Molecular trafficking across the nuclear pore complex Larry Gerace The Scripps

Research

Institue,

La Jolla, California,

USA

The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge. Current Opinion in Cell Biology 1992, 4:637-645 Introduction

( [ 5**] and references therein; Fig. 1). The mass distribution in both rings and spokes has eightfold radial symmetry when viewed UI face (i.e. in a direction perpendicular to the nuclear surface) and the ring-spoke framework has twofold symmetry about an axis parallel to the NE through the center of the NPC. Attached to the inside of the spokes is a central channel complex (also called the plug [j] or transporter [6] ) responsible for mediated transport of macromolecules (Fig. 1). The central channel complex region of the NPC is highly polymorphic within an NPC population, probably reflecting heterogeneit). of intransit ligands and transport-related structural states trapped at the moment of NE isolation. Classification and averaging of images of the central channel complex region of amphibian oocyte NPCs directly supports the notion that the central channel can expand to open configurations during mediated transport [7].

Molecular trafficking between the nucleus and cytoplasm occurs continuously in eukatyotes and is fundamental to the process of cellular metabolism. The boundary of the nucleus is defined by the nuclear envelope (NE), a double membrane system perforated by the large supramolecular structures called nuclear pore complexes ( NPCs ). These form kyssageways for nucleoc7~oplasmic transport (reviewed in (l-31 1. The NPC contains an aqueous channel ( diameter 10 nm) that allows passive diffusion of ions and other small molecules. Most proteins and RN.4.sare too large to diffuse across this channel at significant rates and are transported by saturable. signal-requiring and ATP-dependent mechanisms. This mediated transport occurs through a gated channel that can expand to at least 25 nm in diameter to allow tram&)cation of intact multimeric proteins and protein-nucleic acid complexes (e.g. ribosomal subunits). The NPC is a highly complex machine with the ability to coordinate multiple transport pathways, as individual NPCs carry out both import of protein and export of RNA. This review focuses on recent adKmces in understanding the mechanisms and machinery of nuclear protein import, the most accessible and well studied transport process. It also considers emerging efforts to understand other transport pathways. Architecture

of the nuclear

pore

It is generally assumed that mediated transport of macromolecules across the NPC is a vectorial process, with certain signals specifjing transport in a cytoplasmic to nuclear direction, and others in a nuclear to cytoplasmic direction. This would require biochemical and structural ‘asymmetry to be superimposed on the apparently symmetrical framework of the NPC described above. Recently, a striking demonstration of asymmetric fibrillar structures associated with the NPC has emerged from studies involving amphibian oocytes (Fig. 1). A set of eight 35-50~1 fibrils emanate from the cytoplasmic ring, and another set of eight 50-100 nm fib& come from the nucleoplasmic ring [W,!?] Unlike the cytoplasmic fib&, the nucleoplasmic fib& appear to be attached at their distal ends, forming a basket or cage-like structure. The cytoplasmic and nucleoplasmic fibrils cannot be visualized with many preparative procedures [8*-l, presumably because they are extracted or collapsed on the NPC surface. However, they are likely to be general com-

complex

The NPC has a mass of approximately 125 X 106Da [4], and its structure has been analyzed recently by a number of ultrastructural, image analysis and averaging techniques Its basic framework consists of a central ‘spoke’ assembly connected to two peripheral ‘rings’ situated on its nucleoplasmic and cytoplasmic surfaces

hnRNP-heterogeneous NLSnuclear localization

small sequence; Current

Abbreviations nuclear RNA-protein NPC-nuclear pore Biology

Ltd

ISSN

complex; complex; 0955-0674

NE-nuclear snRNP-small

envelope; nuclear

RNP.

637

638

Membranes

(IN

(a) Cytoplasmic fibri’ Cytoplasmic

‘/

\ >

t

__--

ONM

-__

INM Nucleoplasmic ring

Nucleoplasmic fibril

u

\ Nuc’eus i Nucleus

Fig. 1. A schematic model of the architecture of the INM, inner nuclear membrane; 5, spokes; C, central lumen from the spoke region. (b) Top view showing complex. Adapted from 1641 and work in [8**].

P

120

nm

-

nuclear pore complex (NPC). (a) Side (transverse) view. ONM, outer nuclear membrane; channel complex; R, rings. Also note the ‘knobs’ that extend into the nuclear envelope the mass distribution in the assembly formed by the rings, spokes and central channel

ponents of the NPC, as thin section electron microscop!, has revealed similar fibrillar structures in numerous cell types [lo], albeit substantially less well ordered than the librils seen recently in oocy-tes. The qtoplasmic and nucleoplasmic fibrils could be involved in initial recognition events in mediated transport, and could also participate in translocation of ligands to the central channel complex (see below). While a large hod!, of evidence localizes mediated transport pathways to the central channel complex [6,11,12], the location of the diffusional channelc s) has not been clearly determined. One possible site is an approximately 10~1 aperture found in some conligurations of the central channel [?I. An alternative site for the diffusion channel is suggested by recent studies involving three-dimensional analysis of a large population of negatively stained NPCs of X~~opzrs oocytes [ 5**]. This work has revealed that eight approximately 10nm dia. meter channels occur at a radius of about t0 nm from the NPC center between adjacent spokes. The existence of peripheral channels is also supported by many published images of tangential thin sections of nuclei and NE, which have revealed eight non-staining holes at the periphery of the NPC [ 101. Peripherally situated dilfusional channels would have the conceptual appeal of being physically independent of the mediated transport channel, which might be heavily occupied during periods of intensive transport.

Polypeptides

of the nuclear

pore complex

Only a small number of NPC polypeptides have been described to date, and these comprise less than 5-10 ‘% of the NPC mass. Monoclonal antibodies and the lectin wheat germ agglutinin ha1.e been used to identie a group of proteins of the mammalian NPC that are modifed at up to 10-20 sites with O-linked N-ace~lglucos~unine, :I sugar modifcation found on numerous other cellular proteins as well. At least eight distinct O-linked glycoproteins are present in the mammalian NPC, and se\.era1 related (presumably 0-glycos)4ated) proteins ha\~ been found in Srrc&~~ot~zq~cc.s cewr~bim ( [ 13-l and references therein ). Considerable interest has been focused on the O-linked glycoproteins because at least some of this group appear to be invohved in mediated transport across the NPC. Both wheat germ agglutinin and antibodies to the glycoproteins inhibit mediated import of protein and export of RNA ( [ 13.1 and references therein ), and NPCs assembled it1 ld/rv with Swzopzrs egg extracts that have been depleted of soluble O-linked gl!.coproteins are unable to perfomm mediated protein iniport [ l-11. These studies have been extended recentI!, I~!. the finding that a cytosolic factor required for mediated protein import binds to at least two of the mammalian O-linked glycoproteins and can be depleted from the CTtosol that is required in a cell-free tmnsport assa!~using gl!coprotein finit\. matrices [ 13*]_ This c?Tosolic factor

Molecular

is distinct from the p54/p56 receptor discussed below.

nuclear location sequence

Interestingly, some of the O-linked glycoproteins, including vertebrate ~62 and yeast NSPl and NUPl share up to 15-25 copies of a degenerate pentapeptide repeat that is predicted to form P-sheet secondary structure ( [ 15,161 and references therein). These repeats could give rise to a similar binding site on these different proteins. At least three of the mammalian glycoproteins are contained in a single macromolecular complex that can be released from nuclear envelopes by chemical extraction [ 17.1. In thin section electron microscope localization carried out by a number of laboratories, the O-linked glycoproteins detected by wheat germ agglutinin and antibodies that bind to multiple glycoproteins are concentrated near the nuclear and cytoplasmic rings, and are only seen at low levels near the central channel complex (e.g. [l&19]). The peripheral NPC localization of the O-linked glycoproteins is consistent with a role in early events of nuclear import, possibly as components of the cytoplasmic fibrils (see below). An additional constituent of the NPC that has been characterized recently is the transmembrane glycoprotein, gp210 [20*]. Unlike the O-linked glycoproteins, gp210 bears asparagine-linked high mannose-type oligosaccharide, and most of its mass occurs in the NE lumen. In view of this topology and its relatively high abundance in the NPC, it is likely to be part of the ‘knobs’ or lumenal subunits recently shown to extend into the NE lumen from the spoke region ( [6,8**] ; Fig. 1). An appealing possibility is that gp210 is a membrane anchor involved in NPC organization and assembly. A recent study evaluated this possibility by introducing a I~OIIOclonal antibody into the ER/NE lumen, which bound to the lumenal domain of gp210 in t+tlo [ 20*]. Unexpectedly, antibody binding to this region of gp210 strongly inhibited both mediated protein import and passive dif. fusion without affecting the number of NPCs in the NE. This transmembmne functional effect suggests that the lumenal domain of gp210 may be part of either a static scaffolding or dynamic framework for constituents of the NPC located on the cyto/‘nucleoplasmic surface of nu clear membranes. Signals

for nuclear

import

Mediated import of proteins into the nucleus is specified bv short stretches of amino acids contained in the protein called nuclear location sequences (NLSs; reviewed in [21,22] ). Proteins that lack NISs can be imported by ‘piggybacking’ on an N&containing protein, but this generally is an individualized mechanism based on protein-specific binding. While most NISs are highly enriched in basic residues, they do not conform to a clear consensus. The well studied NIS of the SV40 large T antigen (PKI*%KRKV) contains a single contiguous stretch of basic residues and has been considered as a prototype. However, it was shown recently that Xenopus nucleoplasmin has a second type of basic NLS, characterized by the presence of two essential and interdependent basic clusters (224 residues each) separated by 10 inter-

trafficking

across the nuclear

pore complex

Cerace

vening ‘spacer’ residues that can tolerate mutations and insertions [23”]. As similar bipartite basic clusters are present in many other nuclear proteins, bipartite NLSs like that of nucleoplasmin may be widespread [ 23**]. The NPC also imports certain cytoplasmically assembled RNA-protein (RNP) complexes into the nucleus, such as U small nuclear RNPs (snRNPs). Detailed studies on Ul snRNA demonstrate that its nuclear import requires both the trimethylguanosine cap and binding of Sm proteins [24,25]. While the actual signal(s) on Ul snRNP that is recognized by the nuclear import machinery is unknown, it could be contained in a cap-binding protein and/or an Sm polypeptide. The trimethylguanosine cap also appears to be important for the nuclear import signal of U2, U4 and U5 snRNP, but not for U3 snRNP [26*,27*]. Many viral genomes are imported into the nucleus after entry into cells, including the genomes of influenza virus and SV40. The RNA genome of influenza [28] and the DNA genome of SV40 [29] both cross the NPC during nuclear import. The substrates for import are thought to be a complex of the viral nucleic acid together with associated protein(s) [ 28,29,30**]. Import signals probably occur in the protein(s) attached to the nucleic acids, which contain basic NLSs. While the geometry of influenza RNP (15 nm diameter; up to 130 nm long) should easily allow its transport through the central gated channel, the dicameter of SV40 (approximately 5Onm) would presumably necessitate a conformational change of the virus upon transport. A diversity

of signaling

pathways

The nuclear import function of NLSs is mediated by saturable cellular receptors [2]. Despite structural differences between simple and bipartite basic NISs, kinetic competition studies indicate that the NIS of the T antigen and that of nucleoplasmin interact with the same receptor, or with receptors having similar specificity, and are therefore of the same class [31**,32]. The T antigen signal may associate very eficiently with a portion of a binding site on a receptor that is designed to accommodate bipartite signals [ 23**]. While competition studies indicate that the import of most nuclear proteins involves T antigen-like NLSs (NR Michaud, DS Goldfarb, abstract 1839,31st Annual Meeting of The American Society for Cell Biology, Boston, MA, December 1991, J. Cell Biol.), at least one additional signaling pathway appears to be involved in nuclear import. This pathway is used b). U2 snRNP, whose import is not competed by the T antigen NIS but is competed by trimethylguanosine cap dinucleotide [3100]. The nuclear import of Ul, U4 and U5 snRNPs may involve a signaling pathway similar to U2 snRNP, while the import of ~6 snRNP uses a T antigen-like NIS pathway [26*,27-l. Some data suggests that U3 snRNP may use a third signaling pathway [27’]. The search

for import

receptors

There has been considerable interest in defining receptors that interact with NLSs to carry out nuclear import, which are likely to be pivotal functional elements. In

639

640

Membranes

recent years a variety of strategies using jnthetic peptides containing basic NlSs (e.g. from the SV40 T antigen or nucleoplasmin) have identified numerous proteins in mammalian cells and yeast that bind these NLSs specifically (2,3,22]. Functional studies clearly are required to determine the relevance of these NIS-binding proteins to nuclear import, as some NLS-binding proteins may have other functions (see below). A further caveat concerns the highly basic nature of NLS peptides, which are prone to non-specific electrostatic interactions. Recently, two proteins that bind the SV40 T antigen NLS specifically have been demonstrated to have the functional properties of NLS receptors. These are closely related 54 and 56 kD polypeptides identified in mammalian cells by crosslinking NIS peptides to native protein fractions [ 33**]. These binding proteins have been purified to homogeneity from cytosol and they stimulate nuclear import in a cell-free system, as well as restore transport to a system that is inactivated by prior treatment with Nethylmaleimide. The presence of these proteins in the cy toplasm as well as the nucleus suggests that they may be shuttling carriers (see below). A number of other NLS binding proteins have been characterized in the last year by localization and biochemical studies [34*,35,36], and some of these have a subcellular distribution very different from that of p54 and ~56. Among these, a 14OkD protein identified in mammalian cells is localized specilitally to the nucleolus [35], and a 70 kD protein identilied in S. cerervkiae also has a highly restricted subnuclear localization reminiscent of the nucleolus in this cell type [34*]. While a nucleolar localization is not expected for a NIS receptor involved in targeting cytoplasmic proteins to the NPC, it is conceivable that the nucleolar NE-binding proteins are involved in targeting a subset of NIS-containing proteins to the nucleolus once they have entered the nucleus. It is not unreasonable to expect that some sequences containing NLSs have multiple targeting functions, particularly if NLSs are evolutionarily derived from sequences originally involved in binding to DNA or other functional elements of the nucleus [ 23**]. In this context, it is interesting that a highly basic 20 residue stretch of the Rex protein is responsible both for nuclear import and nucleolar targeting [37]. Pathway

of nuclear

protein

import

The recent work on cytosolic NLS receptors combined with physiological experiments and structural studies on migration of ligand-coated gold particles through the NPC suggests a hypothetical pathway for nuclear import, depicted in Fig. 2. An initial transport step apparently involves interaction of NLScontaining proteins with a cytosolic NIS receptor ( [ 33**,38,39] ; Fig. 2a). This receptor is likely to be the 54 and/or 56 kD cytosolic NLSbinding proteins described recently [33**] and perhaps other related polypeptides. A subsequent transport step is thought to involve docking of the ligand-receptor complex at the cytoplasmic surface of the NPC via a receptor recognition site ( [ 11,39,40]; Fig. 2b). Evidence for a stable interaction of a cytosolic NIS receptor with the NPC comes from the finding that p54 is enriched in isolated rat liver NEs in a salt-resistant association [41]. Follow-

ing docking at the NPC periphery, the &and is evidently translocated to the central channel complex, which can involve a distance of up to 6&80 nm ( [ 11,121; Fig. 2~). Specific recognition of the ligand (either direct or indirect) at the channel complex presumably triggers gating of the central channel and physical translocation to the nucleoplasmic side of the NPC (Fig. 2d). The &and may be translocated from the initial docking site to the central channel complex and subsequently to the nucleoplasm in association with the cytosolic NIS receptor. This is consistent with the presence of p54 in a nuclear contents fraction [41]. However, it also is conceivable that the cytosolic NIS receptor transfers the ligand to another N&binding protein at the NPC subsequent to docking. In either case, the cytoplasmic NLS receptor is likely to be recycled for further rounds of transport (Fig. 2e). Initial docking of ligands at the NPC appears to occur some distance away from the surface of the c). Masking of N& could be accomplished in principle by either intermolecular mechanisms involving heterologous proteins, or intramolecular mechanisms involving protein conformations that alter the accessibility or structure of regions containing NLSs. Intramolecular masking is implicated in regulation of the NLS in a 1lOkD precursor that gives rise to the 50kD subunit of NF-xB [50*]. The NIS occurs in the 50kD subunit, but its activity is repressed by a remote region of the precursor that is ultimately removed. Recent studies have shown that NIS activity can be controlled by phosphorylation of residues near NISs, which clearly could induce protein conformational changes. SWIS, a factor that controls mating type switching in S. cereutiiue, is imported into the nucleus only during the G, phase and is retained in the cytoplasm during the S. Gz and M phases. Its nuclear import is repressed by phosphorylation of three CM328 (cdc2) kinase sites located near the NIS and becomes activated when these sites are dephosphorylated i?z ldzlo (G1 phase) or when they are mutated to alanine [ 51**]. A further example is provided by studies showing that the rate of nuclear import of an NLl!+g~~atosidase fusion protein is strongly influenced by sequences that flank the NIS [ 521 and is repressed b!. in t&-o phosphorylation by p34cdcr kinase of a threonine adjacent to the NIS [47.1. A second mechanism for regulating nuclear import involves reversible anchoring of proteins to cytoplasmic structural elements, as suggested for the catalytic subunit of cAMP-dependent protein kinase, which dissociates from a site near the Go@ and enters the nucleus in the presence of CAMP [2]. A third mechanism may involve regulation of the transport apparatus itself. Recent studies analyzing the import of nucleoplasmincoated gold particles in cultured cells have suggested that substantial differences in the mediated transport capacity of the NPC occur between dividing and confluent cells (53,54*]. While the mediated import of small gold particles (5-8nm in diameter) in the two populations was unchanged, the transport of large gold particles (11-27 nm in diameter) was greatly diminished in confluent cells, possibly due to a decrease in the number of NPCs able to transport large particles [5-i*], Export of RNA from

the nucleus

Similar to nuclear protein import, export of RNA from the nucleus is an ATP-requiring, carrier-mediated process 155,561. However, RNA export has additional complexit) as it is controlled at multiple levels within the nucleus (reviewed in [57]). It is becoming evident that many transcription products of RNA polymerase II (pre-mRNAs) are not freely diffusible in the nucleus, but are found in localized subnuclear regions that can appear as ‘tracks’ [58] or ‘domains’ [ 59**]. These could be areas where splicing and other processing events take place, as well as regions involved in movement of RNA from processing sites to the NE. Striking subnuclear compartmentalization is also seen with the ribosomal RI%+, transcribed by RNA polymerase I, which are anchored in the nucleolus where their processing and assembly

into ribosomal subunits takes place. Nuclear export of RNAS appears to require release from intranuclear substructural domains (e.g. the nucleolus), and perhaps mediated transport to the nuclear envelope within a solidphase matrix before it can become a substrate for the transport machinery of the NPC. The primary structure of RNA may contain signals that can influence transport negativeel),,by specifying binding to nuclear substructures [57]. Other signals are likely to act positively, by determining interaction with the NPC or by facilitating export from intranuclear sites [ 60*]. In \iew of this complexit); it is difficult to discriminate signals that are specifically involved in mediated RNA transport across the NPC. For example, it has been shown that the presence of monomethylated cap structures Etcilitates export of mRNA [ 611, but the nuclear site where these signals exert their effect is unclear. Essentiall!, all nuclear RNAs are packaged with proteins in RNP complexes, which are likely to be the acn~al substrates for nuclear import. In the case of pre-mRNAs (contained in heterogeneous nuclear RNA.s). some intranuclear packaging proteins appear to dissociate from the RNA prior to transport, such as the heterogeneous nuclear RNP (hnRNP) C proteins [62*]. Other packaging proteins, such as the hnRNP Al protein, are apparently transported together with their associated RNAs to the cytoplasm, where they dissociate from the RNA and are rapidl!, imported back into the nucleus [62*]. This shuttling behavior has also been described for the ribosome-associ3ted proteins B23 and nucleolin [63]. It is not unreasonable to expect that the information recognized by the NPC for RNP export resides in RNA-associated proteins, perhaps in some shuttling proteins. In this respect, certain RNPassociated shuttling proteins could haire a role analogous to that proposed for NIS receptors in nuclear protein import (Fig. 2 ). One accessible model for RNP export from the nucleus is provided by the RNA genome of the influenza vims, where it was recently shown that the virus matrix protein is required both for determining nuclear export of the replicated viral RNA-nucleoprotein complex, and for preventing nuclear re-entv of the RNPs once they reach the cytoplasm [30**]. Whether the matrix protein contains a signal recognized by the NPC for mediated transport or simply causes dissociation of the viral RNP from intranuclear anchoring sites remains to be determined. Conclusions Exciting progress has been made recently in describing mechanisms of nuclear import. NISs consisting of two interdependent basic clusters are now thought to be prevalent. Nevertheless, these signals appear to utilize a receptor pathway similar to that of NLSs consisting of a single basic region. A first step toward understanding how these basic NISs mediate transport across the NPC- the identification of relevant transport receptors- has been achieved. It remains to be determined how these and other possible receptors function in the transport pathway. While import of most nuclear proteins appears to involve basic NISs, work with snRNPs indicates the exis-

Molecular

tence of at least one other signaling pathway for import. There is a good likelihood that additional pathways will come to light in the future, some of which could be specific to certain cell types or differentiation stages. Presumably all signaling pathways utilize common machinery of the NPC such as the central channel complex, and it will be important to determine how these different pathways intersect. Cell-free systems that reproduce authentic nuclear import have been described, and will provide invaluable tools to dissect the biochemistry of this process. Major future challenges are to identify the molecular itinerary of ligands at different stages of transport across the NPC, to define components that form the central channel complex, to describe mechanisms of nucleotide utilization, and to identify cytosolic components that collaborate with the NPC to achieve mediated transport. Compared with the process of protein import, the understanding of RNA export through the NPC is substantially less developed. This in part reflects the complexity of the process, as signals are probably utilized at multiple sites within the nucleus for efficient export. The identification of signals in RNP (either protein or RNA) that directly interact with the tr’ansport machinery of the NPC is of great importance. The use of genetic procedures to define components of both import and export pthWayS is in its early stages, but should be an important comp’lement to the biochemical efforts made so far. It will be essential to integrate information on the biochemistry of nuclear import with structural features of the NPC, which clearly is a dynamic transport machine. Recently implemented microscopic and image analysis techniques should be instrumental in this endeavor. A tantalizing new entree to understanding the process has been provided by description of cytoplasmic and nucleoplasmic fibers associated with the surfaces of the NPC, which could have a key role in movement of ligands through the NPC. Considering the structural, biochemical and physiological data that have accumulated recently, it is clear that molecular trafEcking through the NPC involves an enormously complex set of different processes. One can expect that the challenges posed by these problems will be matched by the surprises that emerge from future work. Acknowledgements I am grateful to the members of my lahoraton: for numerous sumulating discussions on the content of this reliew. I also wish to thank Velia Fowler. Roland Foisner. Janice Evans, Joanne Westendorf, Ron Mill&an and Jenny 11inshaw for vex helpful comments on the manuscript.

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26. .

FISCHER U, DARWWWICZ E. TAIIAR~ SM. D,ATtI,%N NA. LL~HRWW R, MATI’AJ IW: Diversity in the Signals Required for Nuclear Accumulation of Ll snRNPs and Variety in the Pathways of Nuclear Transport. / Cell Bid 1991. 113:‘05-‘14 /n virro transcribed U RNAs coma&g different 5’ cap stmctures were injected into Xeie,,oprrs oocytes and nuclear import x~s coal uated. demonstrating that the trimethylguanosine cap is required for import of Ul and U2 RNA. and to a lessrr extent for import of (‘-1 and US RNA In contraSI the y-monomethyl rriphosphate cap of ~‘6 RNA is not essential for its impon. 27. .

IWCHAL~D N, GOlDFARE DS: Microinjected Ll snRNAs Are Imported to Oocyte Nuclei via the Nuclear Pore Complex by Three Distinguishable Pathways. J Cell Hiol 1992. 116:851&l. Studies in oocytes showed rhat the nuclear import of L:l, U2, 1’4 and L:i RNAs is competed by free trimethylguanosine cap dinucleotide, while the import of U3 snRNA is not. Furthermore. nuclear impon of 1’1-C’i snRh% is not competed by BSA conjugated aith the S\‘AO T antigen NLS. U3 snRNP may use a different signaling pdthwdy from that of basic NISs and the other trimeth)lguanosine cap.containing snRNA.s.

MmnN K. HELENI~IS A: Nuclear Transport of Influenza Virus Ribonucleoproteins: The Viral Matrix Protein (Ml) Promotes Export and Inhibits Import. Cell 1991. 67:117-130. The nuclear export of the replicated RNA-nucleoprotein complex of influenza virus wz, shonn fo require association of the M protein with the RNP. Conversely M protein appeared fo inhibit nuclear import of the RNP after virus entry into cells. Import can occur only after the M protein dissociates from the RNP.

30.

..

MICHAIW N, GOIIWW DS: Multiple Pathways in Nuclear Transport: The Import of U2 snRNP Occurs by a Novel Kinetic Pathway. J Cell Eiol 1991. 112:215-223. Injection into Xenoprcc ooc>‘tes of bovine semm albumin conjugated with peptides conraining the S\‘+O T antigen NLS reduced the rate of nuclear import of nucleoplasmin and ~6 snRNA in a saturable fashion. but did not affect 1’2 snRNA import. This indicates that the signaling pathn:iy for nuclear import of 1 12 snRNP differs from that used by basic NLSs and 116 RNP.

31.

..

32.

GOIDI;.WI~ D. MICIWIII) port of Proteins and

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A&W SA, GERACE L: Cytosolic Proteins that Specihcally Bind Nuclear Location Signals Are Receptors for Nuclear Import. Cell 1991. 6683’Wi’. Pol!peptides of it and 56 kD that sprcilically hind the SVqO T antigen NW were isolated from mammalian o’tosol. The proteins were N-cthylmalcimide~sensitive components that stimulate nuclear protein mipon 111 a prrme~hilized cell assay. and restore nuclear transpon to a s?srem ina&-dted by N-erhylmaleimide. Thus, these NIShinding proteins act as functional rcveptors for nuclear import. ..

34.

ILE WC. X1.1: %. MI:IESE T: The NSRI Gene Encodes a Protein that Specifically Binds Nuclear Localization Sequences and Has Two RNA Recognition Motifs. J Cell Rio/ 1331. 113.1-12. The ~quenc‘c of a gene rnctxling a spccilic N&binding protein of .S. ce~c’rvrcte describes a protrin containing two RNA- recognition motifs, a highly acidic amino terminus and a basic carho@ terminus The protein is concenrrdtci 111a discrete domain v.ithin the nucleus. .

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S’KX:II~\I I’. OWORSI: M. KI’RII~ARA T. SII.\~:.R P. A Yeast Protein That Binds Nuclear Localization Signals: Purification. Localization , and Antibody Inhibition of Binding Activity. .I C& Hid 1991. 113:12i3-12%. SKNI II. SIIIDA t I, Hyt’x~ NM S. NOSAK~ T. MAIQ M, ~~T.ANAKA, hl. Sequence Requirements for Nucleolar Localization of Human T Cell Leukemia Virus Type I pX Protein, Which Regulates Viral RNA Processing. Cell 1988, 55:197-209. BRI:EI’WR Al. GomFAm Histone Hl and Other 1990, 60:99$-1008.

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KIct~~)\os WD. tv1ru.s AD. DII.W’OHW SM. L&\ICEY RA, DISG~,.W. C: Nuclear Protein Migration Involves Two Steps: Rapid Binding at the Nuclear Envelope Followed by Slower Translocation through Nuclear Pores. Cell 1988. 52:65%66+ AIXV $A, LOBI. TJ. MITCIW~. XlA. GER~CE L Identification of Specific Binding Proteins for a Nuclear Location Sequence. Nature 1989, 337PG279. HI’HTI.lT doplasmic

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MARTIN K, HEIINIIIS A: Transport of Incoming Influenza Virus Nucleocapsids into the Nucleus. J Iqirol 1991, 65:232-244.

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ClEMR J, YAhlADA M, KhsAhMTsCI H: lmpoft of Simian Virus 40 Viions through Nuclear Pore Complexes. Proc Nut/ AC& Sci USA 1991, 88~7333-7337.

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JANS DA, ACI(ER~VV\IN MJ, BISCHOFF JR, BEACH DH, PETERS R: p3dCdC2-Mediated Phosphorylation at T124 Inhibits Nuclear import of SV40 T Antigen Proteins. / Cell Biol 1991. 115:120~1212. Tne nuclear import of a synthetic constmct containing the NLS region of the SV40 T antigen fused to fl-galactosidase was anat)zed by cell microinjection. When the construct w;1s phosphorylated in 13&o by the p34 kinase on a residue adjacent to thr NLS. the rate of nuclear import was markedly reduced. 48.

49.

NEWMEYER D. Fo~aes

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HENKEI. T. 7~31~1. 11. \‘AN ZEE K. MIYJPR JM. F~NINC. E.B~t~~!ttui P: Intramolecular Masking of the Nuclear Location Signal and Dimerization Domain in the Precursor for the p50 NF-LB Subunit. Cell 1992. 68:1121-1133. Antibodies recognizing the NLS in thr p50 subunit of NF-x13 showed that the NLS was masked when ~50 ~JS part of the 110 kD precursor, but became accessible when ~5 little as 191 rrsidurs were removed from the carbo,Tt terminus of the precursor. 51. ..

Mou. T. TEfsI( G. Stlrus~ I’. Roamcl-t 11. N.%%n-rtj K. The Role of Phosphorylation and the CDC28 Protein Kinase in Cell Cycle-Regulated Nuclear Import of the S. cereoisiae Transcription Factor SWl5. Cell 1991, 66:‘-1.%758 A detailed analysis of in rim) and iu litlo phosphoglanon of the SWl5 transcription factor indicated that inhibition of its nucttzir impon during the S, Gz and M phasrts is clue to phospho~latlon of threr sites nr;u its NL5 hy cdc28 kinase. When these sites became dephosphonlatrd during G,. the protein wz~q imported into the nucleus. This pro\;des a paradigm for cell cycle-regulated nuclear import.

52.

53.

54. .

RIHS H-P. IANS DA. F.&v tl. Pt~niti R: The Rate of Nuclear Cytoplasiic Protein Transport 1s Determined by the Casein Kinase 11 Site Flanking the Nuclear Localization Sequence of the SV40 TAntigen. &HO./ 1991. 10:63$637 FELDHERR C. AKIN D: The Permeability of the Nuclear Envelope in Dividing and Nondividing Cell Cultures. J Cell Riot 1990, 111.1-x.

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trafficking

across the nuclear

pore complex

Gerace

with growing cells, growth-arrested cultures showed up to a 78.fold decreaFe in the rate of import of large gold panicles (1 l-27 nm). In contrast, the rate of import of small nucleoplasmin.coated gold (5-8nm) was unchanged in the arrested cultures.

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ZASLOFF. M: tRNA Transport from the otic Cell: Carrier-Mediated Translocation Acud Sci C.‘SA 1983, 80:643&6440.

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BATAIUE N. HELSER T. FIUED HM: Ribosomal Subunits Microinjected Oocyte Nucleus: a Generalized, Biol 1990, 111:1571-1582.

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CIU~TER KC, TNMJ\EJA KL LAWRENCE JB: Discrete Nuclear Domains of Poly(A) RNA and Their Relationship to the Functional Organization of the Nucleus. / Cell Biol 1991, 115:1191-1202. 111 situ hybridization with a probe to detect poll(A) RNA showed this RNA to be highly concentrated in discrete ‘transcript domains’ in nuclei that coincide wirh the tocalization of snRNPs. These could be specialized nuclear regions involved in RNA splicing.

59.

..

60.

ECWER R. ELL~IEIER W. BIRNSTIEL ML Mature mRNA 3’ End Formation Stimulates RNA Export from the Nucleus. 6WBO .I 1991, 10:351-G-3522. Sequences present on the intron-free histone rnh\A that speci@ 3’.end formation by the endogenous cellular pathway stimulated mRNA expon from the nucleus. In contr;ltit, histone transcripts. whose 3’ ends were generated b!: an artiticial pathnxy utilizing a c&acting ribozyme, were trdnspon-dehcient. .

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355:730-732. Analyses with hrterokaryons showed that the hnRNP Al protein shuttles ben\ren nuctrus and c\Toplasm. while the C proteins do not. When rrdnscnption b!, RNA po$merJse 11 was inhibited by actmomycin D. the Al hut not C protein accumulated in the cytoplasm. 63.

BORER RA. LEHSER CF. EPPENHERCER HM. NIC& EA Major Nucleolar Proteins Shuttle between Nucleus and Cytoplasm. Cell 1989. 56:3“+390.

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L Gerace. Department 10666 North Torrey

of Cell BioloF. The Scripps Pines Road, I;~ Jolla. California

Research Institute, 92037, USA

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