Cell Biology Intemational Reports, VoL 16, No 8, 1992
791
REGULATION OF MACROMOLECULARTRAFHC MEDIATED BY THE NUCLEAR PORE COMPLEX M.W. Miller and J.A. Hanover
Lab. Biochemistry and Metabolism, NIDDK, National Institute of Health, Bethesda, MD 20892, USA
ABSTRACT A sophisticated selective mechanism that regulates nuclear-cytoplasmic traffic has evolved in eukaryotes which circmnvents the formidable barrier presented by the nuclear envelope. The sites of RNA and protein exchanges are the nuclear pore complexes (NPCs), 125 MDa supramolecular assemblies inserted into the envelope (see recent reviews by Dingwall, 1991; Goldfarb and Michaud, 1991; Miller et al., 1991; Nigg et al., 1991). In this article, the role NPCs play in regulating intracellular macromolecular traffic will be discussed. COMPOSrrlON OF THE NPC The ultrastructure of the NPC has several generally accepted features (Akey, 1989; Reichelt et al., 1990; Stewart et al., 1990; see also review by Miller et al., 1991). Each NPC consists of two parallel rings, one on each face of the nuclear envelope, connected to each other by a complex central ring of spokes. A central granule is often seen at the center of the spoke ring. Each NPC appears anchored to the nuclear envelope by a symmetrical array of radial arms embedded within the envelope's lumen. A single NPC is roughly 150 nm in diameter and 70 nm thick and is characterized by an eight-fold rotational symmetry. Many small fibrils emanate from the NPC such that each pore complex is essentially a component of a larger network of NPCs linked to cytoplasmic and nucleoplasmic domains by a complex array of fibers. The nucleoplasmic ring of the pore complex is closely apposed to the discontinuous latticework of nuclear lamina which underlies the nucleoplasmic surface of the nuclear envelope. To date, several components of the NPC have been identified and many are glycoproteins. A 210 KDa high mannose pore glycoprotein localized within the lumen of the nuclear envelope directly abutting the pore complex has been identified and cloned (Wozniak et al., 1989; Greber et al., 1990). The bull of gp210 is contained within the envelope's lumen although 58 amino acids of the c-terminus of gp210 is exposed outside the lumen. Such localization suggests that gp210 may anchor the NPC to the envelope. In addition, a nonmuscle myosin-like ATPase, with similar gel mobility to gp210, has also been localized to the NPC in Drosophila (Berrios et al., 1991). Little about the structure and function of this protein is known. A second class of pore glycoproteins called nucleoporins are peripheral proteins distinguished by O-linked N-acetylglucosamine (GIcNAc) residues (see Miller et al, 1991 for references). These glycoproteins are disposed on the cytoplasmic and
THE STRUCTURE AND
0309-1651 •92/080791-8/$03 00/0
© 1992 Academic Press Ltd
792
Cell Biology International Reports, Vol. 16, No. 8, 1992
nncleoplasmic but not lumenal faces of the NPC. Several nucleoporins have been cloned to date: p62 in vertebrates (Starr et al., 1990) and two essential genes in yeast, NUP (Davis and Fink, 1990) and NSP1 (Nehrbass, 1990). Each is characterized by a highly repeated signature motif of AJGFXFG present in the middle of each protein. The c-termini of NSP1 and p62 also have heptad repeats typical of alpha helical intermediate filament proteins. The c-terminal region of NSP1 encodes the essential requirement for growth and contains the information which targets NSPI to the nuclear envelope (Hurt, 1990; Nehrbass et al., 1990). The ability to reform nuclei in vitro from cellular extracts appears to be a valuable tool for dissecting how NPCs assemble. By limiting the amount of membranous vesicles present in a reformation extract, "pre-pore" complexes can be observed bound to chromatin not associated with membranes (Sheehan et al., 1988). However, it appears that normal NPC formation probably first requires vesicle association with chromatin (Lohka, 1988). In vitro NPC formation requires neither chromatin nor a functional nuclear lamina (as does the nuclear envelope) for a nucleation site (Dabauvalle et al, 1991; Burke and Gerace, 1986; Newport et al, 1990). The nuclear envelope can form without forming functional NPCs (Newport, 1987; DabauvaUe et al., 1990). MECHANISMS FOR CROSSING THE NUCLEAR ENVELOPE
Passive entry. The nuclear envelope is permeated with 9-10 nm diameter aqueous channels which permit the diffusion of most ions and small solutes (Paine et al, 1975; but see Dingwall, 1991). This is in contrast to nuclear macromolecules > 26 nm ill diameter which cross the nuclear envelope by an active transport process (discussed below). Proteins < 4.2 KDa in size introduced into the cytoplasm quickly equilibrate in the nucleus. Larger proteins with masses of 12-67 KDa will also diffuse but at rates inversely proportional to their sizes. The rates of diffusionmediated nuclear accumulation are not necessarily fixed for a given cell type and can be affected by various endogenous and exogenous factors (reviewed by Miller et al., 1991; Feldherr and Akin, 1990). It is not clear whether these effects are due to specific interactions with pore components or to indirect alterations of envelope physiology. Alternatively, proteins may passively localize into the nucleus due to an affinity for a second nuclear substrate (reviewed by Miller et al., 1991). Certain DNA binding protehas retain nuclear localization during mitosis via their association with chromatin rather than spilling into the cytoplasm during mitotic metaphase. ACTIVE TRANSLOCATION For proteins larger than 20-40 KDa, nuclear entry cannot be due to simple diffusion since observed rates of uptake are often substantially greater than the rates predicted by diffusion alone (see reviews by Garcia-Bustos et al., 1991; Goldfarb and Michaud, 1991; Miller et al., 1991). Some potentially diffusible proteins axe also actively transported. Active transport of proteins and RNAs occurs rapidly (roughly 6 x 10 9 molecules/min per nucleus), is time and temperature dependent, and is saturable. Little is known about the mechanism of transport although separate binding and
Cell Biology International Reports, Vol. 16, No. 8, 1992
793
translocation steps can be differentiated (Newmeyer et al., 1988; Richardson et al., 1988). Translocation but not binding requires ATP hydrolysis although it is not clear whether ATP provides the energy for the motive force or if it is required indirectly e.g. to maintain pore integrity. Characterization of the Drosophila NPC associated ATPase could provide insight into this question. Translocation can also be inhibited by binding either exogenous lectins which recognize GlcNAc or certain antinucleoporin antisera (see Miller et al., 1991 for references). The specific nature of these blocks is not clear but they seem not to physically occlude the diffusion channel. NPCs formed in vitro which lack proteins bearing O-linked GlcNAc are deficient in both translocation and binding steps (Finlay et al, 1990; Dabauvalle et al., 1990). Specifically, a multhneric protein complex involving the nucleoporin p62 is implicated (Finlay et al, 1991). Genetic manipulation of the yeast nucleoporin NSP1 shows that the c-terminal region is required for nuclear accumulation of the nucleolar protein NOPI (Nehrbass et al., 1990). Progress has been made in identifying the site of the transport channel within the NPC. Early work speculated that the central granule was actually macromolecules caught in the process of translocation. More recently, this granule has been redeemed as the "transporter," the site to which transportable nuclear proteins ftrst dock, are repositioned over the transport channel and subsequently transported (see Goldfarb and Michaud, 1991). Computer enhanced reconstructions suggest that multiple transporter conformations exist and a multistep translocation process has been inferred (Akey, 1990). However, another report suggests that transported proteins also bind other regions of the NPC, notably the cytoplasmic rings (Stewart et al., 1990). TRANSPORT SIGNALS, SIGNAL RECEPTORS AND SHUTTLES Since most nuclear proteins are released into the cytoplasm and mix with cytoplasmic materials during mitosis, nuclear proteins must contain information which designate these protein.~ for nuclear import. Such sequences, called nuclear localization sequences (NLSs), have been functionally defined in many proteins and are a permanent feature of these proteins (reviewed by Garcia-Bustos et al., 1991; Miller et al., 1991). The canonical NLS, that of the SV40 T-antigen, is characterized by a stretch of basic amino acids adjacent to a structure breaking amino acid thought to ensure that the NLS is exposed on the protein's surface. However, NLSs have no strict sequence consensus, may be simple or complex, and may be present once or more within the same protein. Several different classes of NLSs have been identified. Two different NLSs (T-antigen and snRNPs, see below) do not compete for transport arguing that multiple transport pathways exist(see Goldfarb and Michaud, 1991). Certain cytoplasmic snRNPs are targeted to the nucleus by a composite NLS generated by the 5'-trimethylguanosine cap of the snRNA and the presence of specific snRNP proteins (reviewed by Goldfarb and Michaud, 1991; Miller et al., 1991). Analogously, nuclear accumulation of some hnRNP complexes require polymerase II transcription (Pinol-Roma and Dreyfuss et al., 1991), possibly for the synthesis of a specific RNA which is a component of the hnRNP complex and a part of a composite NLS. Such mechanisms appear to ensure that only properly formed multimeric complexes of proteins and nucleic acids are transported into the nucleus.
794
Cell Biology International Reports, VoL 16, No. 8, 1992
Identification of NLSs has led to the discovery of NLS binding proteins speculated to be carriers or shuttling proteins which mediate binding to the pore complex (reviewed Garcia-Bustos et al., 1991). Adam and Gerace (1991) have recently provided strong evidence that 54 and 56 KDa NLS binding proteins actually function as transport carders. Different NLS-binding proteins exhibit preferential localization within and may shuttle between specific subcellular domains (nucleus, cytoplasm or nucleolus). Consequently, domain specific carders may act to organize and direct intranuclear traffic originating from the N-PC. Signals for nucleolar localization have been identified (Dang and Lee, 1989; Hatanaka, 1990) which operate independent of the NLS in nucleolar proteins. The export of RNAs from the nucleus shares many features of protein and snRNP hnport (reviewed by Goldfarb and Michaud, 1991; Miller et al, 1991). Nuclear RNAs are exported from the same NPCs which hnport proteins, export is saturable and can be blocked by lectin. As with snRNA uptake, RNA export may require specific protein-RNA interactions occur before an RNA is exported. Significantly, it is becoming apparent that the processes of transcription, processing, export and translation are in some way linked. Curiously, transcriptional promoter dements have been shown to affect export (de la Pena and Zasloff, 1987; Braddock et al., 1990). Proper RNA processing is generally a prerequisite for RNA export (e.g. Tobian et al., 1983). In general, most mRNAs possessing introns are not exported until completion of the proper splicing. Several notable exceptions are seen in the course of HIV or HTLV infections where several late viral transcripts harboring introns are exported and translated due to transactivation by the r e v or r e x gene products, respectively (see Miller et al., 1991 for references). Chang and Sharp (1989) have further shown that a nonviral RNA which is not exported due to a mutation which prevents splicing can be exported under conditions where REV is present and able bind the mutated transcript. Finally, Goldfarb and Michaud (1991) have recently reviewed evidence which suggests that the nucleus may be able to assess the translatability of an mRNA and modulate its export accordingly. ANCHORAGE MEDIATED AVAILABILITY OF MACROMOLECULES FOR TRANSPORT
Possibly the most cormnonly used step in the regulation of transport is that which governs the availability of any competent transport substrate to the transport machinery. Anchorage mediated availability is one newly documented means for limiting the transport of many proteins which have regulated localizations in the cytoplasm (reviewed by Nigg, 198; Miller et al, 1991). Analogously, RNAs which are not properly processed similarly appear anchored or trapped within the processing pathways and are unavailable for export. Only if and when such trapped RNAs are released are such RNAs exportable. The nuclear matrix has been shown to be a significant nuclear "compaiament" where RNAs may be anchored (Schroder et al., 1987). INTRANOCLEAR TRAFHC PATI~RNS AND THE PRESENTATION OF TRANSPORTABLE MACROMOLECULES TO AND FROM THE NPC
It is becoming increasingly clear that the nucleus is highly ordered in space and time. Such organization has particular implications concerning the cellular and specifically
CeH Biology International Reports, Vol. 16, No. 8, 1992
795
intranuclear dissemination of m acromolecular information. Leppard and Shenk (1989) have demonstrated that an mRNA transcript passes fltrough several distinct biochemically defined compartments following its transcription to its point of export. Legrain and Rosbash (1989) showed that genetic approaches can track the decisions a transcript makes in following a particular pathway of processing and/or export. During normal HIV or HTLV infection, export of unspliced mRNAs requires nucleolar localization of REV and REX mediated by nuclear and nucleolar targeting signals (see Miller et al., 1991 for references). REX, in turn, localizes unspliced HTLV mRNAs to the nucleolus prior to export (Kalland et al., 1991). Recent evidence further suggests REV-mediated efflux of unspliced HIV mRNAs from isolated nuclei may be partially due to REV affects on a nuclear envelope NTPase involved in RNA efflux (Pfeifer et al., 1991). A critical question remaining to be answered is whether NPCs are physically linked to genomic transcription units and to what extent macromolecular traffic patterns are hardwired within the nucleus. Blobel (1985) has proposed that the nuclear components are highly ordered in space and time such that specific gene products are "gated" to specific NPCs for transport which, in turn, specifies their cytoplasmic destination (see Fig. 1). As gene expression is regulated, different genome-NPC relationships would be created.
tosubcellalar destination
transpor~"~ n I~= NLS
cytoplasm (Translation)
nuclear
envelope lamina subnuclear shuttle [ ~ ? / 0
track~RNA in~,, to nucleolus
an RNP
nucleus (Transcription)
Fig. 1. Macromolecular traffic through the NPC. Significantly, the majority of transcriptional sites tend to be located at the nuclear periphery (e.g. Hutchison and Weintraub, 1985; De Graaf et al., 1991). There is evidence for a reticular network of snRNPs comprising the splicing machinery which connect transcribed D N A with the nuclear periphery (Spector, 1990). Lawrence et al. (1989) have also shown that some mRNAs may be localized onto specific tracks linking internal transcription sites to the nuclear periphery. It is not clear whether these tracks are common and if RNA loaded on these tracks are exported.
796
CellBiology lnternational Reports, Vol. 16, No. 8, 1992
Whether a gating hypothesis is validated or not, it seems clear that the highly organized structures of the nucleus and cytoplasm will demand a highly efficient means for regulating the flow of macromolecular traffic. REFERENCES Adam S.A. and Gerace L. (1991) Cytosolic proteins that specifically bind nuclear location signals and receptors for nuclear import. Cell 66: 837-847. Akey C.W. (1990) Visualization of transport-related configurations of the nuclear pore transporter. Biophys. J. 58: 341-355. Akey C.W. (1989) Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy. Cell Biol. 109: 955-970. Berrios M., Fisher P.A. and Matz E.C. (1991) Localization of a myosin heavy chainlike polypeptide to Drosophila nuclear pore complexes. Proc. Natl. Acad. Sci. 88: 219-223. Blobel G. (1985) Gene gating: a hypothesis. Proc. Natl. Acad. Sci. 82: 8527-8529. Braddock M., Thorbum A.M., Chambers A., Elliott Q.D., Anderson G.J., Kingsman A.J. and Kingsman S.M. (1990) A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction. Cell 62:1123-1133. Burke B. and Gerace L. (1986) A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 44: 639-652. Chang D.D. and Sharp P.A. (1989) Regulation by HIV Rev depends upon recognition of splice sites. Cell 59: 789-795. Dabauvalle M.C., Loos K. and Scheer U. (1990) Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma 100: 56-66. Dabauvalle M.C., Loos K., Merkert H. and Scheer U. (1991) Spontaneous assembly of pore complex-containing membranes ("Annulate lamellae") in Xenopus egg extract in the absence of chromatin. J. Cell Biol. 112: 1073-1082. Dang C.V. and Lee W.M.F. (1989) Nuclear and nucleolar targeting sequences of cerb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J. Biol. Chem. 264: 18019-18023. Davis L.I. and Fink G.R. (1990) The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell 61: 965-978. De Graaf A., Van Hemert F., Linnemans W.A.M., Brakenhoff G.J., De Jong L., Van Renswoude J. and Van Driel R. (1990) Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells. Eur. J. Cell Biol. 52: 135-141. De la Pena P. and Zasloff M. (1987) Enhancement of mRNA transport by promotor elements. Cell 50: 613-619. Dingwall C. (1991) Transport across the nuclear envelope: enigmas and explanations. Bioessays 13: 213-218. Feldherr C.M. and Akin D. (1990) The permeability of the nuclear envelope in dividing and non-dividing cell cultures. J. Ceil Biol. 111: 1-8. Finlay D.R. and Forbes D.J. (1990) Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell 60: 17-29. Finlay D.R., Meier E., Bradley P., Horecka J. and Forbes D.J. (1991) A complex of
Cell Biology International Reports, VoL 16, No. 8, 1992
nuclear pore proteins
797
requiredfor pore function. J. Cell Biol. 114: 169-183.
Garcia-Bustos J.J., Heitman A. and Hall M.N. (1991) Nuclear protein localization. Biochim. Biophys. Acta 107: 83-101. Goldfarb D. and Michaud N. (1991) Pathways for the nuclear transport of proteins and RNAs. Trends Cell Biol. 1: 20-24. Greber U.F., Senior A. and Gerace L. (1990) A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J. 9: 1495-1502. Haaf T. and Schmid M. (1991 ) Chromosome topology in mammalian interphas~ nuclei. Exp. Cell Res. 192: 325-332. Hatanaka M. (1990) Discovery of the nucleolar targeting signal. Bioessays 12:143148. Hesketh J.E. and Pryme I.F. (1991) Interaction between mRNA, ribosomes and the cytoskeleton. Biochem. J. 277: 1-10. Hurt E. (1990) Targeting of a cytosolic protein to the nuclear periphery. J. Cell Biol. 111: 2829-2837. Hutchison N. and Weintraub H. (1985) Localization of DNAse I-sensitive sequences to specific regions of interphase nuclei. Cell 43: 471-482. KaUand K.H., Langhoff E., Bos J.J., Gottinger H. and Haseltine W.A. (1991) Rexdependent nucleolar accumulation of HTLV-1 rnRNAs. New Biol. 3: 389-397. Lawrence J.B., Singer R.H. and Marselle L.M. (1989) Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57: 493-502. Legrain P. and Rosbash M. (1989) Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57: 573-583. Leppard K.N. and Shenk T. (1989) The adenovirus EIB 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. EMBO J. 8: 2329-233..6. Lohka M.J. (1988) The reconstitution of nuclear envelopes in cell-free extracts. Cell Biol. Int. Rep. 12: 833-848. Nehrbass U., Kern H., Mutvei A., Horstmann H., Marshallsay B. and Hurt E.C. (1990) NSPI: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Ceil 61: 979-989. Newmeyer D.D. and Forbes D.J. (1988) Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Ceil 52: 641-653. Newport J. (1987) Nuclear reconstitution in vitro: stages of assembly around proteinfree DNA. Cell. 48: 205-217. Newport J., Wilson K.L. and Dunphy W.G. (1990) A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol. 111: 2247-2259. Nigg E.A. (1990) Mechanisms of signal transduction to the cell nucleus. Adv. Cancer Res. 55: 271-310. Nigg E.A., Baeuede P.A. and Luhrmann R. (1991) Nuclear import-export: in search of signals and mechanisms. Cell 66: 15-22. Paine P.L., Moore L.C. and Horowits S.B. (1975) Nuclear envelope permeability. Nature 254:109-114.
798
Cell Biology International Reports, VoL 16, No. 8, 1992
Pfeifer K., WeBer B.E., Ugarkovic D., Bachmann M., Schroder H.C. and Muller W.E. (199I) Evidence for a direct interaction of Rev protein with nuclear mRNA-translocation system. Eur. J. Biochem. 199: 53-64. Pinol-Roma S. and Dreyfuss G. (1991) Transcription-dependent and Transcriptionindependent nuclear transport of hnRNP proteins. Science 253:312-313. Reichelt R., Holzenburg A., Buhle E.L.J., Jamik M., Engel A. and Aebi U. (1990) Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore eomplex components. J. Cell Biol. 110: 883-894. Richardson W.D.A., Mills D., Dilworth S.M., Laskey R.A. and Dingwall C. (1988) Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52: 655664. Schroder H.C., Bachmarm M., Diehl B., Seifert A. and Muller W.E.G. (1987) Transport of mRNA from nucleus to cytoplasm. Progr. Nucl. Acid. Res. Mol. Biol. 34: 89-142. Sheehan M.A., Mills A.D., Sleeman A.M., Laskey R.A. and Blow J.J. (1988) Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J. Cell Biol. 106: 1-12. Spector D.L. (1990) Higher order nuclear organization: Three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc. Natl. Acad. Sci. 87: 147151. Starr C.M., D'Onofrio M., Park M.K. and Hanover J.A. (1990) Primary sequence and heterologous expression of nuclear pore glycoprotein p62. J. Cell Biol. 110: 1861-1871. Stewart M., Whytock S. and Mills A.D. (1990) Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes. J. Mol. Biol. 213: 575-582. Tobian J.A., Drinkard L. and Zasloff M. (1985). tRNA nuclear transport: defining the critical regions of human tRNAm'tl by point mutagenesis. Cell 43: 415-422. Wozniak R.W., Bartnik E. and Blobel G. (1989) Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J. Cell Biol. 108: 20832092. Zasloff M. (1983) tRNA transport from the nucleus in a eukaryotic cell: carriermediated translocation process. Proc. Natl. Acad. Sci. 80: 6436-644.
791
REGULATION OF MACROMOLECULARTRAFHC MEDIATED BY THE NUCLEAR PORE COMPLEX M.W. Miller and J.A. Hanover
Lab. Biochemistry and Metabolism, NIDDK, National Institute of Health, Bethesda, MD 20892, USA
ABSTRACT A sophisticated selective mechanism that regulates nuclear-cytoplasmic traffic has evolved in eukaryotes which circmnvents the formidable barrier presented by the nuclear envelope. The sites of RNA and protein exchanges are the nuclear pore complexes (NPCs), 125 MDa supramolecular assemblies inserted into the envelope (see recent reviews by Dingwall, 1991; Goldfarb and Michaud, 1991; Miller et al., 1991; Nigg et al., 1991). In this article, the role NPCs play in regulating intracellular macromolecular traffic will be discussed. COMPOSrrlON OF THE NPC The ultrastructure of the NPC has several generally accepted features (Akey, 1989; Reichelt et al., 1990; Stewart et al., 1990; see also review by Miller et al., 1991). Each NPC consists of two parallel rings, one on each face of the nuclear envelope, connected to each other by a complex central ring of spokes. A central granule is often seen at the center of the spoke ring. Each NPC appears anchored to the nuclear envelope by a symmetrical array of radial arms embedded within the envelope's lumen. A single NPC is roughly 150 nm in diameter and 70 nm thick and is characterized by an eight-fold rotational symmetry. Many small fibrils emanate from the NPC such that each pore complex is essentially a component of a larger network of NPCs linked to cytoplasmic and nucleoplasmic domains by a complex array of fibers. The nucleoplasmic ring of the pore complex is closely apposed to the discontinuous latticework of nuclear lamina which underlies the nucleoplasmic surface of the nuclear envelope. To date, several components of the NPC have been identified and many are glycoproteins. A 210 KDa high mannose pore glycoprotein localized within the lumen of the nuclear envelope directly abutting the pore complex has been identified and cloned (Wozniak et al., 1989; Greber et al., 1990). The bull of gp210 is contained within the envelope's lumen although 58 amino acids of the c-terminus of gp210 is exposed outside the lumen. Such localization suggests that gp210 may anchor the NPC to the envelope. In addition, a nonmuscle myosin-like ATPase, with similar gel mobility to gp210, has also been localized to the NPC in Drosophila (Berrios et al., 1991). Little about the structure and function of this protein is known. A second class of pore glycoproteins called nucleoporins are peripheral proteins distinguished by O-linked N-acetylglucosamine (GIcNAc) residues (see Miller et al, 1991 for references). These glycoproteins are disposed on the cytoplasmic and
THE STRUCTURE AND
0309-1651 •92/080791-8/$03 00/0
© 1992 Academic Press Ltd
792
Cell Biology International Reports, Vol. 16, No. 8, 1992
nncleoplasmic but not lumenal faces of the NPC. Several nucleoporins have been cloned to date: p62 in vertebrates (Starr et al., 1990) and two essential genes in yeast, NUP (Davis and Fink, 1990) and NSP1 (Nehrbass, 1990). Each is characterized by a highly repeated signature motif of AJGFXFG present in the middle of each protein. The c-termini of NSP1 and p62 also have heptad repeats typical of alpha helical intermediate filament proteins. The c-terminal region of NSP1 encodes the essential requirement for growth and contains the information which targets NSPI to the nuclear envelope (Hurt, 1990; Nehrbass et al., 1990). The ability to reform nuclei in vitro from cellular extracts appears to be a valuable tool for dissecting how NPCs assemble. By limiting the amount of membranous vesicles present in a reformation extract, "pre-pore" complexes can be observed bound to chromatin not associated with membranes (Sheehan et al., 1988). However, it appears that normal NPC formation probably first requires vesicle association with chromatin (Lohka, 1988). In vitro NPC formation requires neither chromatin nor a functional nuclear lamina (as does the nuclear envelope) for a nucleation site (Dabauvalle et al, 1991; Burke and Gerace, 1986; Newport et al, 1990). The nuclear envelope can form without forming functional NPCs (Newport, 1987; DabauvaUe et al., 1990). MECHANISMS FOR CROSSING THE NUCLEAR ENVELOPE
Passive entry. The nuclear envelope is permeated with 9-10 nm diameter aqueous channels which permit the diffusion of most ions and small solutes (Paine et al, 1975; but see Dingwall, 1991). This is in contrast to nuclear macromolecules > 26 nm ill diameter which cross the nuclear envelope by an active transport process (discussed below). Proteins < 4.2 KDa in size introduced into the cytoplasm quickly equilibrate in the nucleus. Larger proteins with masses of 12-67 KDa will also diffuse but at rates inversely proportional to their sizes. The rates of diffusionmediated nuclear accumulation are not necessarily fixed for a given cell type and can be affected by various endogenous and exogenous factors (reviewed by Miller et al., 1991; Feldherr and Akin, 1990). It is not clear whether these effects are due to specific interactions with pore components or to indirect alterations of envelope physiology. Alternatively, proteins may passively localize into the nucleus due to an affinity for a second nuclear substrate (reviewed by Miller et al., 1991). Certain DNA binding protehas retain nuclear localization during mitosis via their association with chromatin rather than spilling into the cytoplasm during mitotic metaphase. ACTIVE TRANSLOCATION For proteins larger than 20-40 KDa, nuclear entry cannot be due to simple diffusion since observed rates of uptake are often substantially greater than the rates predicted by diffusion alone (see reviews by Garcia-Bustos et al., 1991; Goldfarb and Michaud, 1991; Miller et al., 1991). Some potentially diffusible proteins axe also actively transported. Active transport of proteins and RNAs occurs rapidly (roughly 6 x 10 9 molecules/min per nucleus), is time and temperature dependent, and is saturable. Little is known about the mechanism of transport although separate binding and
Cell Biology International Reports, Vol. 16, No. 8, 1992
793
translocation steps can be differentiated (Newmeyer et al., 1988; Richardson et al., 1988). Translocation but not binding requires ATP hydrolysis although it is not clear whether ATP provides the energy for the motive force or if it is required indirectly e.g. to maintain pore integrity. Characterization of the Drosophila NPC associated ATPase could provide insight into this question. Translocation can also be inhibited by binding either exogenous lectins which recognize GlcNAc or certain antinucleoporin antisera (see Miller et al., 1991 for references). The specific nature of these blocks is not clear but they seem not to physically occlude the diffusion channel. NPCs formed in vitro which lack proteins bearing O-linked GlcNAc are deficient in both translocation and binding steps (Finlay et al, 1990; Dabauvalle et al., 1990). Specifically, a multhneric protein complex involving the nucleoporin p62 is implicated (Finlay et al, 1991). Genetic manipulation of the yeast nucleoporin NSP1 shows that the c-terminal region is required for nuclear accumulation of the nucleolar protein NOPI (Nehrbass et al., 1990). Progress has been made in identifying the site of the transport channel within the NPC. Early work speculated that the central granule was actually macromolecules caught in the process of translocation. More recently, this granule has been redeemed as the "transporter," the site to which transportable nuclear proteins ftrst dock, are repositioned over the transport channel and subsequently transported (see Goldfarb and Michaud, 1991). Computer enhanced reconstructions suggest that multiple transporter conformations exist and a multistep translocation process has been inferred (Akey, 1990). However, another report suggests that transported proteins also bind other regions of the NPC, notably the cytoplasmic rings (Stewart et al., 1990). TRANSPORT SIGNALS, SIGNAL RECEPTORS AND SHUTTLES Since most nuclear proteins are released into the cytoplasm and mix with cytoplasmic materials during mitosis, nuclear proteins must contain information which designate these protein.~ for nuclear import. Such sequences, called nuclear localization sequences (NLSs), have been functionally defined in many proteins and are a permanent feature of these proteins (reviewed by Garcia-Bustos et al., 1991; Miller et al., 1991). The canonical NLS, that of the SV40 T-antigen, is characterized by a stretch of basic amino acids adjacent to a structure breaking amino acid thought to ensure that the NLS is exposed on the protein's surface. However, NLSs have no strict sequence consensus, may be simple or complex, and may be present once or more within the same protein. Several different classes of NLSs have been identified. Two different NLSs (T-antigen and snRNPs, see below) do not compete for transport arguing that multiple transport pathways exist(see Goldfarb and Michaud, 1991). Certain cytoplasmic snRNPs are targeted to the nucleus by a composite NLS generated by the 5'-trimethylguanosine cap of the snRNA and the presence of specific snRNP proteins (reviewed by Goldfarb and Michaud, 1991; Miller et al., 1991). Analogously, nuclear accumulation of some hnRNP complexes require polymerase II transcription (Pinol-Roma and Dreyfuss et al., 1991), possibly for the synthesis of a specific RNA which is a component of the hnRNP complex and a part of a composite NLS. Such mechanisms appear to ensure that only properly formed multimeric complexes of proteins and nucleic acids are transported into the nucleus.
794
Cell Biology International Reports, VoL 16, No. 8, 1992
Identification of NLSs has led to the discovery of NLS binding proteins speculated to be carriers or shuttling proteins which mediate binding to the pore complex (reviewed Garcia-Bustos et al., 1991). Adam and Gerace (1991) have recently provided strong evidence that 54 and 56 KDa NLS binding proteins actually function as transport carders. Different NLS-binding proteins exhibit preferential localization within and may shuttle between specific subcellular domains (nucleus, cytoplasm or nucleolus). Consequently, domain specific carders may act to organize and direct intranuclear traffic originating from the N-PC. Signals for nucleolar localization have been identified (Dang and Lee, 1989; Hatanaka, 1990) which operate independent of the NLS in nucleolar proteins. The export of RNAs from the nucleus shares many features of protein and snRNP hnport (reviewed by Goldfarb and Michaud, 1991; Miller et al, 1991). Nuclear RNAs are exported from the same NPCs which hnport proteins, export is saturable and can be blocked by lectin. As with snRNA uptake, RNA export may require specific protein-RNA interactions occur before an RNA is exported. Significantly, it is becoming apparent that the processes of transcription, processing, export and translation are in some way linked. Curiously, transcriptional promoter dements have been shown to affect export (de la Pena and Zasloff, 1987; Braddock et al., 1990). Proper RNA processing is generally a prerequisite for RNA export (e.g. Tobian et al., 1983). In general, most mRNAs possessing introns are not exported until completion of the proper splicing. Several notable exceptions are seen in the course of HIV or HTLV infections where several late viral transcripts harboring introns are exported and translated due to transactivation by the r e v or r e x gene products, respectively (see Miller et al., 1991 for references). Chang and Sharp (1989) have further shown that a nonviral RNA which is not exported due to a mutation which prevents splicing can be exported under conditions where REV is present and able bind the mutated transcript. Finally, Goldfarb and Michaud (1991) have recently reviewed evidence which suggests that the nucleus may be able to assess the translatability of an mRNA and modulate its export accordingly. ANCHORAGE MEDIATED AVAILABILITY OF MACROMOLECULES FOR TRANSPORT
Possibly the most cormnonly used step in the regulation of transport is that which governs the availability of any competent transport substrate to the transport machinery. Anchorage mediated availability is one newly documented means for limiting the transport of many proteins which have regulated localizations in the cytoplasm (reviewed by Nigg, 198; Miller et al, 1991). Analogously, RNAs which are not properly processed similarly appear anchored or trapped within the processing pathways and are unavailable for export. Only if and when such trapped RNAs are released are such RNAs exportable. The nuclear matrix has been shown to be a significant nuclear "compaiament" where RNAs may be anchored (Schroder et al., 1987). INTRANOCLEAR TRAFHC PATI~RNS AND THE PRESENTATION OF TRANSPORTABLE MACROMOLECULES TO AND FROM THE NPC
It is becoming increasingly clear that the nucleus is highly ordered in space and time. Such organization has particular implications concerning the cellular and specifically
CeH Biology International Reports, Vol. 16, No. 8, 1992
795
intranuclear dissemination of m acromolecular information. Leppard and Shenk (1989) have demonstrated that an mRNA transcript passes fltrough several distinct biochemically defined compartments following its transcription to its point of export. Legrain and Rosbash (1989) showed that genetic approaches can track the decisions a transcript makes in following a particular pathway of processing and/or export. During normal HIV or HTLV infection, export of unspliced mRNAs requires nucleolar localization of REV and REX mediated by nuclear and nucleolar targeting signals (see Miller et al., 1991 for references). REX, in turn, localizes unspliced HTLV mRNAs to the nucleolus prior to export (Kalland et al., 1991). Recent evidence further suggests REV-mediated efflux of unspliced HIV mRNAs from isolated nuclei may be partially due to REV affects on a nuclear envelope NTPase involved in RNA efflux (Pfeifer et al., 1991). A critical question remaining to be answered is whether NPCs are physically linked to genomic transcription units and to what extent macromolecular traffic patterns are hardwired within the nucleus. Blobel (1985) has proposed that the nuclear components are highly ordered in space and time such that specific gene products are "gated" to specific NPCs for transport which, in turn, specifies their cytoplasmic destination (see Fig. 1). As gene expression is regulated, different genome-NPC relationships would be created.
tosubcellalar destination
transpor~"~ n I~= NLS
cytoplasm (Translation)
nuclear
envelope lamina subnuclear shuttle [ ~ ? / 0
track~RNA in~,, to nucleolus
an RNP
nucleus (Transcription)
Fig. 1. Macromolecular traffic through the NPC. Significantly, the majority of transcriptional sites tend to be located at the nuclear periphery (e.g. Hutchison and Weintraub, 1985; De Graaf et al., 1991). There is evidence for a reticular network of snRNPs comprising the splicing machinery which connect transcribed D N A with the nuclear periphery (Spector, 1990). Lawrence et al. (1989) have also shown that some mRNAs may be localized onto specific tracks linking internal transcription sites to the nuclear periphery. It is not clear whether these tracks are common and if RNA loaded on these tracks are exported.
796
CellBiology lnternational Reports, Vol. 16, No. 8, 1992
Whether a gating hypothesis is validated or not, it seems clear that the highly organized structures of the nucleus and cytoplasm will demand a highly efficient means for regulating the flow of macromolecular traffic. REFERENCES Adam S.A. and Gerace L. (1991) Cytosolic proteins that specifically bind nuclear location signals and receptors for nuclear import. Cell 66: 837-847. Akey C.W. (1990) Visualization of transport-related configurations of the nuclear pore transporter. Biophys. J. 58: 341-355. Akey C.W. (1989) Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy. Cell Biol. 109: 955-970. Berrios M., Fisher P.A. and Matz E.C. (1991) Localization of a myosin heavy chainlike polypeptide to Drosophila nuclear pore complexes. Proc. Natl. Acad. Sci. 88: 219-223. Blobel G. (1985) Gene gating: a hypothesis. Proc. Natl. Acad. Sci. 82: 8527-8529. Braddock M., Thorbum A.M., Chambers A., Elliott Q.D., Anderson G.J., Kingsman A.J. and Kingsman S.M. (1990) A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction. Cell 62:1123-1133. Burke B. and Gerace L. (1986) A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 44: 639-652. Chang D.D. and Sharp P.A. (1989) Regulation by HIV Rev depends upon recognition of splice sites. Cell 59: 789-795. Dabauvalle M.C., Loos K. and Scheer U. (1990) Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma 100: 56-66. Dabauvalle M.C., Loos K., Merkert H. and Scheer U. (1991) Spontaneous assembly of pore complex-containing membranes ("Annulate lamellae") in Xenopus egg extract in the absence of chromatin. J. Cell Biol. 112: 1073-1082. Dang C.V. and Lee W.M.F. (1989) Nuclear and nucleolar targeting sequences of cerb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J. Biol. Chem. 264: 18019-18023. Davis L.I. and Fink G.R. (1990) The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell 61: 965-978. De Graaf A., Van Hemert F., Linnemans W.A.M., Brakenhoff G.J., De Jong L., Van Renswoude J. and Van Driel R. (1990) Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells. Eur. J. Cell Biol. 52: 135-141. De la Pena P. and Zasloff M. (1987) Enhancement of mRNA transport by promotor elements. Cell 50: 613-619. Dingwall C. (1991) Transport across the nuclear envelope: enigmas and explanations. Bioessays 13: 213-218. Feldherr C.M. and Akin D. (1990) The permeability of the nuclear envelope in dividing and non-dividing cell cultures. J. Ceil Biol. 111: 1-8. Finlay D.R. and Forbes D.J. (1990) Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell 60: 17-29. Finlay D.R., Meier E., Bradley P., Horecka J. and Forbes D.J. (1991) A complex of
Cell Biology International Reports, VoL 16, No. 8, 1992
nuclear pore proteins
797
requiredfor pore function. J. Cell Biol. 114: 169-183.
Garcia-Bustos J.J., Heitman A. and Hall M.N. (1991) Nuclear protein localization. Biochim. Biophys. Acta 107: 83-101. Goldfarb D. and Michaud N. (1991) Pathways for the nuclear transport of proteins and RNAs. Trends Cell Biol. 1: 20-24. Greber U.F., Senior A. and Gerace L. (1990) A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J. 9: 1495-1502. Haaf T. and Schmid M. (1991 ) Chromosome topology in mammalian interphas~ nuclei. Exp. Cell Res. 192: 325-332. Hatanaka M. (1990) Discovery of the nucleolar targeting signal. Bioessays 12:143148. Hesketh J.E. and Pryme I.F. (1991) Interaction between mRNA, ribosomes and the cytoskeleton. Biochem. J. 277: 1-10. Hurt E. (1990) Targeting of a cytosolic protein to the nuclear periphery. J. Cell Biol. 111: 2829-2837. Hutchison N. and Weintraub H. (1985) Localization of DNAse I-sensitive sequences to specific regions of interphase nuclei. Cell 43: 471-482. KaUand K.H., Langhoff E., Bos J.J., Gottinger H. and Haseltine W.A. (1991) Rexdependent nucleolar accumulation of HTLV-1 rnRNAs. New Biol. 3: 389-397. Lawrence J.B., Singer R.H. and Marselle L.M. (1989) Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57: 493-502. Legrain P. and Rosbash M. (1989) Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57: 573-583. Leppard K.N. and Shenk T. (1989) The adenovirus EIB 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. EMBO J. 8: 2329-233..6. Lohka M.J. (1988) The reconstitution of nuclear envelopes in cell-free extracts. Cell Biol. Int. Rep. 12: 833-848. Nehrbass U., Kern H., Mutvei A., Horstmann H., Marshallsay B. and Hurt E.C. (1990) NSPI: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Ceil 61: 979-989. Newmeyer D.D. and Forbes D.J. (1988) Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Ceil 52: 641-653. Newport J. (1987) Nuclear reconstitution in vitro: stages of assembly around proteinfree DNA. Cell. 48: 205-217. Newport J., Wilson K.L. and Dunphy W.G. (1990) A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol. 111: 2247-2259. Nigg E.A. (1990) Mechanisms of signal transduction to the cell nucleus. Adv. Cancer Res. 55: 271-310. Nigg E.A., Baeuede P.A. and Luhrmann R. (1991) Nuclear import-export: in search of signals and mechanisms. Cell 66: 15-22. Paine P.L., Moore L.C. and Horowits S.B. (1975) Nuclear envelope permeability. Nature 254:109-114.
798
Cell Biology International Reports, VoL 16, No. 8, 1992
Pfeifer K., WeBer B.E., Ugarkovic D., Bachmann M., Schroder H.C. and Muller W.E. (199I) Evidence for a direct interaction of Rev protein with nuclear mRNA-translocation system. Eur. J. Biochem. 199: 53-64. Pinol-Roma S. and Dreyfuss G. (1991) Transcription-dependent and Transcriptionindependent nuclear transport of hnRNP proteins. Science 253:312-313. Reichelt R., Holzenburg A., Buhle E.L.J., Jamik M., Engel A. and Aebi U. (1990) Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore eomplex components. J. Cell Biol. 110: 883-894. Richardson W.D.A., Mills D., Dilworth S.M., Laskey R.A. and Dingwall C. (1988) Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52: 655664. Schroder H.C., Bachmarm M., Diehl B., Seifert A. and Muller W.E.G. (1987) Transport of mRNA from nucleus to cytoplasm. Progr. Nucl. Acid. Res. Mol. Biol. 34: 89-142. Sheehan M.A., Mills A.D., Sleeman A.M., Laskey R.A. and Blow J.J. (1988) Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J. Cell Biol. 106: 1-12. Spector D.L. (1990) Higher order nuclear organization: Three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc. Natl. Acad. Sci. 87: 147151. Starr C.M., D'Onofrio M., Park M.K. and Hanover J.A. (1990) Primary sequence and heterologous expression of nuclear pore glycoprotein p62. J. Cell Biol. 110: 1861-1871. Stewart M., Whytock S. and Mills A.D. (1990) Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes. J. Mol. Biol. 213: 575-582. Tobian J.A., Drinkard L. and Zasloff M. (1985). tRNA nuclear transport: defining the critical regions of human tRNAm'tl by point mutagenesis. Cell 43: 415-422. Wozniak R.W., Bartnik E. and Blobel G. (1989) Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J. Cell Biol. 108: 20832092. Zasloff M. (1983) tRNA transport from the nucleus in a eukaryotic cell: carriermediated translocation process. Proc. Natl. Acad. Sci. 80: 6436-644.
