Table Of ContentMolecular Biology of Saccharomyces
edited by
L.A. Grivell
Molecular Biology Section, University of Amsterdam, The Netherlands
Reprinted from Antonie van Leeuwenhoek 62: 1/2
SPRINGER SCIENCE+BUSINESS MEDIA, B.V.
Library of Congress Cataloging-in-Publication Data
Molecular blology of Saccharomyces / edlted by L.A. Grlvell.
p. cm.
ISBN 978-94-010-5104-0 ISBN 978-94-011-2504-8 (eBook)
DOI 10.1007/978-94-011-2504-8
1. Saccharomyces. 2. Yeast fungl. 3. Fungal molecular blology.
I. Grlvell, L. A. .
OK623.S23M644 1992
589.2·330488--dc20 92-13592
ISBN 978-94-010-5104-0
Prinled 0/1 acid-free paper
AII Rights Reserved
© 1992 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1992
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording Of by any information storage and
retrieval system, without written permission from the copyright owner.
Contents
Editorial
Nehrbass D. & Hurt E.C.: Nuclear transport and nuclear pores in yeast 3
Gasser S.M., Walter R., Dang Q. & Cardenas M.E.: Topoisomerase II: its functions and phosphorylation 15
Diffley J.F,X.: Global regulators of chromosome function in yeast 25
Brown J.D., Plumpton M. & Beggs J.D.: The genetics of nuclear pre-mRNA splicing: a complex story 35
Linder P.: Molecular biology of translation in yeast 47
Kunau W.-H. & Hartig A.: Peroxisome biogenesis in Saccharomyces cerevisiae 63
Gellissen G., Melber K., Janowicz Z.A., Dahlems D.M., Weydemann D., Piontek M., Strasser A.W.M. &
Hollenberg C.P.: Heterologous protein production in yeast 79
Konopka J.B. & Fields S.: The pheromone signal pathway in Saccharomyces cerevisiae 95
Thevelein J.M.: The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae 109
Bolotin-Fukuhara M. & Grivell L.A.: Genetic approaches to the study of mitochondrial biogenesis in yeast 131
Antonie van Leeuwenhoek 62: 1, 1992.
Editorial
The yeast Saccharomyces is one of the simplest charomyces can be regarded as a model eukaryote
eukaryotes known and also one of the world's most in a variety of respects, including the way it regu
important commercial micro-organisms. As an ex lates gene expression, carries out housekeeping
perimental organism, there is much to recommend tasks necessary for cell growth and division, re
it: it grows rapidly; it can be manipulated by classi sponds to signals from the environment and targets
cal genetic and by recombinant DNA techniques; it proteins to various destinations. The Special Issue
is likely to be the first eukaryote for which a com thus not only tells us more about how this versatile
plete genomic sequence will be available and it micro-organism itself 'ticks', but how it can help us
offers unique opportunities for putting predictions better to understand other eukaryotic cells.
of gene function to the test. My thanks are due to the contributors, who re
In this Special Issue, I have brought together a sponded with such enthusiasm to my request to
number of reviews dealing with topics in funda produce at short notice up-to-date readable sur
mental and applied aspects of yeast molecular biol veys of recent developments in their respective
ogy and genetics. Although apparently diverse in fields. I believe that they have succeeded.
scope, these articles strengthen the view held by an
increasing number of research scientists that Sac- University of Amsterdam L.A. Grivell
Antonie van Leeuwenhoek 62: 3--14, 1992.
© 1992 Kluwer Academic Publishers.
Nuclear transport and nuclear pores in yeast
U. Nehrbass & E.C. Hurt
European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstraf3e 1, D-6900 Heidelberg, Germany
Key words: Saccharomyces cerevisiae, nuclear pore complex, nuclear transport,
nuclear localization sequence, nucleoporins
Abstract
The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumu
lation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes.
Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by
receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins
are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore
complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear
import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an
efficient structural and functional analysis of these components. Due to the evolutionary conservation
potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus,
yeast can be considered as a eukaryotic model system to study nuclear transport.
1. Introduction tively recognizes the determinant, and mediates
the translocation across the nuclear membrane.
Nuclear proteins are synthesized in the cytoplasm During the past few years the coding signal of nu
and post-translationally transported across the nu clear proteins, the nuclear localization sequence
clear envelope. Nuclear transport is controlled by (NLS), has been identified, and the site of entry
the nuclear envelope which consists of the outer into the nucleus has been shown to be the nuclear
and inner nuclear membrane, nuclear pore com pore complex. Recent work has focused on the
plexes (NPCs), and an underlying nuclear lamina. 'decoding system' i.e. on NLS receptors in the cy
The double membrane is constituted of two lipid toplasm or at the nuclear envelope. However, the
bilayers that enclose the perinuclear space. The molecular mechanisms underlying nuclear trans
bilayers meet only at sites where nuclear pore com port have remained largely unknown.
plexes, large proteinaceous assemblies, traverse Progress in nuclear transport research on the
the envelope. The outer nuclear membrane is con molecular level requires a system which provides
tinuous with the endoplasmic reticulum and carries fast and systematic approaches to find components
ribosomes, whilst at the inner membrane the nucle of the import machinery. In this respect, yeast has
ar lamina forms attachment points for chromatin advanced into the limelight of scientific interest,
and nucleoskeletal elements. being a versatile experimental system that allows
It has become clear that in order to be specifical for the investigation of nuclear transport using both
ly transported into the nucleus, nuclear proteins in vivo and in vitro approaches. A widely used in
require a common transport determinant. This im vivo approach has been the analysis of the sub
plies the existence of a decoding system that selec- cellular localization of mutated nuclear proteins or
4
fusion proteins in yeast cells with the goal of identi terminal domain was not able to enter the nucleus,
fying NLS. The genes encoding the nuclear pro whereas the short C-terminal domain could medi
teins can be delivered into the cells on yeast shut ate nuclear entry of attached colloidal gold parti
tling plasmids and their expression can be regu cles. However, when the truncated nucleoplasmin
lated exploiting regulable yeast promoters. The lacking the C-terminal domain was injected into
fate of the in vivo expressed proteins is then fol the nucleus directly it was retained, thus ruling out
lowed by indirect immunofluorescence, electron a retention model for nuclear transport. Subse
microscopy, subcellular fractionation, or with the quently, a nuclear localization signal consisting of
help of functional assays. For the identification of the short amino acid sequence Pro-Lys-Lys-Lys
components of the nuclear import machinery, a Arg-Lys-Val was found to be responsible for the
variety of different approaches (genetic, biochem nuclear targeting of SV40 large T antigen, and
ical or immunological) is currently applied. Once a single amino acid exchanges within this signal
component of the import machinery has been (Lys128 to Thr or Asn) rendered a non-nuclear
found, yeast offers a number of powerful tools for protein (Kalderon et al. 1984). Although nuclear
its functional analysis. In cases where it is possible transport is induced by the short heptapeptide, the
to obtain conditionally lethal alleles of such compo context of this NLS within the protein is crucial for
nents, yeast allows for the identification of extra its optimal function. Rhis and Peters (1989) showed
genic suppressors, which themselves could turn out that for efficient nuclear transport of a SV40-lacZ
to encode for proteins of the import machinery. fusion protein, the 15 amino acids preceeding the
To be considered as a model system for nuclear NLS were also required. Furthermore, the NLS
import, yeast should allow for a generalization of has to be exposed on the surface of the protein in
experimental findings. This, however, is only pos order to have access to components of the import
sible if yeast has an evolutionarily conserved im machinery; when burried within the hydrophobic
port machinery sharing components and principal interior of pyruvate kinase, the SV40 large T anti
mechanisms with the nuclear import apparatus of gen NLS was unable to mediate nuclear transport
higher eukaryotes. In this respect, the following (Roberts et al. 1987; Nelson & Silver 1989). Until
chapters will try to provide an up to date picture of recently, the SV40 large T antigen NLS with its
yeast nuclear transport in the context of corre stretch of lysinal amino acid residues was consid
sponding data obtained from higher eukaryotes. ered to be the prototype of a NLS. Recent work by
Robbins et al. (1991), however, showed that NLSs
may have a more complex organization. In a de
2. Targeting nuclear proteins tailed analysis applying site-directed mutagenesis
of the nucleoplasmin NLS, two adjacent basic do
In the original view on the targeting mechanism of mains, separated by 10 amino acids, were found to
nuclear proteins for nuclear entry, specific signal be required for efficient nuclear targeting. The
sequences within the nuclear proteins were sug transport defect caused by a single mutation in one
gested to be responsible for nuclear targeting. domain was amplified by a successive mutation in
These signals were postulated to meet two criteria: the other domain, indicating their interdepend
deleting or mutating them should abolish nuclear ence. Although amino acid exchanges in the spacer
transport and fusing these signal sequences to non sequence did not interfere with the functionality of
nuclear proteins should induce accumulation of the the bipartite NLS, alterations in the length of the
reporter protein in the nucleus. First experimental spacer element impaired nuclear transport. An ex
evidence for the existence of short and confined tensive search in protein data libraries then re
nuclear targeting sequences came from work by vealed that many other nuclear proteins share the
Dingwall et al. (1982) who studied the targeting of principle of a bipartite nuclear targeting motif, as
nucleoplasmin after microinjection into Xenopus initially found in nucleoplasmin.
oocytes. Truncated nucleoplasm in lacking the C- Nuclear localization sequences from higher eu-
5
karyotes are functional in evolutionarily distant ing fusion protein. However, a H2B-lacZ fusion
species such as yeast, showing that the mechanism protein containing a mutated NLS, but in addition
of nuclear transport has been conserved during the H2A dimerization domain, was localized in the
evolution. Nelson & Silver (1989) demonstrated nucleus. This result suggested that H2A and H2B
that the SV 40 large T antigen NLS sequence medi may be co-transported to the nucleus as a hetero
ates nuclear accumulation of non-nuclear proteins dimer. NLS mediated transport of small nuclear
in Saccharomyces cerevisiae. Two yeast ribosomal proteins has also been observed in mammalian cells
proteins, L3 (Moreland et al. 1985) and L29 (Un e.g. in case of histone HI (Breeuwer & Goldfarb
derwood & Fried 1990), were shown in a fusion 1990).
protein approach to contain NLS, which in case of
L3 is contained within the first 21 amino-terminal
amino acids, whilst nuclear transport of L29-lacZ 3. The import machinery
fusion proteins can be mediated by two independ
ent, internal, basic heptapeptides. Some of the nu a) The decoding system: putative receptors
clear localization signals identified in yeast pro
teins, however, reveal unique features. The yeast The existence of short and confined nuclear target
transcription factor GAL4 contains its nuclear lo ing sequences with!n a nuclear protein implies the
calization signal within an extended stretch of the existence of a decoding system, which specifically
amino-terminal domain, not resembling the bipar recognizes the NLS and catalyzes translocation
tite consensus NLS motif (Silver et al. 1984). The through the nuclear pores. It was shown that nucle
transcriptional repressor mata2 contains two inde ar transport may be divided into two steps, with a
pendent NLSs being functionally distinct (Hall et 'quick' binding step of the nuclear protein to the
al. 1984; Hall et al. 1990). In addition to a N nuclear envelope being followed by the 'slow',
terminal sequence that resembles other NLS, a ATP-dependent translocation through the nuclear
distinct and distant amino acid stretch within the pore channel (Newmeyer & Forbes 1988; Richard
homeodomain was able to mediate nuclear trans son et al. 1988). Moreover, nuclear transport is
port of a mata2-lacZ fusion protein. Deletion of saturable, arguing that this is a receptor-mediated
the internal NLS reduces nuclear transport so that process with limiting amounts of receptor. There is
the nuclear protein accumulates around the nucle no experimental data pointing to the exact mecha
ar periphery, most likely the nuclear pore complex nism of receptor recogniti0n. The diversity of NLS
es. Based on these observations, the N-terminal sequences however, requires a complex recogni
NLS signal was suggested to mediate association tion mechanism, unless what is recognized is not
with the nuclear envelope, whilst the internal signal the primary sequence but rather a common deter
triggers translocation through the pore. Whether minant like the secondary s:ructure. Each group of
such a two step import mechanism is also true for NLS could have a specific re ceptor or alternatively
nuclear proteins containing only a single NLS re there could be adaptor mole ~ules in the cytoplasm
mains to be shown in the yeast system. which recognize individual NLS and deliver them
Surprisingly, proteins whose molecular weight is to a common receptor at the pore complex. In vivo
below the exclusion limit of nuclear pore complex studies indicate that NLS may first bind to such
es and thus could enter the nucleus by diffusion, soluble receptors in the cytoplasm before the com
can contain NLSs as shown for the yeast histone plex is delivered to the nuclear pores (Richardson
H2B (Moreland et al. 1987). The amino acids 28 to et al. 1988; Breeuwer & Goldfarb 1990).
33 (Gly-Lys-Lys-Arg-Ser-Lys-Ala) in histone Approaches to identify NLS binding proteins
H2B, which resemble the SV40 large T antigen were mainly biochemical, exploiting an assumed
NLS, will target a non-nuclear protein into the stable association of the receptor with its ligand in
nucleus. A point mutation exchanging Lys31 for cell-free binding assays. Accordingly, ligand over
Met abolishes nuclear transport of the correspond- lays were developed to measure binding of labeled
6
NLS-conjugates to cellular proteins immobilized will have to await further clarification. This, how
on nitrocelluose. In higher eukaryotes two soluble ever, should be possible in yeast if mutants of these
cytoplasmic NLS binding proteins of 54 and 56 kDa genes can be generated. The nsr- mutant e.g. can
have been identified by Adam and Gerace (1991). be tested for a possible correlation of its sick phe
Evidence for an involvement of these proteins in notype to an impairment in nuclear transport, as
nuclear uptake mechanisms comes from in vitro would be expected if NSR1 was functioning as an
studies, where the authors could show that the NLS receptor in vivo.
purified 54 kDa and 56 kDa proteins can stimulate Surprisingly, NSR 1 is not the only nucleolar pro
nuclear import. tein having NLS binding properties. For example,
In yeast, the 'NLS overlay' approach led to the N038 can be purified from nucleolar extracts by
identification of two putative NLS binding proteins affinity chromatography using a synthetic peptide
(Lee & Melese 1989; Silver et al. 1989). Both pro corresponding to the SV40 large T antigen nuclear
teins of 70 and 67 kDa specifically bound to the localization signal (Goldfarb 1988). Another pro
SV40 large T antigen NLS, but not to a mutated, tein, p 140 from rat liver, initially identified in cell
import-incompetent form of this NLS. Although extracts by chemical crosslinking to NLS peptides,
both proteins have been identified by a very similar was later shown to be a nucleolar shuttling protein
approach, and have a similar molecular weight, (Meier & BlobeI1990). At the moment, however,
they are not identical (Silver, personal communi it seems difficult to reconcile these data with a
cation). single cellular process in nuclear transport. It is
The gene for the 67 kDa protein NSR1 has been speculated that these NLS binding proteins might
cloned and sequenced (Lee et al. 1991). In gene function to chaperone karyophiles through the
disruption experiments it was demonstrated that pore complexes, before they return to the cyto
NSRl is not essential, but resulting nsr- mutants plasm. A final evaluation, however, whether N038
grow poorly. NSR1 is tightly associated with nucle and p140 are involved in NLS dependent nuclear
ar structures since it can only be extracted with urea transport. will also have to await experimental evi
or high salt. In indirect immunofluorescence NSR1 dence that these proteins function as NLS recep
gives an intranuclear staining typical of a nucleolar tors in vivo.
localization. Moreover, the amino acid sequence of
NSR1 reveals a glycine/arginine-rich repetitive se
quence and two putative RNA binding motifs. b) Nuclear pores and nucleoporins
Similarly, the 70 kDa NLS-binding protein is a
nuclear protein, but less tightly associated with Proteins destined for the nucleus have to cross the
nuclei (Stochaj et al. 1991). In indirect immunoflu nuclear membrane. Proteins smaller than the func
orescence on whole yeast cells, anti-70 kDa anti tional pore diameter of 9 nm can passively diffuse
bodies give a strong nuclear and only a weak cyto through sites in the double membrane. Moreover,
plasmic staining. Binding of the nuclear reporter these sites of entry have to provide a mechanism
protein SV40-HSA (SV40 large T antigen NLS that allows for larger proteins to be translocated in
coupled to human serum albumin) to purified yeast a specific, signal-dependent manner. The structur
nuclei is reduced both by antibodies against the al entities of the nuclear envelope designed to meet
70 kD protein and by salt stripped nuclei, which these requirements are the nuclear pore complex
removes this NLS binding protein (Stochaj et al. es.
1991). Binding can, however, be reconstituted by
addition of a crude fraction enriched in NLS bind The structure of the nuclear pore complex
ing proteins. Since their discovery in the early fifties, the nuclear
The in vivo role of these two NLS binding pro pores always attracted structural biologists and
teins is not clear so far and accordingly their poten therefore were subjected to extensive structural
tial to participate in nuclear uptake mechanisms studies. Their involvement in nuclear transport,
7
however, remained debated until Feldherr and co chitecture stems from electron microscopic studies
workers devised assays which allowed microinjec of NPC from higher eukaryotes, especially from
tion of colloidal gold and ferritin coated with nucle Xenopus nuclear membranes. Less is known about
ar proteins into the cytoplasm of cells. This ap the structural organization of nuclear pore com
proach led to the identification of pore complexes plexes from yeast. This is partly due to the fact that
as the entry and exit sites of nuclear transport (re yeast is not easily amenable to electron microscopic
viewed by Peters 1986). The structural analysis of techniques.
the pore complexes went far beyond the functional However, the most recent and detailed analysis
characterization. From electron microscopy it be of the yeast nuclear pore complex confirms that the
came clear that that NPCs have a conserved princi blueprint of nuclear pore complex architecture had
pal building device: on the cytoplasmic and the already been elaborated in the early eukaryotic
nucleoplasmic faces of the pore complex are two evolution (Allen & Douglas 1989). Upon subnucle
coaxial rings consisting of eight subunits (Akey ar fractionation of isolated yeast nuclei by sequen
1989). These rings sandwich a large spoke assembly tial extraction with detergent, nucleases and salt,
made up of eight subunits that have attachments to the yeast NPC reveals an organization similar to
both rings at the edge of the complex and include a the NPC of higher eukaryotes. Isolated NPCs are
central structure referred to as the central plug, still associated with a remaining filamentous struc
central granule or transporter. As implied by its ture, that resembles the nuclear lamina of higher
name, this transporter is thought to be part of the eukaryotes. Although putative analogues of the
transport channel. Moreover, there are fibrils ex lamin A, Band C from higher eukaryotes have
tending from the pore rings into the nucleo- and been identified in yeast using immunological ap
cytoplasm (Richardson et al. 1988). The cytoplas proaches (Georgatos et al. 1989), there is, how
mic fibrils have been reported to be involved in a ever, no biochemical evidence for the existence of
possible receptor function for nuclear proteins. nuclear lamina as yet. The organization of pore
Some of these cytoplasmic pore fibrils may also complexes in this preparation resembles a wagon
interact with intermediate filaments (Carmo-Fon wheeL with the pore complex sitting at the hub of
seca et al. 1987). the wheel. One type of filament (5-9 nm) radiates
The nuclear pore complex has an overall dia from the pore complex to the rim of the wheel,
meter of 120 nm, a relative molecular mass of which might be constituted by another filament.
124 MD and is estimated to contain more than 100 The radially emanating filaments seem to be orga
different proteins (Featherstone et al. 1988). The nized by a 30 nm ring that becomes visible in par
number of nuclear pores varies with cell type, but tially disassembled pore complexes lacking the cen
in most cases is quite constant (Franke 1974; Maul tral plug or transporter. These stripped pore com
1977). Yeast nuclei have been found to contain plexes still reveal a set of spokes that are organized
10-15 pores per /Lm2 of nuclear surface, with the by the 30 nm ring in an eight-fold symmetry. In situ,
total number of nuclear pores adding up to 200 per in isolated yeast nuclei, the NPC extend short fil
nucleus (Moor & Miihlethaler 1963; Jordan et al. aments into the cytosol, again in analogy to higher
1977). The distribution of nuclear pores is reported eukaryotes. These filaments could be involved in
to be uneven, particularly in cells derived from mediating an interaction of the NPC with cytoske
starved cultures (Moor & Miihlethaler 1963). The letal elements.
absolute number of pores varies during the yeast
cell cycle with increases in early Gil phase and be Components of the nuclear pore complex
fore nuclear division (Jordan et al. 1977). The pore Despite the large size and structual complexity of
density however remains relatively constant due to the pore complex, only a small number of pore
a rapid growth of the nuclear envelope during the complex proteins were so far identified. The most
peaks of nuclear pore assembly. detailed work on nuclear pore complex compo
Most of our knowledge on the nuclear pore ar- nents and their structural and functional analysis
8
has been done with rat liver nuclei, using immun under the strong GAllO promoter led to cell death
ological, biochemical and recently also recombi in an often multi-budded state, indicating that a
nant DNA techniques. correct stoichiometry of NUPI is of functional rele
One prominent family of 8-10 novel NPC con vance. The localization of the NUPI protein was
stituents of higher eukaryotes are the nucleopo shown by epitope tagging, since all antibodies
rins, which playa crucial part in nuclear transport available against NUPI recognized other yeast nu
(see below). They have in common an O-glycosyla clear pore proteins including NSPI. Therefore, a
tion with N-acetylglucosamine (GIcNAc) on serine short influenza haemagglutinin epitope was insert
and threonine residues and thus can bind the lectin ed into the NUPI protein. In indirect immunofluo
wheat germ agglutinin (Hart et al. 1989). Nucleo rescence, NUPI could be localized to the nuclear
porins were identified with the help of monoclonal periphery, giving a heterogeneous staining pattern
antibodies raised against rat liver nuclear prep highly similar to that obtained with anti-nucleopo
arations and by their ability to bind to WGA (Davis rin antibodies. From the amino acid sequence of
& Blobe11986; Park et al. 1987; Snow et al. 1987; NUP1, a three domain structure was deduced: a
Davis & BlobelI987). In immuno-electron micro charged and very acidic amino-terminal domain, a
scopic studies, the nucleoporins were either found middle portion consisting of 28 degenerate 9 amino
to be labelled along filamentous structures at the acid long repeats separated by short and highly
marginal rings of the pore complex (Snow et al. charged peptide stretches of varying length, and a
1987) or in other studies were found to be part of short and basic C-terminal domain. In the middle,
the central transporter (Akey & Goldfarb 1989). repetitive domain NUPI shares common structural
This contradiction in the localization of nucleopo motifs with the yeast nucleoporin NSPI (Hurt
rins could be reconciled if the central transporter in 1988; Nehrbass et al. 1990). It was shown that
the nuclear pore complex preparations of Akey epitopes within this central, repetitive domain are
and Goldfarb (1989) were the result of collapsed responsible for the cross-reactivity ofthe nucleopo
filamentous structures plugging the pores. rin antibodies (Davis & Fink 1990). So far it has not
Since the basic building device of nuclear pores been investigated whether NUPI is involved in nu
has been conserved during evolution it was reason clear transport mechanisms.
able to assume that yeast may contain the same
class of nuclear pore proteins. In fact, when West The nuclear pore complex protein NSPI
ern blots of whole cell yeast extracts were probed NSP1 was initially identified with the help of anti
with monoclonal antibodies against the rat liver bodies raised against the insoluble nucleoskeleton
nucleoporin p62, three cross-reactive bands of preparation of isolated yeast nuclei (Hurt 1988).
95 kDa, 110 kDa and 130 kDa were recognized One affinity-purified polyclonal antibody present
(Aris & Blobel 1989; Davis & Fink 1990). Immu in these immun-sera recognizes NSPI as a 100 kDa
nofluorescence localization using yeast cells and protein on Western blots. Gene disruption experi
isolated nuclei showed a punctate and patchy stain ments show that NSPI is an essential gene and
ing pattern of the nuclear periphery, reminiscient overexpression of NSPI under the GAllO promo
of nuclear pore staining (Aris & BlobelI989). ter on a high copy number plasmid leads to cell
death (Hurt 1989). In indirect immunofluorescence
The nuclear pore complex protein NUPI on whole yeast cells, NSPI shows a heterogeneous
Using mammalian anti-nucleoporin monoclonal punctate and occasionally blobby distribution at
antibodies for expression screening of a yeast ge the nuclear periphery. Immunoelectron micro
nomic library, the gene NUPl coding for a 130 kDa scopic studies on frozen thin sections of whole yeast
yeast nucleoporin was cloned (Davis & Fink 1990). cells or isolated nuclei clearly demonstrated that
In gene disruption experiments, the NUPJ gene NSPI is localized at nuclear pores (Nehrbass et al.
was found to be essential. Overexpression of NUP 1 1990; Hurt et al. 1992). This finding is in analogy
