Molecular Cell, Vol. 1, 223–234, January, 1998, Copyright 1998 by Cell Press
Three-Dimensional Architecture of the
Isolated Yeast Nuclear Pore Complex:
Functional and Evolutionary Implications
Qing Yang,*‡ Michael P. Rout, †
and Christopher W. Akey*§
* Department of Biophysics
Boston University School of Medicine
Boston, Massachusetts 02118-2526
† The Laboratory of Cellular and Structural
Biology
The Rockefeller University
New York, New York 10021-6399
‡ Endocrinology and Reproduction Research Branch
National Institutes of Health
Bethesda, Maryland 20892-4510
Summary
We have calculated a three-dimensional map of the
yeast nuclear pore complex (yNPC) from frozenhydrated specimens, thereby providing a direct comparison with the vertebrate NPC. Overall, the smaller
yNPC is comprised of an octagonal inner spoke ring
that is anchored within the nuclear envelope by a novel
membrane-interacting ring. In addition, a cylindrical
transporter is located centrally within the spokes and
exhibits a variable radial expansion in projection that
may reflect gating. The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they
are required to form a functional NPC. However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences
in nuclear transport regulation or mitotic behavior.
Introduction
The nuclear pore complex (NPC) spans the nuclear envelope (NE) within a specialized pore membrane domain,
formed by a fusion of the inner and outer nuclear membranes. The NPC functions as a channel for the nuclear
import of proteins and snRNPs, while providing an export path for tRNAs, mRNPs, ribosomal subunits, and
snRNPs (reviewed in Gorlich and Mattaj, 1996; Nigg,
1997). Nuclear import can be divided into three major
steps: targeting/docking, translocation, and substrate
release (Newmeyer and Forbes, 1988; Richardson et al.,
1988; Akey and Goldfarb, 1989; Pante and Aebi, 1996).
Recent work has resulted in a paradigm for nuclear
import and export, in which receptors mediate the binding of transport substrates containing a nuclear localization (NLS) or export sequence (NES) to repeat-containing nucleoporins (GLFG/FXFG) within the NPC. It
is postulated that substrate translocation may utilize
cycles of GTP hydrolysis mediated by Ran (Koepp and
Silver, 1996; Nigg, 1997). In addition, the NPC is thought
to provide passive diffusion channels that give the NE
its characteristic permeability to small molecules (Paine
et al., 1975; Akey and Radermacher, 1993; Akey, 1995;
§ To whom correspondence should be addressed.
Perez-Terzic et al., 1996), while peripheral channels may
facilitate the redistribution of nuclear membrane proteins (Powell and Burke, 1990; Hinshaw et al., 1992,
Akey and Radermacher, 1993; Wiese and Wilson, 1993).
The three-dimensional structure of the vertebrate NPC
has been studied in amphibian oocytes using electron
microscopy and single-particle methods on frozenhydrated (Akey and Radermacher, 1993) and negatively
stained specimens (Unwin and Milligan, 1982; Hinshaw
et al., 1992). These studies have provided a low resolution model of this translocation machine (see Figure
1). The NPC is comprised of an octagonal spoke–ring
complex that is embedded in the NE and that encircles a
central channel complex (termed the transporter; Akey,
1989, 1990). The spoke–ring complex anchors more peripherally associated components, including the cytoplasmic filaments (Richardson et al., 1988; Jarnik and
Aebi, 1991; Ris, 1991) and the nuclear basket (Jarnik
and Aebi, 1991; Ris, 1991; Goldberg and Allen, 1992),
which play a role in the docking of import (Richardson
et al., 1988; Pante and Aebi, 1996; Rutherford et al.,
1997) and export substrates (Kiseleva et al., 1996), respectively.
Nucleocytoplasmic transport utilizes a central channel within the NPC for import and export (Feldherr et
al., 1984; Dworetzky and Feldherr, 1988). However, the
mechanism of translocation across the NPC is currently
unknown. Recently, atomic force microscopy and scanning electron microscopy on Xenopus (Goldberg and
Allen, 1996; Perez-Terzic et al., 1996) and Chironomus
NPCs (Goldberg and Allen, 1996; Kiseleva et al., 1998)
have provided further observations of the NPC transporter (Akey and Radermacher, 1993). The NPC transporter is present in manually isolated NEs in a number
of radially expanded configurations in projection, suggesting that the channel is gated (Akey and Goldfarb,
1989; Akey, 1990). The physical imprint of the transporter is revealed in thin sections of Balbiani ring mRNPs
and nucleoplasmin-gold particles caught traversing the
NPC (Stevens and Swift, 1966; Feldherr et al., 1984;
Mehlin et al., 1992), as these substrates are constrained
within a z200–250 Å diameter channel that extends
across the NPC for about 500–600 Å.
The completion of the Saccharomyces genome sequencing project (Clayton et al., 1997 and references
therein) has resulted in an accelerated identification of
yeast nucleoporins (nups) (Rout and Wente, 1994; Doye
and Hurt, 1997). Moreover, the recent isolation of enriched fractions of yeast NPCs and NEs (Rout and Blobel, 1993; Strambio-de-Castillia et al., 1995) has opened
the door to structural studies, as isolated yNPCs have
dimensions similar to those in cells. However, yeast
NPCs are both smaller and less massive than vNPCs
(z66 MDa versus z125 MDa; Reichelt et al., 1990; Rout
and Blobel, 1993). Hence, a detailed three-dimensional
(3-D) structure of the yNPC will allow an understanding
of this fundamental size difference and provide a framework in which the position of yeast nucleoporins can be
mapped.
In this work, we present the 3-D structure of the yNPC
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224
the method of Rout and Blobel (1993) and utilized computational methods to select the fraction with the highest degree of structural preservation. A representative
subset of 8-fold symmetric yNPCs within this fraction
were identified by rotational power spectra analysis, and
the appropriate two- and three-dimensional maps were
calculated.
Results
Mass and Symmetry of Yeast NPCs
We have used dark-field imaging in the scanning transmission electron microscope (STEM; Wall and Hainfeld,
1986) to estimate the mass average of the enriched
and highly enriched fractions of yNPCs, isolated by the
method of Rout and Blobel (1993). The data for highly
enriched yNPCs is shown in Figure 2A and gives a
Gaussian-fitted peak of 54.5 MDa (s 5 10.1). Similar
mass values were obtained for enriched yNPCs and
cross-linked yNPCs from the highly enriched fraction
(Yang, 1997). The STEM mass data and earlier estimates
obtained by light scattering and sedimentation methods
(Rout and Blobel, 1993) overlap significantly and place
the mass of the yNPC between 55 and 66 MDa. Previous
measurements rely on bulk properties of the sample and
are influenced by “larger” particles (Rout and Blobel,
1993), while the STEM measures individual particles to
form a mass spectrum. Hence, the differences between
the midpoint values may reflect the nonideal behavior
of partially purified yNPCs. However, this mass estimate
is consistent with the smaller size of the yNPC observed
in thin sections of cells and nuclei (Rout and Blobel,
1993; Strambio-de-Castillia et al., 1995) and with our
3-D analysis.
Isolated yeast NPCs are present in a range of masses,
and images reveal varying degrees of elliptical distortion. These observations suggested that many of the
yNPCs have sustained some physical damage during
isolation. Therefore, we have carried out a detailed analysis of the preservation of detergent-solubilized yNPC
fractions using 2-D averages (Frank et al., 1996; Akey,
1989) and rotational power spectra analysis (Crowther
and Amos, 1971; Kocsis et al., 1995). Although the highly
enriched fraction is biochemically cleaner, projection
maps suggested that the enriched yNPCs have less
circumferential distortions within the spokes (Yang,
1997). Hence, we chose the enriched fraction for twoand three-dimensional cryodata collection. After visual
selection, rotational power spectra were calculated from
the untilted yNPCs. This provided a “one-dimensional
diffraction pattern” that was used to evaluate particle
preservation, allowing us to choose the best yNPCs for
further analysis. A histogram of rotational 8-fold power
within the dataset is presented in Figure 2B. Particles
with a low signal-to-noise ratio, strong elliptical distortions, or physical damage were excluded, based on the
lack of a significant 8-fold harmonic in their power spectra (dotted curve in Figure 2C). Roughly half of the particles demonstrated 8- and 16-fold harmonics in their
spectra (Figures 2C–2E), reflecting the 8-fold symmetry
of the yNPC (Rout and Blobel, 1993).
The resolution that is attainable with supramolecular
assemblies in the electron cryomicroscope is often limited by the biochemical stability of the specimen. Therefore, we have characterized yNPC fractions isolated by
Structure of the Isolated Yeast NPC and
Interactions with the Nuclear Envelope
We calculated projection maps for both detergent-solubilized (dform) and nuclear envelope–associated (mform)
Figure 1. A Consensus Model of the Vertebrate NPC
The overall structure is comprised of a wheel-like array of eight
spokes connected to the inner spoke ring (ISR), which encircles the
central channel complex (transporter [T] is pink). The spoke complex
is framed top and bottom by the cytoplasmic (CR) and nuclear (NR)
thin rings that serve as attachment sites for cytoplasmic particles
(CP) and filaments and the nuclear basket. The spokes are embedded in the NE (light blue), are interconnected within the lumen to
form a lumenal ring (LR), and are attached to the lamina (L). The
outer and inner nuclear membranes are labeled (ONM) and (INM),
respectively. A set of inner ring filaments interconnect the top and
bottom of the central transporter to the thin rings and for clarity,
are shown only on the cytoplasmic side (bold lines).
determined by electron cryomicroscopy and image processing. The resulting maps have been compared with
their vertebrate counterparts, to provide insights into
the function and evolution of the NPC and its subassemblies. Overall, the yNPC is smaller than the vertebrate
NPC (vNPC) and maintains a conserved inner spoke ring
and central transporter. In addition, yeast and vertebrate
NPCs both interact with the NE at comparable radii using
similar spoke domains. However, the yNPC does not
have a lumenal spoke ring but rather has evolved a novel
membrane-interacting ring that is comprised of 8 linear
arms and 8 membrane-spanning spoke domains. In projection maps, the central yNPC transporter is visualized
in 3 possible transport-related configurations and demonstrates 2 related “in transit” forms, similar to that
observed in vertebrate transporters (Akey and Goldfarb,
1989; Akey, 1990). The implications of these observations for the mechanism of nucleocytoplasmic transport
are discussed.
Architecture of the Yeast Nuclear Pore Complex
225
Figure 2. Mass and Rotational Power Spectra Analyses of Yeast NPCs
(A) Mass histogram of 506 yNPCs obtained
from dark-field STEM (peak mass and standard deviation are 54.5 Mda and 10.1 Mda).
(B) Histogram of relative 8-fold power for particles within the yNPC dataset, plotted as a
percentile. The distribution is skewed with a
median of z28%.
(C–E) Averaged rotational power spectra from
the bottom 1728, the best 1535 yNPCs
(curves labeled [1] and [2] in [B] and [C]), from
the top 10 (labeled [3] in [B] and [D]), and from
the global transporter dataset ([GBT] in [E]).
yNPCs in frozen-buffer and negative stain, respectively
(Figures 3A and 3B). These maps were then compared
with similar published maps of the vNPC (Figures 3C
and 3D; Akey, 1995) to understand the structural basis
of previously observed size differences and to identify
the membrane-interacting spoke domain within the
yNPC.
In Figure 3A, the dform map of the yNPC has an outer
diameter of z960 Å and is comprised of 8 spokes. Each
spoke has 2 radial domains (circles labeled 1 and 2),
with the proximal domains forming an inner spoke ring.
The inner spoke ring encircles a central blurred disk
that represents a global average of the transporter. An
additional linear arm of density extends radially from
the inner spoke ring and is connected circumferentially
to both of the neighboring spoke 2 domains, to form a
Figure 3. Two-Dimensional Projection Maps
of Yeast NPCs and a Comparison with Vertebrate NPCs
(A) Map of yNPCs without nuclear membranes in frozen buffer. Labels: radial spoke
domains (labeled [1] and [2] with circles), inner spoke ring (ISR), linear arm (LA), central
transporter (T), putative transport substrates
(S). The position of the approximate spoke
2-fold axis is indicated.
(B) Map of NE-associated yNPCs in negative
stain. Two radial spoke domains are labeled
with circles, and the position of the NE is
indicated (*).
(C) Map of vNPCs without nuclear membranes in frozen buffer. The radial arm is indicated (RA), and three radial spoke domains
are labeled (1)–(3) (with circles). The position
of the NE is shown by contours from the appropriate difference map (Akey, 1995).
(D) Map of vNPCs associated with the NE in
frozen buffer (Akey, 1995). The three radial
spoke domains (closed circles) and the NE
(*) are indicated. Protein and membranes are
open; scale bar 5 300 Å.
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226
Table 1. Domain Positions and Dimensions in Yeast and Vertebrate NPCs
Yeast NPC
Structural domains
Transporter
Inner spoke domain
Second spoke domain
Membrane ring
Nuclear membrane pore
Entire NPC
Di (Å)
490
730
780
Vertebrate NPC
Dc
580
840
860
820
Do
Height
340–380
680
960
300
350–380
160–220
50–70
Di (Å)
870
960
480
720
Dc
Do
Height
600
860
320–420
720
940
625
z340a
840–850
350–380
1200–1450b
800–
1000c
Vertebrate NPC dimensions are from Akey (1989), Akey and Radermacher (1993), and Akey (1995). Di/Dc/Do are inner, center and outer
diameters.
a
Measured from Akey and Radermacher (1993).
b
Including the radial arms.
c
Including the cytoplasmic particles.
ring. The spokes display approximate 2-fold symmetry
when mirrored about the arrow labeled 2 (which bisects
the spokes); however, the linear arm is displaced circumferentially in a counter-clockwise direction from a
“local” 2-fold axis (located between the spokes as
shown in Figure 3C), to interact preferentially with an
adjacent spoke. This deviation from 822 symmetry may
have arisen during purification, as similar circumferential
distortions are present in vNPCs (Akey and Radermacher, 1993).
A comparison of 2-D maps from yeast and vertebrate
NPCs suggests a reason for the observed size differences. Vertebrate spokes have 3 radial domains in projection with the positions of the innermost 2 domains
being equivalent to those in yNPCs (Figures 3A and 3C,
Table 1). A second projection map of the yNPC reveals
that the spokes have 2 radial domains when the nuclear
membrane is present (Figure 3B, see circles). Hence, it
seems unlikely that the “best” yNPCs have lost major
spoke domains during solubilization and purification.
We suggest that the yNPC spoke may be intrinsically
smaller than its vertebrate counterpart.
Although yeast and vNPCs differ in lateral diameter
and mass, some features of their interaction with the
NE are conserved. In Figure 3B, the nuclear membrane
is present as a strong band of circumferential density
that interacts with the second spoke domain in yNPCs.
This interaction occurs at a similar radius within vNPCs
(Figure 3D and contoured difference map in Figure 3C;
Akey, 1995). However, there are notable differences between yeast and vertebrates in the details of this interaction. For example, the yNPC has an additional linear arm
that is not observed in the mform yNPC map, because
it is likely buried within the membrane.
Visualization of Multiple Forms
of the Central Transporter
The diameter of the globally averaged central transporter in yNPCs is similar to that observed in vNPCs
(z350–380 Å; Akey, 1989, 1990). The vertebrate transporter appears to have 8-fold symmetry, as 8 internal
ring filaments link the transporter to the cytoplasmic
and nuclear thin rings (see Figure 1; Goldberg and Allen,
1996), and these data are consistent with previous labeling studies using MAb414 (Akey and Goldfarb, 1989).
The similar diameter of the yeast and vertebrate transporters and the shared 8-fold symmetry of the spokes
would argue that the yeast transporter has 8-fold symmetry, and this property was used in the following analysis. Classification was used to eliminate yNPCs with
poorly defined transporters and to sort the remaining
pore complexes into transporter groups (Akey, 1990;
Frank, 1990). This analysis resulted in projection maps
of three transporter classes shown in Figures 4A–4C,
and a global average (see Figure 4D and averaged power
spectrum in Figure 2E).
There are two prominent classes in which the transporter ring encircles central material, that may represent
transport substrates trapped within the channel during
spheroplast lysis (Figures 4B and 4C). The diameter of
the transporter in Figure 4C is z13% larger than in Figure
4B, and this radial expansion was correlated with an
increase in central substrate density (after scaling within
the spokes). Indeed, the intensity of the central substrate
disk was computationally truncated to provide an equivalent display of the spokes in Figures 4B and 4C. Transporters with a similar “in transit” morphology were identified previously in vNPCs (Figure 4E; Akey and Goldfarb,
1989; Akey, 1990). In Figure 4A, the central transporter
ring surrounds an apparently empty central channel.
However, a comparison of the channel density with
background levels suggests that these transporters may
contain some residual material, as this class may have
originated from the two “in transit” classes by the loss of
trapped substrates during isolation. Finally, the central
placement of the transporter within the inner spoke ring
creates a set of 8 low density features that may form
diffusion pores in both “species” (open circles in Figures
4A and 4E). These data suggest that transporters are
general features of all NPCs and reinforce the concept
that the transporter represents a gated channel involved
in nucleocytoplasmic transport (Akey and Goldfarb,
1989; Akey, 1990; Akey and Radermacher, 1993).
Three-Dimensional Structure of the yNPC
Three-dimensional maps were calculated of the yNPC
to provide insights into the architecture of this assembly.
However, we found that 3-D maps required z500–600
yNPCs to provide an acceptable signal-to-noise ratio
with these data. Hence, at this stage we computed a
Architecture of the Yeast Nuclear Pore Complex
227
Figure 4. Classification of yNPCs Reveals the Central Transporter
(A) Transporter class 1 contains a central ring-like transporter encircling a central channel. One of eight diffusion channels is labeled with an
open circle.
(B) Transporter class 2 contains a transporter ring that encircles a central bright density that, by analogy with vNPCs, may be composed of
substrates caught in transit (see Akey, 1990).
(C) Transporter class 3 contains a central ring with a greater radial expansion than observed in class 2.
(D) Global average of yNPC transporters. The spoke domains ([1] and [2]); the transporter ring (T), and the putative substrate disk (S) are
labeled.
(E) A radially expanded vertebrate NPC transporter (Akey, 1990). Note the similar “in transit” morphology in both vertebrate and yeast
transporters. Scale bar 5 300 Å.
3-D map of the global transporter class (GBT class).
In addition, we used classification to obtain the most
homogeneous spoke class (SC), irrespective of the quality of the associated transporter, and computed the
SC map.
Central cross-sections 100 Å thick and oriented parallel to the original position of the NE are shown in Figures
5A and 5C, for the GBT and SC 3-D maps. In general,
the spokes are similar in both maps, but as expected,
the central transporter is rather disordered in the SC
map. In this view, the spokes form an inner spoke ring,
comprised of spoke domain 1 and the proximal end of
the linear arms (inner closed circle, Figure 5C). In addition, the second spoke domains are interconnected with
the distal end of the linear arms (outer closed circle,
Figure 5C) to form a continuous ring. This feature is
located at a radius that would place a majority of the
ring within the nuclear membrane, while the most distal
parts of the ring may protrude into the lumen. Hence,
we have named this novel feature the “membrane ring.”
Additional evidence for this membrane ring was obtained after detergent or heparin extraction of NEs or
yNPCs, respectively (Strambio-de-Castillia et al., 1995).
Extracted NEs show a filamentous meshwork in which
ring structures are embedded, while strongly heparinized yNPCs give rings with a diameter commensurate
with the size of the membrane ring.
Prominent spoke features were revealed by calculating an angular projection that covered an arc of z22.58,
centered on the spoke (Figure 5B). In this view, the spoke
is composed of an inner spoke domain that spans the
entire yNPC and a second domain located at the spoke
midplane (dashed lines). These domains are joined at
the top and bottom of the inner spoke ring by a bridging
density that forms an angle of z508 relative to the central
plane (closed circle in Figure 5B). Weak density present
at the top and bottom of the spokes (open arrows, Figure
5B) may represent disordered filaments and nuclear
baskets, as filaments were observed radiating from the
yNPCs in dark-field STEM images (not shown) and peripheral filament proteins such as Nup159p are retained
in these preparations (Kraemer et al., 1995). The transporter in the GBT 3-D map (Figure 5B) contains a central
bright density that may reflect substrates trapped within
the cylindrical walls of the channel assembly (TW). Overall, the isolated yNPC is a disk with dimensions of 960
Å 3 350–380 Å (Table 1). A strongly thresholded surface
view of the spokes without the central transporter reveals the connectivity of the spoke domains (Figure 5D).
The second radial spoke domain extends from the inner
spoke ring with its attendant bridging density, and the
linear arm is located between adjacent spokes.
The relationship between the central transporter, the
inner spoke ring, and the membrane ring is shown in
surface views of the GBT 3-D map (Figures 6A–6D). In
Figures 6A and 6B, the spoke–transporter assembly is
viewed obliquely from the bottom and top of the 3-D
volume, respectively. Face-on and side-on views of the
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228
circle in Figure 6C), as observed in vertebrate NPCs
(Akey and Radermacher, 1993). Hence, these internal
channels may play a role in the passive diffusion of small
molecules between the cytosol and nucleus.
Discussion
Figure 5. Selected Slices and Surfaces from Two Yeast NPC 3-D
Maps
(A) An average of 5 xy slices taken from the center of the GBT 3-D
map. The spoke domains, transporter, and central substrate are
labeled as before. The position of this slice is indicated in (B) (dashed
lines).
(B) Angular projection of the yNPC calculated from the GBT 3-D
map. The inner spoke ring (1), the outer spoke domain (2), and
the bridging region (closed circle) are marked. White arrowheads
indicate peripheral density attributed to disordered filaments on
both yNPC surfaces. The transporter wall (TW) and the putative
central substrate (S) are labeled.
(C) A central xy slice from the SC 3-D map (n 5 577). The spokes
and membrane ring are labeled as before. Two domains of the linear
arm are marked with circles, and the membrane interacting ring
(MR) is formed by interactions between the linear arm (LA) and
adjacent spoke domains (2).
(D) A highly thresholded surface view of the GBT spoke ring is
shown; spoke domain (2), the inner spoke ring (ISR), and the linear
arm are marked. Scale bar 5 300 Å.
3-D map are shown in Figures 6C and 6D. The inner
spoke ring encircles the central transporter and may
function as a central organizing assembly within the
yNPC. The membrane ring is formed by circumferential
connections between the second spoke domains and
the adjacent linear arm (Figure 6C), and when viewed
from the side, this feature is not flat, but rather forms a
sinusoidal ring (closed circles in Figure 6D).
In the 3-D map, the central transporter is a cylinder
with dimensions of z350 3 300 Å. In addition, weak
densities interconnect the top and bottom surfaces of
the inner spoke ring and transporter; these features may
be below the current resolution or partially disordered
(not shown). Density identified as transport substrate in
2-D maps is visible at the center of the transporter ([S]
in Figures 6A and 6C). Hence, the GBT 3-D map may
represent transporters trapped in a number of differing
configurations during spheroplast lysis. Interestingly,
the transporter differs in the degree of radial expansion
present at either end of the cylindrical channel. These
differences may reflect an intrinsic size asymmetry of
import and export substrates that is maintained in the
averaged map. Finally, internal channels are located between the transporter and the inner spoke ring (open
Rationale for Comparing Yeast
and Vertebrate NPCs
Yeast and vertebrate cells represent different aspects
of the basic eukaryotic cell design. Generally, yeast cells
are smaller, undergo rapid cell division with a closed
mitosis, and have less complex cytoskeletons due to the
external support of a cell wall. Vertebrate cells possess
elaborations not found in yeast: they have a lamina and
larger nuclei, and their NEs disassemble at mitosis. In
addition, they differentiate into many cell types and undergo apoptosis. Given the evolutionary distance between yeast and vertebrates, it is not unexpected that
the structure of their NPCs would differ. However, both
yeast and vNPCs must contain the minimal components
required to build a functional NPC.
As a prelude to this study, it was necessary to prepare
enriched fractions of yNPCs. However, isolated yNPCs
display a range of physical deformations and mass. We
therefore used rotational power spectra to select the
best yNPCs for two- and three-dimensional maps, accounting for z2%–5% of the yNPCs. We suggest that
the maps in this study are representative of the core
yNPC at the current resolution of z80 Å in 2-D and
z120–150 Å in 3-D, excluding the putative peripheral
filaments and baskets that are disordered. First, 16 yeast
nups coenrich quantitatively with isolated NEs and
yNPCs (Rout and Blobel, 1993; M. P. R., unpublished
data). Second, all known nups (z30) have been identified
in the yNPC preparation using mass spectrometry
(M. P. R. et al., unpublished data). Third, filaments were
observed radiating from yNPCs in dark-field STEM images. As filament components such as Nup159p are
retained in these preparations (Kraemer et al., 1995),
the data indicate that peripherally associated nups are
present in the isolated yNPCs. Thus, the best yNPCs
are likely to contain a full (or nearly full) complement of
nups.
All existing evidence suggests that yNPCs are fundamentally smaller than vNPCs. For example, yNPCs have
a simple disk-like appearance in thin sections of whole
cells, nuclei, and NEs but do not contain the distinctive
features at the surface of the NE that correspond to
the thin coaxial rings in vertebrate NPCs (Franke and
Scheer, 1974; Rout and Blobel, 1993; Strambio-de-Castillia et al., 1995). The overall dimensions of our structure
are in excellant agreement with measurements from thin
sections of yeast nuclei (Rout and Blobel, 1993), and
the morphology is similar. Interestingly, the thickness
of the NE lumen is markedly different in these two eukaryotes: in yeast, the NE is z250–300 Å thick (31 measurements from thin sections of 6 yeast nuclei in cells),
while in vertebrates, the NE is z500–600 Å wide (e.g.,
see Goldberg and Allen, 1996; Pante and Aebi, 1996).
In vertebrate NPCs, the inner and outer nuclear membranes are in close apposition to the thin nuclear and
cytoplasmic rings and their connecting spoke domains
Architecture of the Yeast Nuclear Pore Complex
229
Figure 6. Surface Views of a 3-D Map of the
Yeast NPC with a Globally Averaged Transporter
(A and B) Oblique views of the yNPC revealing
the bottom and top surfaces of the yNPC. The
membrane ring (MR) interconnects adjacent
spokes, making contact with the linear arm
(*). The cylindrical transporter resides within
the inner spoke ring (ISR) and contains putative central substrates (S).
(C) An en face view of the yNPC reveals details of spoke domain architecture ([1] and
[2]) and suggests that diffusion channels are
located between the transporter and ISR
(open circle). The membrane ring (MR), transporter (T), and putative central substrate (S)
are labeled.
(D) Side view of the dform yNPC indicates
that the membrane ring interconnects adjacent spokes and the intervening linear arm
in a sinusoidal manner (see closed circles).
Scale bar 5 300 Å.
(Franke and Scheer, 1974; Jarnik and Aebi, 1991; Akey
and Radermacher, 1993; Goldberg and Allen, 1996).
However, the thin coaxial rings are not present in the
yNPC 3-D map (see next section). We suggest that major
differences in the thickness of the NE lumen are correlated with the vertical size of the spokes and their
mode(s) of interaction with the nuclear membrane.
Hence, this observation signals a fundamental change
in the architectural scale of the NPC in the two distantly
related “species.”
Additional evidence for the smaller yNPC is as follows.
There is no lamin homolog in yeast; thus, domains of
the nuclear thin ring and spoke surface that interact
with the lamina in vertebrates (Jarnik and Aebi, 1991;
Goldberg and Allen, 1996) are not required in yNPCs.
Abundant transmembrane nups in vNPCs (e.g., gp210
and Pom121) are not present in yeast; hence, the corresponding lumenal spoke domains are absent in yNPCs.
In addition, initial solubilization studies using both nuclei
and NEs revealed yNPCs with a size and morphology
similar to that found in the enriched preparation (Rout
and Blobel, 1993). Finally, projection maps of both dform
and mform yNPCs show a similar two-domain spoke
morphology; yet these preparations are made very differently, and the nuclear membrane is expected to stabilize the spokes. When taken together, all of the morphological and biochemical data indicate that yNPCs are
fundamentally smaller than vNPCs; hence, our 3-D map
of the isolated yNPC is likely to represent the basic
architecture of the spoke–transporter assembly.
Functional Implications of Differences in
Yeast and Vertebrate NPC Structure
Surface maps of yeast and vNPCs are presented to
scale in Figures 7A/7B and 7C/7D. The yNPC is smaller
in diameter (960 Å versus 1450 Å) and height (z350 Å
versus z800 Å) than the vNPC, and this difference reflects the mass of the respective NPCs (z60 MDa versus
z125 MDa). However, vNPC domains located at high
radius (the coaxial thin rings, cytoplasmic particles, and
lumenal ring) form a more open structure, and this results in an z5-fold volume increase relative to the yNPC.
When viewed from the side, the vNPC is comprised of
four major ring systems surrounding the central transporter, including the inner spoke ring, the cytoplasmic
and nuclear thin rings, and the lumenal ring (Figures
1, 7C, and 7D). The isolated yNPC is simpler and is
comprised of an inner spoke ring that encircles the central transporter and an outer membrane interacting ring.
We did not detect structural equivalents of the thin cytoplasmic and nuclear rings, cytoplasmic particles, or the
lumenal spoke ring in the yNPC (Figures 7A and 7B);
however, the dimensions of our 3-D structure are in
good agreement with measurements of yNPCs from thin
sections of cells.
Why is the vNPC larger than the yeast pore complex?
Yeast and vertebrate NPCs have undergone divergent
evolution from a common ancestral pore complex; hence,
significant changes in morphology may have resulted
from alterations in surface loop size within nup homologs. Thus, vertebrate nups are often larger than their
yeast counterparts (e.g., vNup107p versus yNup84p).
However, each NPC may also have a complement of
nucleoporins that comprise domains not present in the
other.
A more useful comparison of these distantly related
NPCs can be made from diagrammatic cross-sections
of the structures (Figures 8A and 8B; Akey, 1995). Importantly, the equivalence of the inner spoke ring and position of the NE was used to align vertebrate and yNPCs
Molecular Cell
230
Figure 7. A Comparison between 3-D Structures of Yeast and Vertebrate NPCs
(A) The yeast NPC as viewed from the putative
cytoplasmic surface. The transporter is marked
(T) and putative substrates are marked (S).
(B) Side view of the yeast NPC showing the
marked difference in height relative to the
vNPC and the lack of thin rings on either
surface.
(C) The dform vNPC as viewed from the cytoplasmic surface. The transporter (T) is partly
obscured by a ring of collapsed cytoplasmic
filaments (CF) that emanate from the cytoplasmic particles ([CP], Akey and Radermacher, 1993). The radial arms are labeled
(RA).
(D) Side view of the vNPC reveals the cytoplasmic and nuclear thin rings (CR/NR) that
are integral to the the spoke complex. A lumenal ring (LR) is formed by the lumenal spoke
domain and adjacent radial arms (see closed
circles). Scale bar 5 300 Å.
as shown in Figure 8A and inset. In this alignment, the
inner and central spoke domains of vNPCs have equivalent domains in yNPCs, with the central or second spoke
domains making a similar transmembrane insertion. In
addition, the radial and vertical dimensions of these
two domains are similar (Table 1). As cytoplasmic and
nuclear thin rings are not present in yNPCs, the spoke
domains that support the thin rings are missing. These
include the inner and outer vertical supports that form
a part of the respective surfaces of the vertebrate spoke.
As suggested, the thin coaxial rings and lumenal ring
may stabilize the larger vertebrate spokes and function
in transport or its regulation (Hinshaw et al., 1992; Akey
and Radermacher, 1993; Akey, 1995).
Cytoplasmic filaments and the nuclear basket provide
transport substrate docking sites (Richardson et al.,
1988; Kiseleva et al., 1996; Pante and Aebi, 1996) and
are attached to the cytoplasmic and nuclear thin rings
in vNPCs (Jarnik and Aebi, 1991; Ris, 1991; Goldberg
and Allen, 1992). Biochemical and morphological data
suggest that yNPCs also have these peripheral assemblies (Rout and Blobel, 1993; Kraemer et al., 1995;
M. P. R., unpublished data). Presumably, the primary
attachment sites for these assemblies have been conserved, but may be augmented by the thin rings in vertebrates. For example, Nup88p has been implicated in the
attachment of the cytoplasmic filament protein CAN/
Nup214p to the vNPC (Bastos et al., 1997; Fornerod
et al., 1997). However, there is no yeast protein with
significant sequence similarity to Nup88p, although
Nup159p is a yeast homolog of CAN/Nup214p; hence,
Nup88p may represent a vertebrate-specific protein of
the cytoplasmic rings or particles.
Yeast and vNPCs differ in their interactions with the
NE, although they share conserved transmembrane
spoke domains. Notably, yNPCs have a novel linear arm
located between adjacent spokes that, together with
spoke domain 2, forms a membrane ring (Figure 8B,
left). The membrane ring may function to anchor the
inner spoke ring of yNPCs into the NE and provide circumferential stabilization. Recently, Pom152p has been
immunolocalized near the outer surface of the yNPC
(Wozniak et al., 1994); hence, we suggest that Pom152p
may comprise part of the membrane ring (Strambiode-Castillia et al., 1995). Moreover, many nup mutants
display an altered NE morphology (Doye and Hurt, 1997)
that may reflect the close proximity of the spoke domains to the nuclear membrane in the smaller yNPC.
A feature unique to the vNPC is a ring present within
the NE lumen, comprised of the lumenal spoke domains
and adjacent radial arms (Figure 8B, right). Gp210 is a
major membrane protein of the vNPC and is thought to
be a component of the lumenal ring (Greber et al., 1992;
Wozniak and Blobel, 1992; Akey and Radermacher,
1993). Our data are consistent with the lumenal position
of gp210, as yNPCs do not have a lumenal ring and
there is no gp210 homolog in the yeast. During open
mitosis, vNPCs disassemble into soluble subcomplexes
in response to phosphorylation (Macaulay et al., 1995;
Favreau et al., 1996). As gp210 is specifically phosphorylated in mitosis (Favreau et al., 1996), this nup may play
a role in NPC disassembly. Hence, the lack of gp210
and a lumenal ring may reflect the absence of NPC
disassembly during closed mitosis in yeast.
Recently, it was postulated that spoke conformational
plasticity may mediate the down-regulation of both active transport and the diffusion of small molecules, in
response to calcium depletion from the endoplasmic
Architecture of the Yeast Nuclear Pore Complex
231
Figure 8. A Comparison between Models of
Vertebrate and Yeast NPCs Suggests that
Evolutionary Divergence Has Resulted in Dramatic Changes in the Architectural Scale of
This Organelle
(A) An idealized vertical section of the yeast
(left) and vertebrate (right) NPCs, cut through
the middle of the spokes. The yNPC is comprised of two radial spoke domains ([1] and
[2]) with a bridging density between them;
domain 1 encircles the cylindrical transporter.
Putative cytoplasmic filaments and a nuclear
basket are shown on the yNPC. Components
of the vNPC are: cytoplasmic filaments (CF)
and particles (CP), and the inner ring filaments (IRF) that connect the transporter with
a central channel (CC) to the cytoplasmic (CR)
and nuclear (NR) thin rings. Inset: Aligned
spoke and transporter cross-sections from
yeast and vertebrates. The yeast spoke is
missing the vertebrate domains highlighted
in pink, which include the vertical inner and
outer domains (Vi/Vo) that support the cytoplasmic and nuclear thin rings (CR/NR) and
frame the lumenal spoke domain (LS). The
inner spoke ring and central spoke domains
are conserved in both species. The outer and
inner nuclear membranes (ONM/INM) make
similar contacts with the C or second spoke
domains; however, the overall thickness of
the NE lumen mimics the vertical dimension
of the NPC. The yeast transporter appears to
be missing a central cylinder (pink) that gives
the vertebrate transporter its hourglass shape.
The yeast transporter may retain two possible
gating assemblies (shown in gray).
(B) An idealized cross-section of the yeast
(left) and vertebrate (right) NPCs cut within
the central plane, parallel to the NE. The NPCs
show conserved and divergent interactions
with the nuclear envelope (NE). The lumenal
ring components (highlighted in pink) of the
vNPC are missing in the yNPC, while the latter
has evolved a novel membrane ring (dark
blue). Yeast NPC domains labeled as in (A)
with the addition of the linear arm (LA). Vertebrate NPC: inner (IS), central (CS), and lumenal spoke (LS) domains, radial arms (RA),
transporter (T), and central channel (CC).
reticulum (Akey, 1995; Greber and Gerace, 1995;
Stehno-Bittel et al., 1995). Perhaps Ca21 depletion from
the NE lumen may play a role in the earliest stages
of mitosis and programmed cell death, by triggering a
conformational change within the spokes that modulates the transport activity of the central transporter. As
yeast do not undergo open mitosis or apoptosis, they
may not require the larger spoke design.
The Functions of Conserved Domains in NPCs
Many nup homologs are shared between yeast and
vNPCs (e.g., yeast and human Nic96p; yNup170p/
yNup157p and vNup155p; yNup84p and vNup107p;
Doye and Hurt, 1997), and the transport factors are conserved (Koepp and Silver, 1996). Therefore, yeast and
vNPCs are expected to share a common structure that
mediates nuclear transport. A comparison between
yeast and vNPCs reveals a conserved inner spoke ring
(Figure 8), which may function as the central organizing
framework of the pore complex. The inner spoke ring is
attached to similar second/central spoke domains that
penetrate the nuclear membrane. In addition, the proximal region of the linear arm in the yNPC (see Figures
5A and 5C) either forms part of the inner spoke ring or
is attached to an ISR component. The inner spoke ring
provides a conserved framework in which the central
transporter is suspended (Figures 1 and 8). Moreover,
these conserved spoke domains may play a role in seeding NPC assembly.
How are substrates translocated through the NPC and
peripheral assemblies? Structural data suggest that nuclear transport may span two different environments.
First, substrate complexes that are bound to the cytoplasmic filaments or nuclear basket must move to the
transporter (Richardson et al., 1988; Kiseleva et al., 1996;
Pante and Aebi, 1996; Rutherford et al., 1997). In a second step, substrates traverse a central channel within
the NPC (Feldherr et al., 1984; Akey and Goldfarb, 1989;
Molecular Cell
232
Akey, 1990). In our studies, both yeast and vertebrate
transporters demonstrate a similar ring-like morphology
and an intrinsic ability to expand radially, when viewed
in projection. This conserved feature may reflect an inherent gating property of the NPC, as macromolecules
without NLSs or NESs must be excluded from the central
channel.
The yeast transporter is a shortened cylinder, while
the vertebrate transporter is more elongated with a tripartite hourglass shape (Akey and Radermacher, 1993).
Recent STEM images from Chironomus NPCs (Goldberg
and Allen, 1996; Kiseleva et al., 1998) are consistent
with a tripartite transporter and suggest that there are
two gate assemblies localized at the cytoplasmic and
nuclear faces of the transporter, which may restrict access to the central channel (Akey, 1990; Akey and Radermacher, 1993). As the translocation mechanism is probably similar in yeast and vNPCs, we suggest that the
yeast transporter is formed from two opposite-facing
gate assemblies, without the central extension that
gives the vertebrate transporter its hourglass shape (Figure 8A, inset). The reasons for the central extension in
the vertebrate transporter are unclear, but may reflect
adaptations to allow transport regulation, a more efficient translocation of large substrates, or compensation
for the wider NE lumen. Finally, it has been suggested
that vaults and transporters may be related, given their
shared 8-fold symmetry (Rome et al., 1991). However,
vaults have not been reported in yeast, and a search
of the complete genome does not detect vault protein
homologs, indicating that transporters are distinct from
vaults.
The Evolution of NPCs
Our data suggest that NPCs have undergone a fundamental change in their architectural scale between yeast
and vertebrates. Furthermore, a comparison of the two
NPCs enforces new boundaries on our understanding
of what is required to build a functional NPC. What sort
of NPC was directly ancestral to both yeast and vNPCs?
Two evolutionary scenarios could account for these differences. In the first, the yNPC may have been streamlined from a larger common ancestral NPC, to function
in this small and rapidly dividing eukaryote. In a second
model, early eukaryotes possessed a smaller and more
primitive NPC, which was retained by yeast while animal
cells expanded and elaborated their NPCs. Future studies of NPCs in eukaryotes that diverged from the common ancestral cell before animals and fungi may distinguish between these possibilities. However, yeast NPCs
are likely to have retained or recapitulated features from
an earlier and smaller NPC that is ancestral to all modern
NPCs. With a completed yeast genome sequence in
hand, the smaller yNPC will now serve as a focus for
future labeling and transport studies, which may provide
positional and functional information on individual nups
and their roles in nucleocytoplasmic transport.
Experimental Procedures
Specimen Preparation and Electron Microscopy
NPCs were isolated from Saccharomyces uvarum (NCYC74) in detergent-extracted and membrane-associated forms. The fractions
(crude, enriched, and highly enriched) were isolated with the protocol of Rout and Blobel (1993). To minimize damage, the final sample
used for 3-D analysis was frozen only twice. Sample preparation:
50–100 ml of enriched yNPCs were dialyzed against 100–200 ml of
10 mM Bis-Tris (pH 6.5)/0.1 mM MgCl2 /20% DMSO/0.2% TX-100/
1:1000 solution P (2 mg of pepstatin, 90 mg of PMSF in absolute
ethanol) at 48C for 8–12 hr; highly enriched yNPCs used 0.01%
Tween 20. To prepare frozen specimen, 10 ml of sample was incubated for 40 min on carbon-coated and air glow–discharged copper
grids. Grids were manually double-blotted and plunged into liquid
ethane in a humidity-controlled chamber (Dubochet et al., 1988).
Cryogrids were loaded into a Gatan cryoholder (model 626-DH) and
images recorded at 21768C at 13,0003 on a Philips CM12 with low
dose kit and specimen relocation hardware. The first zero of the
contrast transfer function was at 1/70 Å, and 50/08 tilt image pairs
were collected for random conical tilt 3-D reconstruction (Radermacher, 1988).
Yeast NPCs associated with NEs were prepared by a modification
of the method in Strambio-de-Castillia et al. (1995; M. P. R., unpublished data). Bound ribosomes were stripped from the NEs by 0.5
M KCl and 1.2 3 10 25 M puromycin treatment in 1.75 M sucrose/
10 mM Bis-Tris (pH 6.5)/0.1 mM MgCl2/20% DMSO for 10 min at
208C, followed by dialysis for 4 hr at 48C in buffer without KCl/
puromycin. Stripped NEs were spun onto air glow–discharged grids
and negatively stained with 2% uranyl acetate. Low dose images
were recorded at 13,0003 with an appropriate defocus.
Image Processing
Tilt and zero micrograph pairs (n 5 42) were densitometered on an
Eikonix with an image step size of 20 Å and yNPCs chosen with the
WEB particles option (Frank et al., 1996), based on the circular
morphology of the zero tilt particles. Initially, z10%–15% of the
possible yNPCs were chosen (n 5 3264), and rotational power spectra were calculated to allow a nonbiased selection of particles
(Crowther and Amos, 1971; Kocsis et al., 1995). The ratio of the
8-fold harmonic for each particle relative to the best particle was
used to create a percentile distribution, which indicated that 1565
yNPCs had detectable 8-fold signal. Image processing was carried
out on VMS/Alpha workstations using SPIDER (Frank et al., 1996).
Alignment of untilted yNPCs utilized a multireference approach, in
which the 10 best particles were aligned separately against the top
866 particles to generate new references; subsequently, a final best
reference was calculated and used to realign the dataset. Resolution
was calculated using the differential phase residual for projections
from half-datasets. Classification of the yNPCs into transporter and
spoke classes used dynamic clouds and hierarchical ascendant
classification (Frank, 1990; Akey, 1995), and final classes were
merged using the dendrogram and visual inspection. The 3-D maps
were calculated using the R-weighted back projection method (Radermacher, 1988) and refined using projection onto convex sets (Akey
and Radermacher, 1993), to correct for the missing cone. The 3-D
maps in the text are comprised of the global transporter class (GBT,
n 5 844) that contains all yNPCs with well-defined transporters, and
the best spoke class (SC, n 5 577). Final 3-D maps were Fourierfiltered to 120 Å resolution, and surface models were thresholded
to give a reasonable connectivity. The calculated volume of the GBT
3-D map used in the surface views corresponds to z150% of the
expected mass of 60 MDa, similar to thresholds used for other low
resolution maps (Akey and Radermacher, 1993).
Mass Analysis of yNPCs
The dform yNPCs were subjected to mass analysis by scanning
transmission electron microscopy using a wet film sample preparation method (Wall and Hainfeld, 1986), with TMV as a mass standard.
In some cases, fixation with 0.1% formaldehyde/0.2% glutaraldehyde in sample buffer was done for 25 min. Images were recorded
on the STEM1 at Brookhaven National Laboratory at 40 kV with a
2.5 Å diameter beam. The intensity of yNPCs was integrated after
background correction and multiplied by the TMV scale factor to
obtain the correct mass. Programs were provided by Dr. G. Sosinsky.
Architecture of the Yeast Nuclear Pore Complex
233
Acknowledgments
new regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. J. Cell Biol. 119, 1429–1440.
This work was supported by grants from the NIH (C. W. A.).
M. P. R. thanks G. Blobel and the HHMI for support. Q. Y. received
partial support from a Boston University GSRF. We thank I. V. Akey
and R. Nolte for help with figures, J. Wall and M. Simon for assistance
with STEM data collection, E. Bullitt for critical comments, J. Kilmartin for images of serial sectioned yeast cells, M. Radermacher for
helpful discussions, and L. Rome and P. Stewart for sharing the
results of a vault database search.
Goldberg, M.W., and Allen, T.D. (1996). The nuclear pore complex
and lamina: three dimensional structures and interactions determined by field emission in-lens scanning electron microscopy. J.
Mol. Biol. 257, 848–865.
Received September 24, 1997; revised November 13, 1997.
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