesting where indicated; otherwise, they were grown at 23°C. Proteins were extracted from equal numbers of cells, separated by
SDS-PAGE, and transferred to nitrocellulose. The blot was probed with an affinity-purified polyclonal antibody raised against the C-terminal
region of Nup145p. Bands were visualized using the ECL system. (A) NUP145 (SWY294), nup145∆NS (SWY690), and nup145∆N::LEU2
(SWY122) strains were used. (B) NUP145 (SWY294) and nup145-R4 (SWY394) strains were used.
nificantly less C-terminal polypeptide than that in the wild-type
sample.
Because the nup145∆NS strain was temperature-sensitive, it
was also assayed for nuclear export capacity. The localization
of poly(A)+ RNA was monitored by in situ hybridization with
a digoxigenin-labeled poly(dT)30 oligonucleotide probe after
shifting to growth at 37°C. The staining in wild-type cells was
diffuse and cytoplasmic (Fig. 2C). However, in nup145∆NS
cells, the fluorescent signal became predominantly nuclear,
reflecting a block in poly(A)+ RNA export. By immunoblotting analysis, the protein level of the C-terminal region was not
altered with growth of the nup145∆NS cells at 37°C (Fig. 3A).
Thus, the nup145∆NS cells exhibited an NPC clustering
phenotype, a lowered level of the C-terminal region, and a temperature dependent RNA export defect.
Isolation of temperature-sensitive C-terminal
Nup145p truncations
To further dissect the regions of Nup145p necessary for NPC
function, a panel of temperature-sensitive nup145 mutations
was generated. Gap repair analysis revealed that all the temperature sensitive mutations resided in the sequence encoding
the C-terminal region. The new alleles were sequenced and all
were mutations which led to premature truncations, removing
200 to 300 amino acids from the C terminus (Fig. 1B). The
phenotypes of a 424 amino acid C-terminal truncation
(nup145-100) and a 766 amino acid C-terminal truncation
(nup145-58) were also examined. The nup145-100 allele
sustained the viability of a nup145 null strain, but only on a
high copy 2µ plasmid. In contrast, the nup145-58 allele on a
2µ plasmid did not rescue a nup145 null strain. The nup145100 strain was strongly temperature-sensitive lethal and growth
was impaired even at 23°C.
The doubling time and viability of cells harboring the nup145
temperature-sensitive alleles were examined at 37°C (Fig. 1C).
The mutations fell into 2 classes: those whose growth actually
ceased, and those whose growth merely slowed dramatically
and were still dividing after 25 hours. The degree of growth
impairment at 37°C correlated with the extent of the truncation.
nup145-100, nup145-R2 and nup145-R4 strains contained the
largest truncations, and correspondingly, their growth ceased.
As determined by plating cells at the permissive temperature,
<5% of nup145-R2 and nup145-R4 cells were viable after 16
hours at 37°C, versus ~25% for the milder mutants.
Characterization of the nup145 C-terminal truncation
mutants
The distribution of NPCs in cells harboring the C-terminal
truncations was examined by indirect immunofluorescence
microscopy. Wild-type and nup145-O1 cells exhibited even,
punctate, nuclear rim staining (see Fig. 4), as did nup145-L2
and nup145-E6 (not shown). However, the NPCs in nup145V8 cells were localized into discrete foci at the permissive
growth temperature. Alleles with more extensive truncations
also showed a constitutive NPC clustering phenotype (for
example, nup145-A5 and nup145-R4 in Fig. 4). Therefore, the
absence of 227 or more of the C-terminal residues resulted in
distinct NPC clustering.
To analyze the ultrastructure of NPC clusters in cells with
extensive C-terminal truncations, thin section electron
microscopy was conducted on nup145-R4 and nup145-100
cells. In wild-type cells (Fig. 5A), the NPCs were distributed
around the entire circumference of the nuclear envelope crosssection. In contrast, both mutants contained clusters of NPCs.
The NPCs were often grouped in a single layer of the nuclear
envelope (Fig. 5B,C). These may represent NPCs concentrated
in a patch of nuclear envelope (appearing as linear in the
section). In other sections, the clusters appeared in grape-like
aggregates in regions where the nuclear envelope was highly
convoluted (Fig. 5E,F,H,G). Both types of clustered structures
could result in the concentrated anti-NPC signal detected by
immunofluorescence microscopy.
918
J. L. T. Emtage and others
most severe nup145-R4 allele (Fig. 3B). A cross-reactive
polypeptide with a molecular mass of ~55 kDa corresponded
directly to the product predicted for the nup145-R4 allele, and
a band at the wild-type position (90 kDa) was absent. A direct
comparison to the levels of the C-terminal region in wild-type
cells could not be made because of the polyclonal nature of the
antibody. However, the signal was not reduced dramatically.
After growth at 37°C for 1 hour, the truncated polypeptide in
the nup145-R4 cells was present but the level was lower
relative to cells grown at 23°C.
Fig. 4. The NPCs cluster constitutively in strains carrying Cterminally truncated Nup145p. NUP145 (SWY294), nup145-O1
(SWY393), nup145-V8 (SWY395), nup145-A5 (SWY390), and
nup145-R4 (SWY394) strains were grown at 30°C and processed for
immunofluorescence with antibodies against the NPCs as in Fig. 2A.
The effect of representative C-terminal truncations on
poly(A)+ RNA export was also examined. Cells were shifted
to 37°C for 30 minutes and probed with poly(dT). In contrast
to wild type, nuclear accumulation of poly(A)+ RNA was
detected in a significant fraction of all four mutants tested
(nup145-V8, nup145-A5, nup145-R4, and nup145-100 in Fig.
6). However, at no time point was the export block at 37°C
observed in 100% of the cells. This lack of penetrance suggests
either that the export block is an indirect effect, or that the
accumulated poly(A)+ RNA is not stable over time.
To test whether the C-terminally truncated polypeptides
were expressed, immunoblot analysis was conducted with the
The severe N- and C-terminal truncations result in
pleiotropic defects
In addition to their strong temperature sensitivity, cells carrying
alleles with the most severe truncations (nup145-100, nup145R4, and nup145∆NS) displayed numerous morphological
defects. Cells of all three mutants were noticeably larger than
wild-type cells at all growth temperatures, and DAPI staining
and thin section electron micrographs revealed that the mutant
nuclei were large and irregularly-shaped (data not shown). In
addition, nup145-100 and nup145∆NS strains had an increased
tendency to be anucleate or binucleate, and in the case of
nup145∆NS, even trinucleate. The mutant cells were also
analyzed by indirect immunofluorescence microscopy with
antibodies against the nucleolar Nop1p and tubulin. At 30°C,
the nucleolus in the mutant strains had a wild-type appearance
(cresent-shaped body occupying one edge of the nucleus).
However, after growth at 37°C, the nucleolar staining in
nup145-100 or nup145∆NS cells fragmented into multiple foci
(data not shown). The tubulin staining revealed a markedly
lower percentage of mutant cells with very long spindles and
many of the cells had short spindles spanning their nuclei, with
possible orientation defects in regard to bud position (data not
shown). The growth defect of the nup145-R4 strain was suppressed at 37°C on media of high osmolarity (containing 1.2
M sorbitol or 0.9 M NaCl) (data not shown). Similar
pleiotropic defects have been reported in several nucleoporin
mutant strains (Bogerd et al., 1994; Aitchison et al., 1995a;
Heath et al., 1995; Iovine et al., 1995). These are probably
secondary defects arising from the inability to properly
transport substrates through the NPCs, and not directly related
to any single nucleoporin. Interestingly, neither nup145-100
nor nup145∆NS cells showed a protein import defect at 37°C
(data not shown).
Defining the essential region of Nup145p
The results from the N-terminal and C-terminal truncation
mutations suggest that an internal region from amino acid 593
to 893 may be essential for Nup145p function. The differing
phenotypes between the nup145∆NL and nup145∆NS mutants
suggest that the region from amino acid 563 to 593 may also
be important. Peptide sequencing analysis of the two Nup145p
fragments places the cleavage site between amino acids 524
and 606 (Wente and Blobel, 1994). Therefore, both of these
potentially important spans are in the region of Nup145p where
a cleavage event may be occurring. To map the essential region
of Nup145p and to assess whether cleavage is important for
function, three different in frame internal deletion mutations
were constructed, as diagrammed in Fig. 7A: nup145∆524/592
removed residues 524 to 592 (inclusive), nup145∆592/608
removed residues 592 to 608, and nup145∆592/893 removed
Mapping the functional regions of Nup145p
919
Fig. 5. Examination by electron
microscopy of NPC clusters in
nup145-R4 and nup145-100 cells.
NUP145 (SWY294), nup145-R4
(SWY540), and nup145-100
(SWY513) cells were grown at
30°C and processed for thin
section electron microscopy.
Small arrowheads indicate single
NPCs; large arrowheads point to
clusters of NPCs. (A) NUP145;
(B,D-F) nup145-R4; (C,G-I)
nup145-100. n, nucleus; c,
cytoplasm. Bar, 0.25 µm.
residues 592 to 893. These alleles were expressed behind the
NUP145 promoter on a CEN plasmid and transformed into a
nup145 null strain containing NUP145 on a URA3 vector. The
resulting strains were tested for their ability to lose the
NUP145/URA3 vector by growing them on medium containing 5-FOA, which selects against Ura+ cells. Cells with either
nup145∆524/592 or nup145∆592/608 were viable at all tested
temperatures (Fig. 7B). In contrast, cells with the
nup145∆592/893 allele were not viable at any growth temperature. To confirm this result, we expressed amino acids 593
through 893 behind the NUP145 promoter on a high copy
vector and assayed its ability to complement the nup145 null
allele. As shown in Fig. 7C, cells were viable when this
construct was the sole source of Nup145p. Therefore, a 300
amino acid span at the beginning of the C-terminal region is
essential for Nup145p function.
Nup145p cleavage occurs in vivo but is not required
for function
The nup145∆524/592 and nup145∆592/608 alleles were
920
J. L. T. Emtage and others
Fig. 6. mRNA export is compromised in mutants carrying C-terminal
truncations of Nup145p at the non-permissive temperature. NUP145
(SWY294), nup145-V8 (SWY541), nup145-A5 (SWY535), nup145R4 (SWY540), and nup145-100 (SWY513) cells were grown to early
logarithmic phase at 30°C and shifted to 37°C for 30 minutes before
processing as for Fig. 2C..
further analyzed for perturbations of NPC distribution and for
synthesis of Nup145p. By indirect immunofluorescence localization, the nup145∆524/592 cells had distinct NPC clusters
while the nup145∆592/608 cells appeared like wild type (data
not
shown).
Immunoblot
analysis
revealed
that
nup145∆524/592 cells expressed a cleaved ~90 kDa Cterminal polypeptide at a lower protein level relative to wild
type (Fig. 8A, lanes 1 and 2). Interestingly, a ~90 kDa Cterminal polypeptide was absent in the nup145∆592/608 cell
lysates, and instead a polypeptide of ~140 kDa was observed
(Fig. 8A, lane 3). This suggested that the cleavage site resides
between residues 592 and 608, but that cleavage is not
necessary for Nup145p function.
To further investigate the physiological significance of
Nup145p cleavage, an assay was designed to test whether
cleavage actually occurs in vivo or whether it is a consequence
of cell breakage. If cleavage occurs in vivo, a fusion protein
with the C-terminal region of Nup145p replaced by the IgG
binding region of Protein A should show differential subcellular localization depending on whether the cleavage site is
present. If the cleavage site is absent, the Protein A domain
should be targeted to the NPC via attachment to the N-terminal
region. Alternatively, if cleavage occurs in vivo and the
cleavage site is present, the Protein A domain should be
separated from the N-terminal region and thus found throughout the cell. Nup145p fusion proteins with Protein A inserted
either after residue 594 (before the cleavage site) or after
residue 626 (after the cleavage site) were expressed in wildtype cells and analyzed by immunoblotting for the Protein A
domain (Fig. 8A). Nup145p(1-594)-ProtA appeared uncleaved
and migrated with the predicted molecular mass of ~80 kDa
(lane 7), whereas Nup145p(1-626)-ProtA showed a band for
cleaved Protein A at ~30 kDa (lane 8). Indirect immunofluorescence analysis for the Protein A domain showed that the
Protein A localization for the uncleaved construct was predominantly nuclear/nuclear rim (Nup145p(1-594)-ProtA; Fig.
8B, lower left). Interestingly, the signal was localized throughout the cell for Protein A expressed from the construct that was
cleaved (Fig. 8B, lower right). Therefore, cleavage of the
Nup145p(1-626)-ProtA fusion was occurring in vivo.
To test whether the cleavage site of Nup145p was sufficient
for conferring cleavage of heterologous protein, chimeric
reporter proteins were constructed and assayed for differential
subcellular localization. Sequence encoding amino acids 512
to 627 of Nup145p was fused in frame between the DNA
binding region of Gal4p (Gal4BD) (encoding the first 147
residues of Gal4p) and the green fluorescent protein (GFPS65T).
If the Nup145p region can mediate cleavage of this chimera in
vivo, the GFP fluorescent signal should localize throughout the
cell because nuclear localization of GFP is presumably
dependent on fusion to the Gal4BD region and the nuclear localization signal therein (Silver et al., 1984). As expected, the
GFP signal from the chimera protein without the Nup145p
insert (Gal4BD-GFPS65T) was localized to the nucleus (Fig. 8B,
upper left). However, the chimera with the cleavage site
(Gal4BD-Nup145-GFPS65T) also showed GFP signal predominantly localized in the nucleus (Fig. 8B, upper right).
Immunoblotting with antibodies recognizing the GFP domain
confirmed that the Gal4BD-Nup145-GFPS65T chimeric fusion
was not cleaved (Fig. 8A, lane 5). Thus, the region of Nup145p
from residues 592-604 is not sufficient for conferring cleavage.
Because the N-terminal and C-terminal regions of Nup145p
are apparently cleaved in vivo into separate molecules, we used
the two-hybrid assay to assess whether the two halves physically interact. The N-terminal region was fused to Gal4BD and
the C-terminal region was fused to the activation domain of
Gal4p (Gal4AD). If the two regions interact, transcriptional
activation of a lacZ gene with upstream Gal4p DNA-binding
sites will occur via coincident juxtaposition of the Gal4BD and
Gal4AD domains (Bartel and Fields, 1995). The Gal4BD-Snf1p
and Gal4AD-Snf4p fusions were used as a positive interaction
control (Yang et al., 1992), and also as specificity controls
Mapping the functional regions of Nup145p
921
A
Fig. 7. An internal span of 300 amino acids is essential. (A) Diagram
of the polypeptides encoded by the nup145 internal deletion
mutations. Notations as in Fig. 1A, including: alive +, complements
the nup145 null mutation at 23°C; alive −, does not complement.
Cleaved +, the ~90 kDa fragment is formed; cleaved −, uncleaved
Nup145p observed. (B) Growth phenotypes of the internal deletion
mutants. nup145 null strains carrying a NUP145/URA3 plasmid plus
an empty HIS3 vector or the NUP145, nup145∆524/592,
nup145∆592/608, or nup145∆592/893 alleles in a HIS3 vector
(SWY1333-1337) were streaked on SD-ura-his and 5-FOA plates at
23°C. Those which grew on 5-FOA (now SWY1349-1351 because
the URA3 plasmid is gone) were streaked on a SD-his plate at 37°C,
along with a nup145-R4 strain (SWY394) as a temperature sensitive
control. (C) An internal span of 300 amino acids is sufficient to
support growth. NUP145 (SWY1334), nup145-33 (SWY1364), and
control (SWY389) strains were streaked on a 5-FOA plate at 23°C.
when combined with the Nup145p fusions. The plasmids were
co-transformed into a reporter yeast strain, and the level of βgalactosidase expression was measured using a color filter
assay (Fig. 9). The Gal4BD-N-term and Gal4AD-C-term fusions
did not show detectable activation capacity in combination
with the respective Snf fusions. Interestingly, blue signal was
detected when the N-terminal and C-terminal fusions were
combined, suggesting that the two halves of Nup145p interact
in this assay. However, a direct protein-protein interaction
remains to be demonstrated.
DISCUSSION
To define the structural regions of the nucleoporin Nup145p
responsible for the mRNA export and clustering defects
observed in previous studies (Fabre et al., 1994; Wente and
Blobel, 1994), we have characterized a series of deletion
mutations. We report that a distinct 300 amino acid region from
residues 593 through 893 at the beginning of the C-terminal
domain is essential for cell viability. At least two other regions
are also important for function. Interestingly, it appears that the
cleavage of Nup145p into N- and C-terminal fragments occurs
because in vivo cleavage of a Nup145p N-terminal fusion to
Protein A was observed. However, there was no phenotype
when the breakpoint between the two halves of Nup145p was
eliminated and full length product was synthesized. Therefore,
the conversion of Nup145p into two polypeptides is not
required for NPC function. This is not surprising in light of our
118
86
Mr
**
86
34.1
1
2
34.1
29
3
Nup145p C-term
81
*
*
51.6
51.6
Mr
Nup145p (1-626) - Prot A
*
Nup145p (1-594) - Prot A
203
Gal4BD - GFPS65T
∆592-608
∆524-592
Mr
WT
A
WT
J. L. T. Emtage and others
Gal4BD - Nup145 - GFPS65T
922
*
48.3
33.6
29
4
**
5
6
7
8
Protein A
GFP
512
627
Nup145
p
B
GAL4BD
GFP-S65T
GAL4BD
GFP-S65T
GFP
626
594
Prot A
Prot A
Protein A
DAPI
finding that the two halves of Nup145p can interact with each
other.
Previous studies of Nup145p have suggested two different
hypotheses for the observed fragmentation into N- and Cterminal polypeptides: that Nup145p biogenesis requires
cleavage, or that the cleavage is an indirect consequence of cell
breakage. The experiments in this report have demonstrated
that cleavage does in fact occur in vivo, and it is not simply a
Fig. 8. Characterization of Nup145p
cleavage. (A) Immunoblot analysis of
cleavage was conducted as for Fig. 3
except that the left and middle blots
were visualized using alkaline
phosphatase-linked secondary
antibodies. The blot on the left shows
samples from strains WT (SWY1349),
∆524-592 (SWY1350), and ∆592-608
(SWY1351) grown at 23°C, and
probed with an affinity-purified
polyclonal antibody against the Cterminal region of Nup145p. The
single star marks the ~140 kDa
uncleaved nup145∆592/608 protein
(lane 3), and the double star marks the
cleaved ~90 kDa wild-type (lane 1)
and nup145∆524/592 protein (lane 2).
The middle blot shows samples from
strains Gal4BD-GFPS65T (SWY1360)
and Gal4BD-Nup145-GFPS65T
(SWY1361) grown at 30°C, and
probed with a polyclonal anti-GFP
antibody. The single stars mark the
respective positions of full length,
uncleaved chimeric fusion proteins. A
band of ~30 kDa for cleaved GFP is
not present in lane 5. The blot on the
right shows samples from strains WT
(W303), Nup145p(1-594)-ProtA
(SWY1396), and Nup145p(1-626)ProtA (SWY1397) probed with a
polyclonal rabbit anti-mouse antibody
recognizing Protein A. Non-specific
bands are present in all samples, the
single star marks the position of the
uncleaved Nup145p(1-594)-ProtA
fusion (lane 7), and the double star
marks the cleaved product from the
Nup145p(1-626)-ProtA fusion (lane
8). (B) Fluorescence analysis of in
vivo cleavage. For the GFP images
(upper set), SWY1360 (left) and
SWY1361 (right) were grown at 30°C
in SD-trp medium to early logarithmic
phase, fixed for 10 minutes in 75 mM
NaCl/ 3.7% formaldehyde/ 0.25 µg/ml
DAPI, and washed with 100 mM
potassium phosphate, pH 6.5. For the
Protein A immunofluorescence fields
(lower set), SWY1396 (left) and
SWY1397 (right) were grown at 30°C
and processed for immunofluorescence
with antibodies against the Protein A
domain as in Fig. 2A. Bar, 5 µm.
protein extraction artifact. However, the fact that the product
of nup145∆592/608 allele is not cleaved and supports wildtype growth and NPC distribution reveals that processing is not
required for Nup145p function in vivo. In terms of the site of
cleavage, although the region spanning residues 593-607 was
necessary, it was not sufficient for processing. This is
evidenced by the lack of cleavage in the fusion between Gal4BD
and GFP, and by the published characterization of two different
Mapping the functional regions of Nup145p
Gal4 BD
Gal4AD
Snf1
Snf4
N-term
C-term
Snf1
C-term
N-term
Snf4
filter assay
Fig. 9. Nup145p N-terminal and C-terminal regions interact in the
two hybrid assay. Yeast strain HF7c was cotransformed with the
plasmids expressing the indicated Gal4BD and Gal4AD constructs. βgalactosidase activity was detected with a color filter assay (Breeden
and Nasmyth, 1985).
ProteinA-Nup145p fusions (Fabre et al., 1994). The previous
study reported that when Protein A replaced the GLFG region
of Nup145p (residues 1-247), the resulting fusion protein was
cleaved. However, when Protein A replaced most of the Nterminal region (residues 1-551) the fusion was not cleaved.
Considering our internal deletion of residues 524-592 was
cleaved, additional information for cleavage may reside in the
N-terminal region between residues 247 and 524. Alternatively, the fusion of the heterologous GFP and Protein A
domains immediately N-terminal to the cleavage site may nonspecifically inhibit proteolysis.
Preliminary conclusions regarding the analysis of the
nup145∆N::LEU2 phenotype attributed the clustering
phenotype to the lack of the N-terminal region of Nup145p
(Wente and Blobel, 1994). The phenotypes of the N-terminal,
C-terminal, and internal deletion mutations in this report
further suggest that at least two different regions (between
563-593 and 893-1090) are important for maintaining NPC
distribution. The contrasting phenotypes of the nup145∆NL
and nup145∆NS alleles implicate the region between residues
563 and 593; this is corroborated by clustering in the strain
lacking residues 524 to 592. However, it appears that the
absence of the N-terminal region per se is not responsible for
the clustering phenotype. First, the strain carrying the
nup145∆NL allele lacks the initial 562 amino acids, or most
of the N-terminal region, but displayed a wild-type distribution of NPCs. Second, all three of the clustering mutations
from N-terminal and internal deletion analysis coincidentally
lowered the protein level of the C-terminal region in yeast
cell lysates. Finally, expression of the N-terminal region
(nup145-58) in nup145∆N::LEU2 cells did not rescue the
clustering phenotype, while expression of the C-terminal
region (in the form of nup145∆NS) does (unpublished observations). We have not characterized an N-terminal deletion
mutation that clusters and has wild-type levels of the Cterminal region. These results suggest that lowered levels of
the C-terminal region contribute to the NPC clustering
phenotype. Truncations from the C terminus of Nup145p also
resulted in a constitutive NPC clustering phenotype, indicat-
923
ing that the entire C-terminal region must be present for
properly spaced NPCs.
Because the nup145∆NS and nup145∆524/592 alleles were
expressed under the control of the endogenous NUP145
promoter, the lower levels of C-terminal region probably reflect
an instability of the translated product. The same instability
probably applies to the nup145∆N::LEU2 product, and the
even lower protein level in the nup145∆N::LEU2 strain may
be attributed to the lack of a true promoter. Comparison of the
mutations suggests that the initiation of translation in the
nup145∆N::LEU2 mutant occurs before amino acid 592;
otherwise, the strain would have the pleiotropic defects associated with the nup145∆NS allele. Because the nup145∆NL
allele which initiates at amino acid 562 also behaves essentially
as wild type, the methionine residues at positions 564, 583, and
586 are all possible initiation sites for the nup145∆N::LEU2
C-terminal region. It is not likely that translation initiates
within the LEU2 fragment, because a similar deletion which
removes the same residues but instead has inserted the URA3
gene confers the same phenotype (Wente and Blobel, 1994).
Interestingly, high-copy expression of nup145-58 (encoding
the N-terminal 551 residues) does not complement the
nup145∆NS growth defect (unpublished observations). Thus,
the amino acid sequences between residues 551 and 593
(removed in the nup145∆NS and nup145∆524/592 alleles) may
be required for stabilizing the C-terminal region.
Mutations in genes encoding six different nucleoporin genes
result in NPC clustering: NUP145 (Wente and Blobel, 1994),
NUP133 (Doye et al., 1994; Pemberton et al., 1995; Li et al.,
1995), NUP159 (Gorsch et al., 1995), NUP120 (Aitchison et
al., 1995a; Heath et al., 1995), NUP84, and NUP85 (Siniossoglou et al., 1996). Two distinct types of NPC clustering have
been reported in these mutant strains; clusters with convoluted
membranes, and clusters in linear arrays. In all the previously
reported clustering mutants except nup159, both types of
clusters have also been shown to coexist. It is possible that
more than one mechanism may lead to clustering. However, it
is more likely that the two different types of clusters merely
represent different severities of the same perturbation.
For the above listed nucleoporin genes, there are also
mutated alleles which block RNA export. The RNA export
defects do not reflect a general collapse of NPC function, since
protein import is unaffected (see above references). Thus, these
six nucleoporins may act together in mediating RNA export
and maintaining proper NPC distribution. Mutational studies
of these other NUPs have also not clearly separated the structural regions required for the NPC clustering and RNA export
phenotypes. In most cases, clustering is constitutive while
RNA export is temperature sensitive, which may mean
clustered NPCs can only export RNA at the permissive temperature. However, two exceptions exist. nup159 mutants
display both NPC clustering and a mild RNA export defect at
the permissive temperature (Gorsch et al., 1995). By 15
minutes after the shift to 37°C, RNA export is blocked. After
an hour at 37°C, the clustered NPCs largely return to a normal
configuration. However, at this time, Nup159p is no longer
detectable (Gorsch et al., 1995). Thus, the two nup159 phenotypes do not become separable until Nup159p is absent. Cells
expressing an N-terminal deletion allele of nup133 have
clusters but are only mildly temperature sensitive with a mild
RNA export defect (Doye et al., 1994). The data from this
924
J. L. T. Emtage and others
Nup145p study imply that the full C-terminal region is required
to maintain viability and mRNA export capacity at all temperatures. Interestingly, lower C-terminal protein levels alone do
not affect growth and export capacity, because the
nup145∆N::LEU2 strain is not compromised.
It is possible that NPC clustering and RNA export defects
are both manifestations of failure of some underlying process
which this group of nucleoporins mediates. We have previously
speculated that the formation of clusters may be due to changes
in the attachment of NPCs (and the nuclear envelope) to an
underlying nuclear scaffold which maintains their fairly even
spacing (Wente and Blobel, 1994). RNA may utilize the same
intranuclear structures to be transported from their sites of
synthesis to the NPCs (reviewed by Xing et al., 1993). Such
coincident perturbation in mutants of nucleoporins that
maintain NPC-nuclear interactions would therefore be
expected.
In conclusion, the results from mutational analysis suggest
that all essential Nup145p functions are mediated by its Cterminal region, with the N-terminal region possibly mediating
a non-essential cleavage event. The essential region spanning
residues 593 and 893 of Nup145p can now be targeted for
future analysis. It will also be critical to determine both
Nup145p’s nearest neighbor protein-protein interactions and its
substructural localization within the NPC.
We thank G. Raczniak and A. Wilson for helping with the production of the antibody specific for the Nup145p C-terminal region; J.
Aitchison, M. Rout, and G. Blobel for pProtA/HU; R. Murphy for
pSW545; M. Levi and L. LaRose for technical assistance with EM
experiments; H. Piwnica-Worms for use of her Olympus fluorescence
microscope. We are grateful to C. Hardy and members of the Wente
laboratory for critical discussion of the results, and to C. Cole for communicating unpublished results. J. L. T. Emtage performed this work
as a Fellow of the Missouri Affiliate of the American Heart Association. This work was supported by an RO1 grant from the National
Institutes of Health, No. GM51219-02, to S. R. Wente.
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(Received 30 September 1996 – Accepted 17 January 1997)