.
Control
of nucleocytoplasmic
transport
Laura I. Davis Howard
Hughes
Medical
Institute,
Duke
University
Medical
Center,
Durham,
North
Carolina,
USA
The movement of macromolecules between the nucleus and cytoplasm is tightly controlled. In the past few years it has become increasingly apparent that nuclear traffic is regulated not only by recognition of specific signals on proteins and RNAs, but also by cellular factors that modulate the efficacy with which these signals are recognized.
Current
Opinion
in Cell Biology
Introduction The intraceUular membrane systems of eukaryotic cells act as barriers that divide the cell into functional compartments. How these compartments are established and maintained is an area of intense study in cell biology. While our understanding of the processes by which proteins are transported into the endoplasmic reticulum and mitochondria has grown rapidly in the past several years, we still know relatively little about the control of macromolecular movement between the nucleus and cytoplasm. Recently it has become clear, however, that transport of proteins, such as transcription factors, into the nucleus is not a constitutive process, but can change in response to external stimuli and cell cycle variations. Thus, nucleocytoplasmic transport may be an important regulatory point in the control of gene expression. This review will focus on recent contributions to our understanding of the signals required for both RNA and protein transport, and the cellular machinery that controls the transport process. Recent reviews [ 1,2] provide more extensive coverage of this subject.
RNA transport Nuclear and cytoplasmic RNA populations are very different. Unprocessed mRNAs, for example, are generally not found in the cytoplasm, but remain nuclear until processing is complete. Considerable debate has arisen as to whether these distinct populations reflect a specific transport step that recognizes only mature RNA, or whether unprocessed RNA is tethered within the nuclear interior and released upon processing to diffuse towards the nuclear pore complex. In the latter case transport can be viewed as a constitutive, rather nonspecific, step in the pathway. While this question is still unresolved, there is growing support for a model that lies somewhere in between these two extremes.
1992, 4:424429
A relation between splicing and export was established through analysis of the export of mRNAs containing splice site mutations. Mutant mRNAs that were incapable of forming a complex with small nuclear ribonucleoprotein (snRNPs) that committed them to the splicing pathway were exported to the cytoplasm as unspliced transcripts, whereas mutants that formed a commitment complex but were incapable of being further processed were retained within the nucleus [ 31. These results suggest that binding to the splicing machinery sequesters pre-mRNA within the nucleus until splicing is complete. Analysis of the fate of inefficiently spliced RNAs further suggests that competition between export from the nucleus and formation of a splicing complex may govem the proportion of unspliced RNA in the cytoplasm [4-T]. Changes in this ratio could be achieved either by increasing the rate of export of unprocessed RNA or by decreasing the rate of formation of the commitment complex. The viral regulatory protein Rev, which causes cytoplasmic accumulation of unspliced and singly spliced viral RNA [5,6]. could act via either mechanism (reviewed in [8]). The model suggests that unspliced RN& are tethered within the nucleus but does not preclude an active and specific transport process downstream. The splicing and transport pathways may be linked, so that processed RNAs are fed into a mediated transport system. Several recent studies support this model. Eckner et nf. [9*] have looked at histone mRNA, processed artificially at its 3’ end by a cis-acting riboqme instead of through the normal snRNP-mediated pathway. The rate of transport of this message into the cytoplasm was found to be significantly decreased, suggesting that transit through the normal processing pathway is required for efficient export. Also, in situ hybridization experiments have shown that newly synthesized messages are found in discrete tracks leading from the interior of the nucleus to the nuclear envelope [ lO**], contradicting the idea that RNAs are free to diffuse as soon as they are processed. Evidence that a specific signal is required for export comes from studies in which the monomethyl cap
Abbreviations snRNP-small 424
nuclear
ribonucleoprotein;
@ Current
NLGnuclear
Biology
localization
signal;
Ltd ISSN 0955-0674
snRNA-small
nuclear
RNA.
Control
of nucleocytoplasmic
transport
Davis
structure of mutant pre-mRNAs was substituted with a trimethyl cap [ 111. Upon injection into oocyte nuclei, mRNAs that had been mutated so as not to enter the splicing pathway were exported to the cytoplasm, provided they contained a normal (monomethyl) cap; if they had a trimethyl cap they were retained in the nucleus. This result strongly suggests that cap structure affects the ability of free mRNAs to leave the nucleus and may provide the actual signal for mediated export.
tor. The recent observation that SV40 T antigen bearing a mutant NLS can be properly localized by expression of heat-shock protein 70 [17-l further supports this model by implying that heat-shock proteins can prevent misfolding of mutant NJ..%, and could be required to stabilize some wild-type proteins in a state that exposes the NLS.
A role for the cap structure in transport of small nuclear RNAs (snRNAs) has also been demonstrated [ 11,12*,13]. Ul snRNA export requires the correct cap, and can be inhibited by microinjecting cap analogues [ 11 I. This suggests that recognition of the cap by a saturable receptor is required for snRNA export. Import of the assembled Ul snRNP complex also requires the correct cap structure (in this case the hypermethylated cap modified in the cytoplasm), but a second signal provided by the common Sm protein is also necessary [ 131.
Modulation
Two different approaches suggest that microinjected U6 and Ul RNPs are imported via different pathways. Michaud and Goldfarb [14-l showed that ~6 import, which is cap-independent and may require binding to a protein subunit, could be inhibited by co-injecting saturating concentrations of nuclear protein, whereas Ul import was unaffected. Similarly, Fischer et al. [ 12.1 showed that ~6 but not Ul import could be inhibited by wheat germ agglutinin, a lectin that binds to nuclear pore complex proteins and inhibits protein import. These results suggest that Ul snRNPs use a novel pathway in which cap structure plays an important role, whereas ~6 enters the nucleus via the same pathway as nuclear proteins (discussed below).
The signals import
required
for nuclear
protein
Although the pore complex is permeable to macromolecules as large as 40 k~, the observation that histone Hl enters the nucleus through a receptor-mediated transport pathway, rather than by simple diffusion [ 151, suggests that import of both large and small nuclear proteins is a mediated process. Import is dependent on the presence of a short basic tract of amino acids, referred to as the nuclear localization signal (NIS). The consensus sequence for the NLS is quite loose, and was originally defined (largely through the analysis of viral nucleoproteins) as a tract of approximately four basic amino acids often flanked by a proline residue (reviewed in [ 11). The nucleoplasmin NLS, however, consists of two interdependent domains, a run of four lysines as well as two basic residues that are found 10 amino acids upstream [ l6*]. A search of a protein database showed that approximately half of all sequenced nuclear proteins have a similar motif. The observation that the NIS can be composed of non-contiguous regions adds to existing evidence that in some cases the conformation of the region around the NLS is crucial for recognition by the import recep-
of nuclear
protein
transport
Transport of numerous transcriptional activators can be regulated by cytoplasmic factors that mask the NLS and prevent its recognition by the import apparatus (see [2] for review). Masking can be achieved either by the formation of inhibitory complexes with other proteins, or by post-translational modification (usually phosphorylation) of the NLS itself. Transcription factors sequestered in this manner are thought to be substrates for mediators of various signal transduction pathways, which presumably ‘release’ them by reversing the inhibitory modifications [ 18°,19,20*,21,22-o,23,24*,25,26]. This ensures a rapid transcriptional response to various extracellular signals Recent work on the multisubunit transcriptional activator, Nl-AT, illustrates this. NF-AT is required for early T-cell activation in response to external stimulation. It is a specific target of the immunosuppressive drugs cyclosporin and FK506, which inhibit NF-AT-dependent transcription in uiuo. The use of a stimulation-dependent in oitro transcription assay showed that transcription could be restored to nuclear extracts from stimulated, drug-treated cells by adding cytosol from unstimulated cells [20*]. This suggested that translocation of an existing NF-AT component from cytoplasm to nucleus occurs in response to extracellular stimulation, and that this step is blocked by cyclosporin. Further results provided indirect evidence that activation of NF-AT translocation occurs via a pathway requiring calcium mobilization. A speculative model would be that calcineurin, a phosphatase that is inactivated by cyclosponn [ 27,281 ,is required to dephosphorylate the cytoplasmic subunit of NF-AT and allow its translocation to the nucleus. Nuclear localization can also be regulated during the cell cycle. This is exemplified by the behavior of the yeast transcriptional factor WI5 [22-l, which moves from the cytoplasm to the nucleus during Gr. Three serine residues within and around the NLS are phosphoty lated in a cell-cycle-dependent fashion. The degree of phosphotylation is inversely correlated with the level of nuclear SWIS, and mutation of the serines to alanine caused constitutive nuclear entry, arguing strongly that dephospholyiation allows nuclear translocation. Interestingly, the signal for cell cycle-dependent nuclear entry resides within a stretch of 41 amino acids that includes the NIS, as these residues were sufficient to confer cell cycle-specific nuclear localization when fused to a reporter protein. Thus, it appears that phosphotylation within and around the NIS directly masks the signal from the import apparatus, rather than affecting association with other proteins.
425
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stitute for cytoplasmic extracts. Thus, these polypeptides appear to be necessary but not sufficient for import. Two yeast NLS-binding proteins have also been characterized [35*,36*] and the gene for one of them (NSRI) has been cloned [35*]. NSRl has RNA recognition motifs and is probably localized to the nucleolus. Nucleolar localization has also been observed for a mammalian NLS-binding protein [37]. It is somewhat difficult to reconcile intranuclear localization with a role in transport of proteins from the cytoplasm into the nucleus, and it may be that a number of nucleolar proteins simply bind non-specifically to NISs. Alternatively, an NLS receptor could shuttle between the nucleus and cytoplasm - as has already been demonstrated for the nucleolar proteins nucleolin and B23 [ 311 - and simply reside in the nucleolus most of the time, perhaps because the majority of its substrates are nucleolar proteins. Mutational analysis of the yeast NE-binding proteins should resolve this question.
of nucleocytoplasmic
Davis
transport
Conclusions The available evidence suggests that control of nucleocytoplasmic transport is exerted both by the transport apparatus, which recognizes only a specific subset of cellular proteins and RNAS, and by cytoplasmic and nuclear factors that affect the availability of these macromolecules for recognition. As yet there is no evidence that the ability of the nuclear pore complex to catalyze transport can be directly regulated, but this may simply reflect our lack of knowledge concerning this organelle. What is obvious is that the cell has evolved numerous ways to control what reaches the pore complex. Perhaps of most interest is that mediators emanating from several different signal transduction pathways have now been shown to control the import, and thus the activity, of specific transcription factors. Thus, nucleocytoplasmic transport may provide a new link between early events in signal transduction and the control of gene expression.
Acknowledgements The nuclear
pore complex
Nuclear pore complexes form large, proteinaceous channels through the nuclear envelope (Fig. l), and are assumed to provide the only avenue for transport between nucleus and cytoplasm. Although the pore complex has an estimated mass of 108 kD [38], few of its constituents have been identified. The nuclear pore proteins that serve as binding sites for nuclear import are unknown. Akey and Goldfarb [39] have shown that nuclear proteins dock at a region of the pore complex called the central transporter, and that a group of related glycoproteins, the nucleoporins (reviewed in [2] ), are located within this structure. Immunodepletion of the nucleoporins from extracts prior to in r&-o reconstitution of nuclei prevents docking at the pore complex [40*,41], suggesting that one or more of the nucleoporins either serves as the receptor (directly or through cytoplasmic NE-binding proteins) or is stably associated with it. Two genes that encode yeast nucleoporin homologues NUPl [42] and NSPZ [43] have been identified. Temperature-sensitive mutants of NSPl show defects in protein import at the non-permissive temperzcure [43], providing in zdzjoevidence that these proteins play a role in transport. The mechanism by which macromolecules are translocated through the pore complex is not understood. It has been suggested that this step, which requires ATP, involves dilation of an iris-like structure in the central transporter in response to docking of nuclear proteins [ 441, but this model is speculative. No ATPase has been definitively linked to transport, but the presence of mycsin in the nuclear pore complex has been suggested by immunocytochemistry [45]. Further characterization of the constituents of the nuclear pore complex should provide the answers to many of these questions.
The author would like to thank D Miller, T M&se, Goldfarb for critical reading of the manuscript.
References Papers of view, have . of .. of
1.
and recommended
particular interest, published been highlighted as: special interest outstanding interest
SILVER
PA: 6h489-497.
3.
LEGRAIN
How
within
Proteins
Enter
P, ROSBASH M: Some
for Splicing 57:573-583.
Target
pre-mRNA
the annual
the
cis and
to the
period
Nuclear
Protein
Lo-
Cell
1991,
Nucleus. transacting Cytoplasm.
Mutants Cell 1989,
ALONSO-CAPLEN FV. KRUG RM: Regulation Splicing of Influenza Viis NSl mRNA of Splicing and of the Nucleocytoplasmic mRNk ,V/o/ Cell Biol 1991. 11:1092-1098.
5.
CHANC DD, SHARP PA: Regulation by HIV Rev Depends Recognition of Splice Sites. Cell 1989, 59~789-795.
6.
M, mUBER J, m s-y, mL7.EL jv, 1 rev Crawactivator Acts Through Sequence to Activate Nuclear Export mRNA N&we 1989, 338:254-257. MALIhI
of re-
1071:8>101.
4.
of the Extent of Role of the Rates Transport of NSl
CUUEN
BR:
The
a Structured of Unspliced
upon Hw-
Target Viral
I, HOWES Ew: Exon Recognition and Nucleocytoplasmic Partitioning Determine AMPDl Alternative Transcript Production. Afo! Cell Biol 1991, 11:5356-5363.
7.
MINEO
8.
CULLEN BR. MALI&I MH: The a Novel Class of Eukaryotic Trends
9. .
and D
reading
GARCL+B~ISTOS J, HE~TMAN J, HALL MN: calization. Biochim Biopkys Acta 1991,
2.
B Cullen
Bibem
Sci 1991,
ECKNER R, ELL~~EIER
Formation Stimulates / 1991, 10:3513-3522.
W,
HIV-1 Rev Protein: Post-transcriptional 16346350.
BWWIEL
RNA
Prototype of Regulators.
Export
ML: Mature mRNA from the Nucleus.
3’ end EMBO
Histone mRNA 3’ processing through the snRNP.mediated pathway is required for efficient export. Transcripts processed artificially through the action of a &acting ribozyme are exported more slowly.
427
428
Nucleus
and gene expression
10.
XING Y. IAWRENCE IB: Preservation of Soe&ic RNA Distribution Within the Chromatin-depleted Nuclear Substructure Demonstrated by in Sihr Hybridization Coupled with Biochemical Fractionation. / Cell Biol 1991, 112:1055-1063. ‘Tracks’ of specific mRNAs are visualized within the nucleus, leading to the nuclear envelope. These tracks persist after depletion of chromatin, suggesting that they are independent of surrounding chromatin StfWNre.
..
Cell Cycle-regulated Nuclear Import of the S. cerevisiae Transcription Factor SW15. CeN 1991, 66:743-758. NUCkdr protein localization can be regulated during the cell cycle. Cell cycle-dependent phosphorylation of a region close to the SWI5 NIS is necessary and sufficient to inhibit import.
23.
PICARD D. YAMAMOTO KR: Two Signals dependent Nuclear Localization of the ceptor. ECIBO J 1987. 6:333%3340.
24.
RLISHLO~ S, STEIN D, NIISSIEIN-VO~HARD C: The Graded Distribution of the dorsal Morphogen is Initiated by Selective Nuclear Transport in Drosophila. Gel/ 1989, 59:1165-l 177.
25.
S~FWARI, R: Relocalization Cytoplasm to the Nucleus 1989, 59:1179-1188.
26.
ROTH S. SI-IZIN D, N~ISS~IN.VOLHARD Localization of the Dorsal Protein Pattern in the Drosophila Embryo.
IW: The is a ComCell lm.
27.
1~~1J. FAR~IER JD. LUI’E WS. FRIEDMAF: J, WEISS&IAN I. .SCHHEIHEK SL; Calcineurin is a Common Target of CycIophilin-Cyclosporin A and FKBP-FK506 Complexes. Cell 1991, 66:807815.
MICHAUD N, GOIDFARH DS: Multiple Pathways in Nuclear Transport the Import of U2 snRNP Occurs by a Novel Kinetic Pathway. J Cell Biol 1991, 112:215-223. Import of U2 snRNP can be inhibited by co-injection of saturating amounts of nuclear protein. whereas impon of U6 snRNP cannot. suggesting that there are two distinct pathways for nuclear impon.
28.
MCK;EON F: When Worlds Meet Protein Phosphatases.
11.
I&MM J, MA’I-I’AJ NV: Monomethylated itate RNA Export from the Nucleus.
Cap Structures FacIlCell 1990, 63:109-118.
12. .
FIXHER U, DARZYNIUEWICZ E. TAHARA SM. DATHAN NA, WHR~WVN R, MA’I-I’AJ IW: Diversity in the Signals Required for Nuclear Accumulation of U snRNPs and Variety in the Pathways of Nuclear Transport. J Cell Biol 1991, 113:705-714. ~6 snRNP import is cap-independent and can be inhibited by wheat germ agglutinin. Other U snRNPs require the correct cap structure for import and their import is not inhibited by WGA This paper sugestz that there are two distinct pathways for nuclear import.
13.
HAMM J, DAI?ZYNKIEWICZ E, TAHARA SM. MA~AJ Trimethylguanosine Cap Structure of Ul snRNA ponent of a Biptite Nuclear Targeting Signal. 62:56%577.
14. .
15.
BREELRWER M, GOIDFARB DS: Facilitated Nuclear Transport of Histone Hl and Other Small Nucleophilic Proteins. Ccl/ 1990,60:99%1008.
ROBBINS J, DILWOR~~ SM. &KEY u DINC;\Y’AU C: Two Interdependent Basic Domains in Nucleoplasmin Nuclear Targeting Sequence: Identification of a Class of Bipartite Nuclear Targeting Sequence. Cell 1991, 64:615-623. The nucleoplasmin NIS consisrs of two basic elements separated b! a 10 amino acid ‘spacer‘. Many other nuclear proteins were found to contain a similar motif. 16. .
17. .
JEONC D-I. CHECK S. WINDSOR J, POUACR RE: Human Major HSP70 Protein Complements the Localization and Functional Defects of Cytoplasmic Mutant SV40 T Antigen In Swiss 3T3 Mouse Fibroblast Cells. Get&s &z/l 1991, 5:2235-2244. Human, but not mouse, cells can efficiently localize an SV40 T antigen NLS mutant (CT3) to the nucleus. Human heat-shock protein 7O expression in mouse cells confers nuclear localization of CT3. suggesting that Hsp70 can complement srructural alterations in the NIS.
SO~~~+ER L, HAGE~UCHLE 0. WEUAUER PK, STRLIUIN M: Nuclear Targeting of the Transcription Factor PTFl is Mediated by a Protein Subunit that does not Bind to the PTFl Cognate Sequence. Ceil 1991, 67:987-99-i. Identifies a transcription factor subunit that may be required solely for transport into the nucleus. This subunit contains terminal amino-acevl glucosamine residues. BAELIER~E PA, BALTlMORE D: Activation of DNA-binding ity in an Apparently Cytoplasmic Precursor of the Transcription Factor. Cell 1988. 53:211-217.
ActivNK-xB
20. .
FIANAGAN WM, C0ftTtH3~ B, BRA,\! RJ, C~I-RREF: GR: Nuclear Association of a T-cell Transcription Factor Blocked by FK506 and Cyclospotin A Nurure 1991. 352:803-807. T.cell stimulation acti\ates NF-AT by causing nuclear accumulation of an existing cytoplasmic NF.AT component, Cyclosporin and FK.506 bl[Kk this accumulation. 21.
METZ R, ZIFF E: CAMP Stimulates the C/EBP-related Transcription Factor rNFIL-6 to trunolocate to the Nucleus and Induce c-Jos Transcription. Genres Del’ 1991. 5:1754-1766.
22. ..
MOU. T, TERB G. S~WANA U. Role of Phosphorylation and
RODITSCH H, N,&xnm the CDC28 Protein
K: The Kinase in
of the dorsal Correlates with
Protein from Its Function.
the Cell
C: A Gradient of Nuclear Determines Dorsoventral Cell 1989. 59:1185rl202.
Collide: Immunosuppressants Cell 1991, 66:82$826.
29. .
J.%vs DA, ACKEIL\!ANN MJ. BISCHOI~F JR, BEACH DH. PET~;.K~ R: pW’d’2-mediated Phosphorylation at Tl2-r Inhibits Nuclear Import of SV-40 T Antigen Proteins. J Cell Biol 1991. 115:1203-1212. S\‘rO T.antigen phospho~lated in ~+l,-o does noi accumulate In the nucleus. Kinetic experiments show that the final amount trdnsponed. rdthrr than the rdte of trdnspon. is fiectrd by l~hosl,ho~latit)n.
30.
RIHS H-P, JANS DA, FAN H, PL?I%& R: The Rate of Nuclear Cytoplasmic Protein Transport is Determined by the Casein Kinase II Site Flanking the Nuclear Localization Sequence of SV40 T-Antigen. T;IIRO .I 1991. 10:633639.
31.
BOKE.K RA. ~~IINER CF. EIJI’I:NHI:H~;I;R Flbl, clear Proteins Shuttle Between Nucleus 1989. 56:379-390.
Nlcc; EA: Major and Cytoplasm.
NuCc/l
32.
G~:IOC~ION-IMA~\T~L A, L~:hcol~ 1’. CHRisni+kh-mE S. L~OSI;EI.T H. PIIKHOT~APPIA~‘~\-I- M. MIL~;KOL~ E: Nucleocytoplasmic Shuttling of the Progesterone Receptor. Ellf3O.I 1991. 10:3851-3859. Cinder conditions thar inhibit nucltrsr import (lo\v remperaturr or ATP depletion ), the progesterone receptor redistributes to the c~~opl;~sm. suggesting rhat specific nuclear impon is countered by cons1;mt Jill% sion back into the c)~)sol.
.
33.
18. .
19.
Mediate HormoneGlucocorticoid Re-
HAN lation 19X9.
GW. HAl:wA.UCEH in the Nucleus 58:8-1-H74.
RS. HOI:I’ GD. and Cytoplasm.
Kl:.u.y WC: Glycosy.,lrf?rrr Kc,!, Hioill?ettl
AL)A\I S, GERAC~ L: Cytosolic Proteins that Specifically Bind Nuclear Location Signals are Receptors for Nuclear Import. Cell 1991, 66:837-X-17. C!Toplasmic f;lctors idrntilicd pre\iousl\ as NE-binding pro’eins are required for SV40 large T antigen impon in an in Ihn assAy. ProQdes the first functional evidence that NL‘-binding proteins function in nuclnlr prottzin localization.
34. ..
L!ZE WC. Xilti Z. M~ii:%: T: The NSRI Gene Encodes a Protein that Specifically Binds Nuclear Localization Sequences and has Two RNA Recognition Motifs. .I Cell f3iol 19931. 1 l3:1-12. The gene ( NSh’l) encoding a ye%t NLS-hinding protein is cloned: the protein is localized IO rhe nucleolus. ji. .
SWC~IAJ I’. OSH~RNE M. KI~RIHARI\ T, SIISER P: A Yeast Protein that Binds Nuclear Localization Signals: Purification Localization, and Antibody Inhibition of Binding Activity. ./ Cell Biol 1991. 113:12i3~1254. Antibodies against :1 yezs~ NL%binding protein inhibir binding of nu clear proteins 10 isolated nuclei.
36. .
3’.
MI:IEH I’T. 13m1~1. G. A Nuclear Localization Protein in the Nucleolus. ,/ Cell Viol 1990.
Signal Binding 11 12235-22-15.
Control 38.
39.
REICHELT
R, HOIZENBURG
A, BUHLE
EL, JARNIK
MAE,
AEBI
.
43.
NEHRBASS U, KERN H, MLJTV!ZI A, HURT E: NSPl: a Yeast Nuclear
44.
AKEY
FINU\Y DR, MEIER E, BRADLF/ P, HOERCKA J, FORBES DJ: A Complex of Nuclear Pore Proteins Required for Pore Function.
45.
41.
42.
DR, FORBES DJ: Reconstitution of Biochemically Altered Nuclear Pores: Transport Can be Eliminated and Restored. Cell 199Q, 60:17-29. DAVIS Ll. FINK GR: The NUPI Gene Encodes an Essential Component of the Yeast Nuclear Pore Complex. Cell 1990,
FINIAY
61:365-978.
CW: Visualization of Transport-related of the Nuclear Pore Transporter. Biophys J
46.
Davis
ConIigurations 1990, 58:341-355.
MAIZ EC: Localization of a MyosIn Polypeptide to Drosophila Nuclear Pore
BERIUOS M, FISHER PA,
Heavy Chainlike Complexes. Proc
J Cell Biol 1991, 114:169-183. Three of the nucleoporins form a stable complex. hnmunodepletion of the complex from nuclei prevents nuclear transport in t&o.
transuort
HOASTMANNH, MANWUAY B, Envelope Protein Localized at the Nuclear Pores Exerts Its Essential Function by Its Carboxy-terminal Domain. Cell 1990, 61:979-989.
U:
Correlation Between Structure and Mass Distribution of the Nuclear Pore Complex and of Distinct Pore Complex Components. J Cell Rio1 1990, 110883-894. AKEY CW, &XDFARB DS: Protein Import Through the Nuclear Pore Complex is a Multi-step Process. J Cell Biol1989, 109:971-982.
40.
of nucleocvtoulasmic
Nat1 Acad
Sci USA 1’991. 88:21%223.
AKEY CW: Probing the Structure and Function of the Nuclear Pore Complex. Semin Cells Biol 1991, 2:167-177.
Ll Davis. Howard Hughes Medical Center, Durham,
Medical Institute. Box North Carolina 27710,
3646 Duke USA
University
429
Control
of nucleocytoplasmic
transport
Laura I. Davis Howard
Hughes
Medical
Institute,
Duke
University
Medical
Center,
Durham,
North
Carolina,
USA
The movement of macromolecules between the nucleus and cytoplasm is tightly controlled. In the past few years it has become increasingly apparent that nuclear traffic is regulated not only by recognition of specific signals on proteins and RNAs, but also by cellular factors that modulate the efficacy with which these signals are recognized.
Current
Opinion
in Cell Biology
Introduction The intraceUular membrane systems of eukaryotic cells act as barriers that divide the cell into functional compartments. How these compartments are established and maintained is an area of intense study in cell biology. While our understanding of the processes by which proteins are transported into the endoplasmic reticulum and mitochondria has grown rapidly in the past several years, we still know relatively little about the control of macromolecular movement between the nucleus and cytoplasm. Recently it has become clear, however, that transport of proteins, such as transcription factors, into the nucleus is not a constitutive process, but can change in response to external stimuli and cell cycle variations. Thus, nucleocytoplasmic transport may be an important regulatory point in the control of gene expression. This review will focus on recent contributions to our understanding of the signals required for both RNA and protein transport, and the cellular machinery that controls the transport process. Recent reviews [ 1,2] provide more extensive coverage of this subject.
RNA transport Nuclear and cytoplasmic RNA populations are very different. Unprocessed mRNAs, for example, are generally not found in the cytoplasm, but remain nuclear until processing is complete. Considerable debate has arisen as to whether these distinct populations reflect a specific transport step that recognizes only mature RNA, or whether unprocessed RNA is tethered within the nuclear interior and released upon processing to diffuse towards the nuclear pore complex. In the latter case transport can be viewed as a constitutive, rather nonspecific, step in the pathway. While this question is still unresolved, there is growing support for a model that lies somewhere in between these two extremes.
1992, 4:424429
A relation between splicing and export was established through analysis of the export of mRNAs containing splice site mutations. Mutant mRNAs that were incapable of forming a complex with small nuclear ribonucleoprotein (snRNPs) that committed them to the splicing pathway were exported to the cytoplasm as unspliced transcripts, whereas mutants that formed a commitment complex but were incapable of being further processed were retained within the nucleus [ 31. These results suggest that binding to the splicing machinery sequesters pre-mRNA within the nucleus until splicing is complete. Analysis of the fate of inefficiently spliced RNAs further suggests that competition between export from the nucleus and formation of a splicing complex may govem the proportion of unspliced RNA in the cytoplasm [4-T]. Changes in this ratio could be achieved either by increasing the rate of export of unprocessed RNA or by decreasing the rate of formation of the commitment complex. The viral regulatory protein Rev, which causes cytoplasmic accumulation of unspliced and singly spliced viral RNA [5,6]. could act via either mechanism (reviewed in [8]). The model suggests that unspliced RN& are tethered within the nucleus but does not preclude an active and specific transport process downstream. The splicing and transport pathways may be linked, so that processed RNAs are fed into a mediated transport system. Several recent studies support this model. Eckner et nf. [9*] have looked at histone mRNA, processed artificially at its 3’ end by a cis-acting riboqme instead of through the normal snRNP-mediated pathway. The rate of transport of this message into the cytoplasm was found to be significantly decreased, suggesting that transit through the normal processing pathway is required for efficient export. Also, in situ hybridization experiments have shown that newly synthesized messages are found in discrete tracks leading from the interior of the nucleus to the nuclear envelope [ lO**], contradicting the idea that RNAs are free to diffuse as soon as they are processed. Evidence that a specific signal is required for export comes from studies in which the monomethyl cap
Abbreviations snRNP-small 424
nuclear
ribonucleoprotein;
@ Current
NLGnuclear
Biology
localization
signal;
Ltd ISSN 0955-0674
snRNA-small
nuclear
RNA.
Control
of nucleocytoplasmic
transport
Davis
structure of mutant pre-mRNAs was substituted with a trimethyl cap [ 111. Upon injection into oocyte nuclei, mRNAs that had been mutated so as not to enter the splicing pathway were exported to the cytoplasm, provided they contained a normal (monomethyl) cap; if they had a trimethyl cap they were retained in the nucleus. This result strongly suggests that cap structure affects the ability of free mRNAs to leave the nucleus and may provide the actual signal for mediated export.
tor. The recent observation that SV40 T antigen bearing a mutant NLS can be properly localized by expression of heat-shock protein 70 [17-l further supports this model by implying that heat-shock proteins can prevent misfolding of mutant NJ..%, and could be required to stabilize some wild-type proteins in a state that exposes the NLS.
A role for the cap structure in transport of small nuclear RNAs (snRNAs) has also been demonstrated [ 11,12*,13]. Ul snRNA export requires the correct cap, and can be inhibited by microinjecting cap analogues [ 11 I. This suggests that recognition of the cap by a saturable receptor is required for snRNA export. Import of the assembled Ul snRNP complex also requires the correct cap structure (in this case the hypermethylated cap modified in the cytoplasm), but a second signal provided by the common Sm protein is also necessary [ 131.
Modulation
Two different approaches suggest that microinjected U6 and Ul RNPs are imported via different pathways. Michaud and Goldfarb [14-l showed that ~6 import, which is cap-independent and may require binding to a protein subunit, could be inhibited by co-injecting saturating concentrations of nuclear protein, whereas Ul import was unaffected. Similarly, Fischer et al. [ 12.1 showed that ~6 but not Ul import could be inhibited by wheat germ agglutinin, a lectin that binds to nuclear pore complex proteins and inhibits protein import. These results suggest that Ul snRNPs use a novel pathway in which cap structure plays an important role, whereas ~6 enters the nucleus via the same pathway as nuclear proteins (discussed below).
The signals import
required
for nuclear
protein
Although the pore complex is permeable to macromolecules as large as 40 k~, the observation that histone Hl enters the nucleus through a receptor-mediated transport pathway, rather than by simple diffusion [ 151, suggests that import of both large and small nuclear proteins is a mediated process. Import is dependent on the presence of a short basic tract of amino acids, referred to as the nuclear localization signal (NIS). The consensus sequence for the NLS is quite loose, and was originally defined (largely through the analysis of viral nucleoproteins) as a tract of approximately four basic amino acids often flanked by a proline residue (reviewed in [ 11). The nucleoplasmin NLS, however, consists of two interdependent domains, a run of four lysines as well as two basic residues that are found 10 amino acids upstream [ l6*]. A search of a protein database showed that approximately half of all sequenced nuclear proteins have a similar motif. The observation that the NIS can be composed of non-contiguous regions adds to existing evidence that in some cases the conformation of the region around the NLS is crucial for recognition by the import recep-
of nuclear
protein
transport
Transport of numerous transcriptional activators can be regulated by cytoplasmic factors that mask the NLS and prevent its recognition by the import apparatus (see [2] for review). Masking can be achieved either by the formation of inhibitory complexes with other proteins, or by post-translational modification (usually phosphorylation) of the NLS itself. Transcription factors sequestered in this manner are thought to be substrates for mediators of various signal transduction pathways, which presumably ‘release’ them by reversing the inhibitory modifications [ 18°,19,20*,21,22-o,23,24*,25,26]. This ensures a rapid transcriptional response to various extracellular signals Recent work on the multisubunit transcriptional activator, Nl-AT, illustrates this. NF-AT is required for early T-cell activation in response to external stimulation. It is a specific target of the immunosuppressive drugs cyclosporin and FK506, which inhibit NF-AT-dependent transcription in uiuo. The use of a stimulation-dependent in oitro transcription assay showed that transcription could be restored to nuclear extracts from stimulated, drug-treated cells by adding cytosol from unstimulated cells [20*]. This suggested that translocation of an existing NF-AT component from cytoplasm to nucleus occurs in response to extracellular stimulation, and that this step is blocked by cyclosporin. Further results provided indirect evidence that activation of NF-AT translocation occurs via a pathway requiring calcium mobilization. A speculative model would be that calcineurin, a phosphatase that is inactivated by cyclosponn [ 27,281 ,is required to dephosphorylate the cytoplasmic subunit of NF-AT and allow its translocation to the nucleus. Nuclear localization can also be regulated during the cell cycle. This is exemplified by the behavior of the yeast transcriptional factor WI5 [22-l, which moves from the cytoplasm to the nucleus during Gr. Three serine residues within and around the NLS are phosphoty lated in a cell-cycle-dependent fashion. The degree of phosphotylation is inversely correlated with the level of nuclear SWIS, and mutation of the serines to alanine caused constitutive nuclear entry, arguing strongly that dephospholyiation allows nuclear translocation. Interestingly, the signal for cell cycle-dependent nuclear entry resides within a stretch of 41 amino acids that includes the NIS, as these residues were sufficient to confer cell cycle-specific nuclear localization when fused to a reporter protein. Thus, it appears that phosphotylation within and around the NIS directly masks the signal from the import apparatus, rather than affecting association with other proteins.
425
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stitute for cytoplasmic extracts. Thus, these polypeptides appear to be necessary but not sufficient for import. Two yeast NLS-binding proteins have also been characterized [35*,36*] and the gene for one of them (NSRI) has been cloned [35*]. NSRl has RNA recognition motifs and is probably localized to the nucleolus. Nucleolar localization has also been observed for a mammalian NLS-binding protein [37]. It is somewhat difficult to reconcile intranuclear localization with a role in transport of proteins from the cytoplasm into the nucleus, and it may be that a number of nucleolar proteins simply bind non-specifically to NISs. Alternatively, an NLS receptor could shuttle between the nucleus and cytoplasm - as has already been demonstrated for the nucleolar proteins nucleolin and B23 [ 311 - and simply reside in the nucleolus most of the time, perhaps because the majority of its substrates are nucleolar proteins. Mutational analysis of the yeast NE-binding proteins should resolve this question.
of nucleocytoplasmic
Davis
transport
Conclusions The available evidence suggests that control of nucleocytoplasmic transport is exerted both by the transport apparatus, which recognizes only a specific subset of cellular proteins and RNAS, and by cytoplasmic and nuclear factors that affect the availability of these macromolecules for recognition. As yet there is no evidence that the ability of the nuclear pore complex to catalyze transport can be directly regulated, but this may simply reflect our lack of knowledge concerning this organelle. What is obvious is that the cell has evolved numerous ways to control what reaches the pore complex. Perhaps of most interest is that mediators emanating from several different signal transduction pathways have now been shown to control the import, and thus the activity, of specific transcription factors. Thus, nucleocytoplasmic transport may provide a new link between early events in signal transduction and the control of gene expression.
Acknowledgements The nuclear
pore complex
Nuclear pore complexes form large, proteinaceous channels through the nuclear envelope (Fig. l), and are assumed to provide the only avenue for transport between nucleus and cytoplasm. Although the pore complex has an estimated mass of 108 kD [38], few of its constituents have been identified. The nuclear pore proteins that serve as binding sites for nuclear import are unknown. Akey and Goldfarb [39] have shown that nuclear proteins dock at a region of the pore complex called the central transporter, and that a group of related glycoproteins, the nucleoporins (reviewed in [2] ), are located within this structure. Immunodepletion of the nucleoporins from extracts prior to in r&-o reconstitution of nuclei prevents docking at the pore complex [40*,41], suggesting that one or more of the nucleoporins either serves as the receptor (directly or through cytoplasmic NE-binding proteins) or is stably associated with it. Two genes that encode yeast nucleoporin homologues NUPl [42] and NSPZ [43] have been identified. Temperature-sensitive mutants of NSPl show defects in protein import at the non-permissive temperzcure [43], providing in zdzjoevidence that these proteins play a role in transport. The mechanism by which macromolecules are translocated through the pore complex is not understood. It has been suggested that this step, which requires ATP, involves dilation of an iris-like structure in the central transporter in response to docking of nuclear proteins [ 441, but this model is speculative. No ATPase has been definitively linked to transport, but the presence of mycsin in the nuclear pore complex has been suggested by immunocytochemistry [45]. Further characterization of the constituents of the nuclear pore complex should provide the answers to many of these questions.
The author would like to thank D Miller, T M&se, Goldfarb for critical reading of the manuscript.
References Papers of view, have . of .. of
1.
and recommended
particular interest, published been highlighted as: special interest outstanding interest
SILVER
PA: 6h489-497.
3.
LEGRAIN
How
within
Proteins
Enter
P, ROSBASH M: Some
for Splicing 57:573-583.
Target
pre-mRNA
the annual
the
cis and
to the
period
Nuclear
Protein
Lo-
Cell
1991,
Nucleus. transacting Cytoplasm.
Mutants Cell 1989,
ALONSO-CAPLEN FV. KRUG RM: Regulation Splicing of Influenza Viis NSl mRNA of Splicing and of the Nucleocytoplasmic mRNk ,V/o/ Cell Biol 1991. 11:1092-1098.
5.
CHANC DD, SHARP PA: Regulation by HIV Rev Depends Recognition of Splice Sites. Cell 1989, 59~789-795.
6.
M, mUBER J, m s-y, mL7.EL jv, 1 rev Crawactivator Acts Through Sequence to Activate Nuclear Export mRNA N&we 1989, 338:254-257. MALIhI
of re-
1071:8>101.
4.
of the Extent of Role of the Rates Transport of NSl
CUUEN
BR:
The
a Structured of Unspliced
upon Hw-
Target Viral
I, HOWES Ew: Exon Recognition and Nucleocytoplasmic Partitioning Determine AMPDl Alternative Transcript Production. Afo! Cell Biol 1991, 11:5356-5363.
7.
MINEO
8.
CULLEN BR. MALI&I MH: The a Novel Class of Eukaryotic Trends
9. .
and D
reading
GARCL+B~ISTOS J, HE~TMAN J, HALL MN: calization. Biochim Biopkys Acta 1991,
2.
B Cullen
Bibem
Sci 1991,
ECKNER R, ELL~~EIER
Formation Stimulates / 1991, 10:3513-3522.
W,
HIV-1 Rev Protein: Post-transcriptional 16346350.
BWWIEL
RNA
Prototype of Regulators.
Export
ML: Mature mRNA from the Nucleus.
3’ end EMBO
Histone mRNA 3’ processing through the snRNP.mediated pathway is required for efficient export. Transcripts processed artificially through the action of a &acting ribozyme are exported more slowly.
427
428
Nucleus
and gene expression
10.
XING Y. IAWRENCE IB: Preservation of Soe&ic RNA Distribution Within the Chromatin-depleted Nuclear Substructure Demonstrated by in Sihr Hybridization Coupled with Biochemical Fractionation. / Cell Biol 1991, 112:1055-1063. ‘Tracks’ of specific mRNAs are visualized within the nucleus, leading to the nuclear envelope. These tracks persist after depletion of chromatin, suggesting that they are independent of surrounding chromatin StfWNre.
..
Cell Cycle-regulated Nuclear Import of the S. cerevisiae Transcription Factor SW15. CeN 1991, 66:743-758. NUCkdr protein localization can be regulated during the cell cycle. Cell cycle-dependent phosphorylation of a region close to the SWI5 NIS is necessary and sufficient to inhibit import.
23.
PICARD D. YAMAMOTO KR: Two Signals dependent Nuclear Localization of the ceptor. ECIBO J 1987. 6:333%3340.
24.
RLISHLO~ S, STEIN D, NIISSIEIN-VO~HARD C: The Graded Distribution of the dorsal Morphogen is Initiated by Selective Nuclear Transport in Drosophila. Gel/ 1989, 59:1165-l 177.
25.
S~FWARI, R: Relocalization Cytoplasm to the Nucleus 1989, 59:1179-1188.
26.
ROTH S. SI-IZIN D, N~ISS~IN.VOLHARD Localization of the Dorsal Protein Pattern in the Drosophila Embryo.
IW: The is a ComCell lm.
27.
1~~1J. FAR~IER JD. LUI’E WS. FRIEDMAF: J, WEISS&IAN I. .SCHHEIHEK SL; Calcineurin is a Common Target of CycIophilin-Cyclosporin A and FKBP-FK506 Complexes. Cell 1991, 66:807815.
MICHAUD N, GOIDFARH DS: Multiple Pathways in Nuclear Transport the Import of U2 snRNP Occurs by a Novel Kinetic Pathway. J Cell Biol 1991, 112:215-223. Import of U2 snRNP can be inhibited by co-injection of saturating amounts of nuclear protein. whereas impon of U6 snRNP cannot. suggesting that there are two distinct pathways for nuclear impon.
28.
MCK;EON F: When Worlds Meet Protein Phosphatases.
11.
I&MM J, MA’I-I’AJ NV: Monomethylated itate RNA Export from the Nucleus.
Cap Structures FacIlCell 1990, 63:109-118.
12. .
FIXHER U, DARZYNIUEWICZ E. TAHARA SM. DATHAN NA, WHR~WVN R, MA’I-I’AJ IW: Diversity in the Signals Required for Nuclear Accumulation of U snRNPs and Variety in the Pathways of Nuclear Transport. J Cell Biol 1991, 113:705-714. ~6 snRNP import is cap-independent and can be inhibited by wheat germ agglutinin. Other U snRNPs require the correct cap structure for import and their import is not inhibited by WGA This paper sugestz that there are two distinct pathways for nuclear import.
13.
HAMM J, DAI?ZYNKIEWICZ E, TAHARA SM. MA~AJ Trimethylguanosine Cap Structure of Ul snRNA ponent of a Biptite Nuclear Targeting Signal. 62:56%577.
14. .
15.
BREELRWER M, GOIDFARB DS: Facilitated Nuclear Transport of Histone Hl and Other Small Nucleophilic Proteins. Ccl/ 1990,60:99%1008.
ROBBINS J, DILWOR~~ SM. &KEY u DINC;\Y’AU C: Two Interdependent Basic Domains in Nucleoplasmin Nuclear Targeting Sequence: Identification of a Class of Bipartite Nuclear Targeting Sequence. Cell 1991, 64:615-623. The nucleoplasmin NIS consisrs of two basic elements separated b! a 10 amino acid ‘spacer‘. Many other nuclear proteins were found to contain a similar motif. 16. .
17. .
JEONC D-I. CHECK S. WINDSOR J, POUACR RE: Human Major HSP70 Protein Complements the Localization and Functional Defects of Cytoplasmic Mutant SV40 T Antigen In Swiss 3T3 Mouse Fibroblast Cells. Get&s &z/l 1991, 5:2235-2244. Human, but not mouse, cells can efficiently localize an SV40 T antigen NLS mutant (CT3) to the nucleus. Human heat-shock protein 7O expression in mouse cells confers nuclear localization of CT3. suggesting that Hsp70 can complement srructural alterations in the NIS.
SO~~~+ER L, HAGE~UCHLE 0. WEUAUER PK, STRLIUIN M: Nuclear Targeting of the Transcription Factor PTFl is Mediated by a Protein Subunit that does not Bind to the PTFl Cognate Sequence. Ceil 1991, 67:987-99-i. Identifies a transcription factor subunit that may be required solely for transport into the nucleus. This subunit contains terminal amino-acevl glucosamine residues. BAELIER~E PA, BALTlMORE D: Activation of DNA-binding ity in an Apparently Cytoplasmic Precursor of the Transcription Factor. Cell 1988. 53:211-217.
ActivNK-xB
20. .
FIANAGAN WM, C0ftTtH3~ B, BRA,\! RJ, C~I-RREF: GR: Nuclear Association of a T-cell Transcription Factor Blocked by FK506 and Cyclospotin A Nurure 1991. 352:803-807. T.cell stimulation acti\ates NF-AT by causing nuclear accumulation of an existing cytoplasmic NF.AT component, Cyclosporin and FK.506 bl[Kk this accumulation. 21.
METZ R, ZIFF E: CAMP Stimulates the C/EBP-related Transcription Factor rNFIL-6 to trunolocate to the Nucleus and Induce c-Jos Transcription. Genres Del’ 1991. 5:1754-1766.
22. ..
MOU. T, TERB G. S~WANA U. Role of Phosphorylation and
RODITSCH H, N,&xnm the CDC28 Protein
K: The Kinase in
of the dorsal Correlates with
Protein from Its Function.
the Cell
C: A Gradient of Nuclear Determines Dorsoventral Cell 1989. 59:1185rl202.
Collide: Immunosuppressants Cell 1991, 66:82$826.
29. .
J.%vs DA, ACKEIL\!ANN MJ. BISCHOI~F JR, BEACH DH. PET~;.K~ R: pW’d’2-mediated Phosphorylation at Tl2-r Inhibits Nuclear Import of SV-40 T Antigen Proteins. J Cell Biol 1991. 115:1203-1212. S\‘rO T.antigen phospho~lated in ~+l,-o does noi accumulate In the nucleus. Kinetic experiments show that the final amount trdnsponed. rdthrr than the rdte of trdnspon. is fiectrd by l~hosl,ho~latit)n.
30.
RIHS H-P, JANS DA, FAN H, PL?I%& R: The Rate of Nuclear Cytoplasmic Protein Transport is Determined by the Casein Kinase II Site Flanking the Nuclear Localization Sequence of SV40 T-Antigen. T;IIRO .I 1991. 10:633639.
31.
BOKE.K RA. ~~IINER CF. EIJI’I:NHI:H~;I;R Flbl, clear Proteins Shuttle Between Nucleus 1989. 56:379-390.
Nlcc; EA: Major and Cytoplasm.
NuCc/l
32.
G~:IOC~ION-IMA~\T~L A, L~:hcol~ 1’. CHRisni+kh-mE S. L~OSI;EI.T H. PIIKHOT~APPIA~‘~\-I- M. MIL~;KOL~ E: Nucleocytoplasmic Shuttling of the Progesterone Receptor. Ellf3O.I 1991. 10:3851-3859. Cinder conditions thar inhibit nucltrsr import (lo\v remperaturr or ATP depletion ), the progesterone receptor redistributes to the c~~opl;~sm. suggesting rhat specific nuclear impon is countered by cons1;mt Jill% sion back into the c)~)sol.
.
33.
18. .
19.
Mediate HormoneGlucocorticoid Re-
HAN lation 19X9.
GW. HAl:wA.UCEH in the Nucleus 58:8-1-H74.
RS. HOI:I’ GD. and Cytoplasm.
Kl:.u.y WC: Glycosy.,lrf?rrr Kc,!, Hioill?ettl
AL)A\I S, GERAC~ L: Cytosolic Proteins that Specifically Bind Nuclear Location Signals are Receptors for Nuclear Import. Cell 1991, 66:837-X-17. C!Toplasmic f;lctors idrntilicd pre\iousl\ as NE-binding pro’eins are required for SV40 large T antigen impon in an in Ihn assAy. ProQdes the first functional evidence that NL‘-binding proteins function in nuclnlr prottzin localization.
34. ..
L!ZE WC. Xilti Z. M~ii:%: T: The NSRI Gene Encodes a Protein that Specifically Binds Nuclear Localization Sequences and has Two RNA Recognition Motifs. .I Cell f3iol 19931. 1 l3:1-12. The gene ( NSh’l) encoding a ye%t NLS-hinding protein is cloned: the protein is localized IO rhe nucleolus. ji. .
SWC~IAJ I’. OSH~RNE M. KI~RIHARI\ T, SIISER P: A Yeast Protein that Binds Nuclear Localization Signals: Purification Localization, and Antibody Inhibition of Binding Activity. ./ Cell Biol 1991. 113:12i3~1254. Antibodies against :1 yezs~ NL%binding protein inhibir binding of nu clear proteins 10 isolated nuclei.
36. .
3’.
MI:IEH I’T. 13m1~1. G. A Nuclear Localization Protein in the Nucleolus. ,/ Cell Viol 1990.
Signal Binding 11 12235-22-15.
Control 38.
39.
REICHELT
R, HOIZENBURG
A, BUHLE
EL, JARNIK
MAE,
AEBI
.
43.
NEHRBASS U, KERN H, MLJTV!ZI A, HURT E: NSPl: a Yeast Nuclear
44.
AKEY
FINU\Y DR, MEIER E, BRADLF/ P, HOERCKA J, FORBES DJ: A Complex of Nuclear Pore Proteins Required for Pore Function.
45.
41.
42.
DR, FORBES DJ: Reconstitution of Biochemically Altered Nuclear Pores: Transport Can be Eliminated and Restored. Cell 199Q, 60:17-29. DAVIS Ll. FINK GR: The NUPI Gene Encodes an Essential Component of the Yeast Nuclear Pore Complex. Cell 1990,
FINIAY
61:365-978.
CW: Visualization of Transport-related of the Nuclear Pore Transporter. Biophys J
46.
Davis
ConIigurations 1990, 58:341-355.
MAIZ EC: Localization of a MyosIn Polypeptide to Drosophila Nuclear Pore
BERIUOS M, FISHER PA,
Heavy Chainlike Complexes. Proc
J Cell Biol 1991, 114:169-183. Three of the nucleoporins form a stable complex. hnmunodepletion of the complex from nuclei prevents nuclear transport in t&o.
transuort
HOASTMANNH, MANWUAY B, Envelope Protein Localized at the Nuclear Pores Exerts Its Essential Function by Its Carboxy-terminal Domain. Cell 1990, 61:979-989.
U:
Correlation Between Structure and Mass Distribution of the Nuclear Pore Complex and of Distinct Pore Complex Components. J Cell Rio1 1990, 110883-894. AKEY CW, &XDFARB DS: Protein Import Through the Nuclear Pore Complex is a Multi-step Process. J Cell Biol1989, 109:971-982.
40.
of nucleocvtoulasmic
Nat1 Acad
Sci USA 1’991. 88:21%223.
AKEY CW: Probing the Structure and Function of the Nuclear Pore Complex. Semin Cells Biol 1991, 2:167-177.
Ll Davis. Howard Hughes Medical Center, Durham,
Medical Institute. Box North Carolina 27710,
3646 Duke USA
University
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