Luận án tiến sĩ: Regulation of nuclear transport and mitosis by Ran GTPase

This RanGTP gradient is essential for many cellular processes, including nuclear transport during interphase, mitotic spindle formation, andnuclear envelope assembly during mitosis.. I s

Ting ChenChangsha, Hunan, P.R China

B.S., Xiamen University, 2001

A Dissertation presented to the Graduate Faculty of the University of Virginia in

Candidacy for the Degree of Doctor of Philosophy

Department of Microbiology

University of VirginiaJanuary, 2007

ma A

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One of the hallmarks of the eukaryotic cell is the possession of a nuclear envelope Transport of macromolecules between the nuclear and cytoplasmic compartments is mainly regulated by the small GTPase, Ran The nucleotide exchange factor RCC1 catalyzes formation of RanGTP, whereas the hydrolysis of RanGTP is stimulated by RanGAP1 and RanBPI Because RCC1 is chromatin-bound throughout the cell cycle while RanGAP1 and RanBPI are cytoplasmic, the concentration of RanGTP is high near the chromatin This RanGTP gradient is essential for many cellular processes, including nuclear transport during interphase, mitotic spindle formation, and

nuclear envelope assembly during mitosis

During the initial phase of my project I examined the nucleocytoplasmic shuttling

of an unusual protein called JAZ, which does not contain classical dsRNA binding domains but instead binds RNA with high affinity through C2H2 zinc fingers I showed that JAZ is a nuclear protein at steady state but is highly dynamic within the nucleus and undergoes nucleocytoplasmic shuttling JAZ associates with Exp-5 in the presence of RanGTP and a hairpin RNA, and nuclear export of JAZ requires Exp-5 However, JAZ also binds to ILF3, in an RNA-independent manner, and JAZ and ILF3 can form a heteromeric complex with Exp-5 and RanGTP Unlike ILF3, JAZ does not contain a classical NLS In principle it could diffuse passively through the nuclear pores, and | showed that import is indeed independent of soluble transport factors However, import

is inhibited by wheat germ agglutinin and by low temperatures, which do not inhibit

passive diffusion.

In the second phase of my project, I discovered a unique modification on RCC1,

in which the N-terminal Ser/Pro residue of mammalian RCC1 is methylated on its amino group A methyltransferase activity for N-terminal methylation of RCC1 is present in soluble nuclear extracts from HeLa cells Methylation-defective mutants of

œ-RCC1 are unable to bind as effectively as wild type protein to chromatin during mitosis, —

which results in supernumerary centrosomes and spindle formation defects These

defects are additive to those caused by a mutation that disrupts exchange activity, and

may result from decreased binding to DNA Coupling RCC1 to histone H2A, to force chromatin attachment, reverses the mitotic defects.

This dissertation is dedicated to my mom Guihua Li and my husband Mingda Hang.Without their constant love and support, this dissertation would never have been possible

First, I owe my deepest thanks to my dissertation mentor, lan Macara lan's enthusiasm for science, breadth of knowledge, willingness to share his time, and high stands of scientific merit made great impact upon my graduate study This work would not have been possible without his guidance and support.

I also thank all members of my dissertation committee— Ann Beyer, Mitch Smith,

Lucy Pemberton, Marty Mayo, and Lou Hammarskjold Their advice was always helpful, and they were extremely supportive throughout my graduate studies.

I’d like to thank all members of the Macara laboratory, past and present, for their support to my graduate study In particular, I thank: Amy Brownawell, Kendra Plafker,

Scott Plafker, Quansheng Du, Lin Gao, Greg Riddick, Christine Schaner-Tooley, Yi Qin,Julia Dorfman, Claudia Low, Brandon Kremer, Chris Capaldo, Huaye Zhang, XinyuChen, and Jim Crawford I hope that they enjoyed our time together as much as I did.

I am grateful to many individuals within the Center for.Cell Signaling for their help: Dr Brautigan, Dr Pemberton, and Dr Lannigan I also thank Mary Beth, Patricia

Arkhurst, Drew Thomas, Flora Terry, and Karen Neale for their daily assistance in the

Center for Cell Signaling I would like to thank Martha Campbell and Sandy Weirich for

their kindness and help throughout my study within the Microbiology Department.

Finally, I would like to thank my family for all their love and support Without them this would not be worthwhile.

ABSTRACT | I

DEDICATION m

ACKNOWLEDGEMENTS IV

TABLE OF CONTENTS M LIST OE FIGURES VII

LIST OF ABBREVIATIONS : xX

CHAPTER I GENERAL INTRODUCTION 1

RANGTP AND NUCLEAR TRANSPORT 0 HH TH nh ng TH ng Tu HT TT TH ng g0 2 The Nuclear Localization SiBTIQÌ «cty TH HH TH TT 1T 016 KT 4 The Nuclear Pore COTHDÏ6X Ăn HH HH HH Hà Hà 112111112110 H0 tre 8

The Nuclear Transport ReC€jÍOFS LH HH HH HH HH HH 01H HH 13

ý, 8 8, 0) 2.86 nh nh 20

;£ì64850.))00 i009)000070707Ẻ8Ẻ 78 25 Detection of RanGTP Gradients on Mitotic Chromosomes in vitro and in VÌVO c.c 26

Function Of Ran in Spindle FOTiidfiOH ch HH ng HH nh 28

Function of Ran in Centrosome FOTI[ÍO cà ch HH HH HT HH HT Là HH 32 102p vo 34

CHAPTERIHL THE NUCLEOCYTOPLASMIC SHUTTLING OF JAZ 36

.):xy: tan a 37 INTRODUCTION 000007070777 38 TRESULTT (5G <4 H91 T9 10T Ầ TT HT TT T9 040041 1813 T4 E04 900.00 4106 41 JAZ is a nucleocytoplasmic shuttling DFOÍGH ch HH HH ng HH r 41

Nuclear export of JAZ is dependent on Exp-5 in both digitonin-permeabilized cells and intact cells 44

JAZ, ILF3 and Exp-5 can form heterotrimeric COTIDÍGX Ăn HH HH ghg 62 JAZ is imported into the nucleus by a nonclassical meCh@HÌSTH cành re, 65

DISCUSSION 00105777 74

MATERIALS AND METHODS HH HT Họ ch TL HT 0 016 2H 0.0301 11m 76 Cloning, antibody and recombinant protein €XDT€SSỈOH, à s cnhnHnnHnH Hà H Hg 76 Yeast dihybrid and conjugation (5S/3 Gà LH nu HH TH TH g2 77 Cell culture, transfection, and pFOCGSSÍH ch HH1 2 1 HH HH rệt 77 RNA binding assays ceesnatatntiatsteetnene ¬— 78

2 2.2.827,.11)-0 2 200 nẼnBẼn0Ẽn0nẺ000 0n .e.e 79

MICrOINJCCEION EEEEEEEETE Sổ 0n CỐ ca na cố cố nan 80

FateroKar yon fusiOn ASSAYS 0000000808080 060 nh ố ố 81

Permeabilized cell transport GSSÿ ST HH HH HH HH TH Tà HH Hi TH 001011 ä] CHAPTER II N-TERMINAL œ-AMINO METHYLATION OF RCCI 83 9): am na 84

I6ÿ990/ x99) 10017Ẽ212n78 §5

RESULT (HH TH TH TH TH TT HT TT T1 Tre nhẾ ng re 89 Mammalian RCC] is N-terminally mefHyÏqfed.L «teen k1 1g ri 89 N-terminal methylation motif o.c.cccccccccccccccsssscsseccesecsscessseeveseseseseesensseceseseseesecesseqesneasatseeeseqsetesssesseaseensaseens 94 N-terminal methyltransferase đC[[VỈDJ ánh HH TH KH H0 11111101 HH H110 0 rau 98 Anti-d-N-me2/anti-œ-N-me2/3Ser2-RCC1 antibOdÌlớg ng rong 101 N-terminal methylation facilitates RCC1 interaction with chrOmdfÏH cccceieeierierree 104 N-terminal methylation on RCC] is necessary for normal THÍÍOSÏS cv LH HH xe, Ill N-terminal methylation likely facilitates RCC1 bind to DÌNÀ ch HH1 re 121 N-terminal tail of RCC1 alone binds chromatin in a N-terminal methylation dependent manner 124

DISCUSSION 0000101057 127

)UP902:3/.98-0.9)008 0290:9910 130 Constructs And aHfÌPỌÌð$ HH TH TH HT 01111 HH go 130

Mass spectrometric analysis of RCC] modjficafiOH các sọc HH 11211212 rrea 13]

Tmmunofluorescence THỈCFOSCODD Ăn Là HH1 TT HH Ho ko TH TH Tà HỘ kg nh HH 132

In vitro methylation ASSQV ecccccsecccssssesessscieveseseiesesesececssnsesssecensaeneseneseucsdedeaeseneneesetecssusaaraneteresetenssberaees 133 Live cell imaging, fluorescence recovery after photobleaching, and fluorescence loss in

PHOLODL CACHING oo eececscsssscscesesesenssessssesensscsneseaseasacsassscsaesssesesenseneaensecesseceuenssaeeesaeiauceasaanecaesasaeeteeseaeneeasaaeees 133

CHAPTER IV GENERAL DISCUSSION 135

JAZ AND DSRNA BINDING PROTEINS 010777 136 RCC1 AS A CHROMATIN MARKER \ cccssccsssssssscecesseccsssssncesesssesesaccesecsssasesseseseessceesscseeaesacecesssssseeseseeesteasstease 141

N-TERMINAL METHYLATION 2002127 146 REFERENCE 149

List of Figures

FIGURE 1.1 NUCLEAR TRANSPORT SIGNALS .scsccsssssssseccsscsscsssceesscescssecsseaeesesnssssssncesseasasesneceseeeseaseassneensenees 6 FIGURE 1.2 SCHEMATIC REPRESENTATION OF THE NUCLEAR PORE COMPLEX csscsssssccesessesceeacenstecsseesneeess 11 FIGURE 1.3 A MODEL OF NUCLEAR IMPORT PATHWAY Q.1 110110901 vn ng ng ng ng 011012589 16 FIGURE 1.4 A MODEL OF NUCLEAR EXPORT PATHWAY ong ng HH T4 Hàng 0101 ke 18 FIGURE 1.5 THE GUANINE NUCLEOTIDE CYCLE OF THE RAN OTPASE Q.90 101 tt H14 1 re23 FIGURE 1.6 A MODEL OF HOW THE RANGTP GRADIENT REGULATES SPINDLE FORMATION, 30 FIGURE 2.1 JAZ 1S ANUCLEOCYTOPLASMIC SHUTTLING PROTEIN - G01 SH HH1 1n g0 1 11c42 FIGURE2.2 EXPORTIN-5 BINDS TO JAZ AND STIMULATES JAZ SHUTTLING IN INTACT CELLS - 45 FIGURE 2.3 EXPORTIN-5 IS REQUIRED FOR JAZ SHUTTLING IN INTACT CELLS 55 2< 5< srse 48 FIGURE 2.4 EXP-5 MEDIATES EXPORT OF JAZ IN DIGITONIN-PERMEABILIZED CELLS ccscssssssessesneeseenssetsats 31 FIGURE2.5 JAZ IS A BINDING PARTNER OF ILLE Q0 911 1 TH HH ng ng 44 15 T1 KH 1401 14 35 FIGURE2.6 JAZ INTERACTS WITH THE DSRNA BINDING DOMAINS (DSRBDS) OF ILF3 - 57

FIGURE 2.7 JAZ BINDS DIRECTLY TO ILF3, INDEPENDENTLY OF DSRNA Ăn n4, HH re, 60

FIGURE 2.8 JAZ CAN INTERACT WITH ILF3 AND EXP-5 SIMULTANEOUSLY ccccscscssesessesesstsscseesrerssesereseesees 63

FIGURE 2.9 CELLULAR LOCALIZATION OF DIFFERENT TRUNCATIONS OF JÁ Z Án, 66FIGURE 2.10 NUCLEAR IMPORT OF JAZ ccccccscsscsssssessesseeccesseaesnesseeans secseteseeeee 69

FIGURE 2.11 JAZ IS IMPORTED INTO NUCLEI BY A NON-CLASSICAL PATHWAY Gà HH HH 1418111 72 FIGURE 3.1 REPRESENTATION OF THE INTERACTION BETWEEN RCC1 AND CHROMATTM << 87 FIGURE 3.2 RCCI N-TERMINAL METHYLATION HH TH TH TT TH TT 1304001140 90 FIGURE 3.3 RCC1 N-TERMINAL PHOSPHORYLA TION con HH H491 1g TH HH0 011031010314 1010101 1x9 92 FIGURE 3.4 IDENTIFICATION OF N-TERMINAL METHYLATION MOTIF - Ă Ác 1111 199 4111111101181 1x etxez 96 FIGURE 3.5 IDENTIFICATION OF œ-N-TERMINAL METHYLTRANSFERASE ACTIVITY ào Q HT ST se,99 FIGURE 3.6 DETECTION OF ENDOGENOUS RCCI N-TERMINAL METHYLATION 5505555 <<<<csxsss 102 FIGURE 3.7 METHYLATION OF RCC] REGULATES ITS INTERACTION WITH CHROMOSOMES 106

FIGURE 3.8 METHYLATION OF RCC1 REGULATES ITS INTERACTION WITH CHROMOSOMES IN LIVE CELLS 109

FIGURE 3.9 METHYLATION OF RCCI IS REQUIRED FOR CORRECT SPINDLE ASSEMBLY AND CHROMOSOME SEGREGATHION c9 ng 4g 0 0 4g 0 000918000180 0111 8807018 00001014018002400180100700100000186 113 FIGURE 3.10 ‘TIME-LAPSE IMAGES OF NORMAL AND DEFECTIVE MITOSIS .ccsscsecsssessesceeessaseesessesssesenessescanes 115 TABLE 1, QUANTIFICATION OF MITOTIC DEFECTS IN TRANSFECTED MDCK CELLS .ccscssssesssssessseseesecssssseees 117 FIGURE 3.11 TETHER RCC1 TO CHROMATIN BY FUSION TO HISTONE H2A vn, 119 FIGURE 3.12 METHYLATION OF RCC] FACILITATES ITS INTERACTION WITH DNA «- 122

FIGURE 3.13 METHYLATION PROMOTES BINDING OF THE RCC1 TAIL TO MITOTIC CHROMOSOMES IN LIVING

UNFIXED MDCK CELLS ssscccccccsssssssssssssssssssssccsseseesecessessssssssssssssevavesessssesssasssssuvesssceessanssvessccesssssssssasessssen 125 FIGURE 3.14 MODEL FOR RCC1 INTERACTION WITH NUCLEOSOME HH H010 110 n0 ko 128

Bovine-Serum Albumin Cysteine

Aspartate Dalton 4’ ,6-diamidino-2-phenylindole 3,3’-dihexyloxacarbocyanine

Dulbecco’s Modified Eagle Medium

Deoxyribonucleic Acid Dithiothreitol

Glutamate Ethylenediaminetetraacetic Acid Phenylalanine

Fluoroscein Isothiocyanate Glycine

Guanosince 5’-Diphosphate

Guanosine 5’-Triphosphate

Histidine

Nhydroxyethyl)piperazine-N’

-(2-Six-Histidine Tag; Sequence: HHHHHH

Heterogeneuos Nuclear Ribonucleoparticle

Isoleucine Immunoglobulin-y

Isopropylthio-B-D-galactoside

Lysine

Potassium Chloride hnRNP K Nuclear Shuttling Signal Potassium Acetate

kilo-Dissociation Rate Constant

Methionne Megadalton

Mitogen-Activated Protein Kinase

Methanol Mitogen-Activated Protein Kinase KinaseMagnesium Chloride

Nuclear Factor of Activated T-Cells

Nuclear Localization Signal Nuclear Pore Complex Nucleoplasmin Core Domain

Ras-like Nuclear Protein

Ran-Binding Protein 1, 2, or 3

Ran-Binding DomainRegulator of Chromosome Condensation 1Rabbit Reticulcyte Lysate

Ribonucleic Acid

Ribonucleoparticle

Ribosomal RNA Serine

Sodium Dodecyl Sulfate

Tris(hydroxymethyl)aminomethaneTransfer RNA

Triton X-100 Unit

Valine Tryptophan Wheat Germ Agglutinin Wild-Type

TyrosineIgG-Binding Domain from Protein A

One of the defining features of a eukaryotic cell is the possession of a nuclearenvelope The nuclear envelope is a continuous double membrane structure that spatially

and temporally separates DNA replication and transcription from protein synthesis This

separation provides a high level of regulation but at the same time demands a system of

regulated transportation of macromolecules between the cytoplasm and the nucleus The

complexity and magnitude of nucleocytoplasmic transport is remarkable Rough

estimation suggests 1 million macromolecules traffic cross the nuclear envelope each

minute (Gorlich and Mattaj, 1996) Nuclear proteins, such as histones, transcriptionfactors, and polymerases, must be imported into the nucleus after synthesis in thecytoplasm In contrast, mRNA, tRNA, rRNA, microRNA and ribosomal subunits need

be exported into the cytoplasm to function in translation or mature into functional roles.

Besides these fundamental functions, nuclear transport also plays a crucial role in

regulating the cellular activity of many proteins For example, the tumor suppressor

protein p53 is retained in the nucleus by tetramerization, which mask its nuclear export

signal (NES) (Stommel et al., 1999) As another example, upon activation thephosphatase calcineurin promotes import of the transcription factor NF-AT into thenucleus by dephosphorylating and unmasking a nuclear localization signal (NLS) in NF-

AT (Zhu et al., 1998) At the same time calcineurin also blocks nuclear export by binding to a NES in NF-AT (Zhu and McKeon, 1999) Nuclear accumulation of the transcription factor Yap1p under oxidative stress is triggered by inhibition of the binding between Yaplp and the export receptor Crm1, by oxidation of one of the three cysteine

example, nuclear accumulation of CyclinB/cdc2 at the beginning of mitosis is induced by

phosphorylation of serines in the vicinity of the NES of cyclin B that decreases itsaffinity for the export receptor Crml (Yang et al, 2000) In summary,nucleocytoplasmic transport is fundamental to eukaryotic cells, and regulating the traffic

in response to intra- and extra-cellular cues is a pivotal task for a eukaryotic cell

The translocation of proteins into and out of the nucleus is a signal-mediatedprocess Protein sequences that function as import or export signals are termed nuclearlocalization signal (NLS) or nuclear export signal (NES) respectively.

The first NLS was identified in the simian virus 40(SV40) large-T antigen(Dingwall et al., 1982; Kalderon et al., 1984; Lanford and Butel, 1984) This sequence,PKKKRK, and the NLS found in nucleoplasmin, KRPAATKKAGQAKKKKLD (Robbins

et al., 1991), are the prototypes of the classical monopartite and bipartite NLS (Figure1.1) They are enriched in basic amino acids and are sufficient to trigger nuclear

localization of non-transported proteins Both monopartite and bipartite NLS are known

to be present in many different proteins (Macara, 2001) Other functional but classical NLSs have also been discovered These protein sequences differ from the

non-classic NLSs, but often possess many basic amino acids For example the basic NLSs

found in viral proteins Rev and Rex are composed of basic, arginine-rich stretches

(Palmeri and Malim, 1999) (Figure 1.1) More recently it was discovered the NLSs imported by transportin (Karyopherin B 2) encompass large (>-30-residue) elements with overall basic residues, and contain consensus motifs including a central hydrophobic or basic motif followed by a C-terminal R/K/HX » 3, PY motif (Lee et al., 2006) However not all NLSs are simple protein sequences containing basic residues For example, the NLS of uridine-rich small nuclear ribonucleoproteins (U snRNPs) comprises both the m3G cap on the RNA of the U snRNP and the protein sequences within the Sm core

protein of the RNP, which together are recognized by adapter protein called snurportin(Huber et al., 1998; Palacios et al., 1997) (Figure 1.1).

The consensus sequence for NESs is less well specified than the classical NLS.The prototypical NES (LxxxLxxLxL) was deduced from sequences in the humanimmunodeficiency virus protein Rev (Fischer et al., 1995) and the PKA-inhibitor (PKI)(Wen et al., 1995), and defined by sequence comparison and mutational analysis (Bogerd

et al., 1996) (Figure 1.1) This signal is enriched in leucine residues separated by shortstretches of other residues, but other hydrophobic residues can substitute for several ofthe Leu residues, and the number of intervening residues is also variable (Bogerd et al.,1996) Other functional NESs that do not match this consensus also have beendiscovered, such as the NES in the NFAT transcription factor (Klemm et al., 1997)

Sequences that are able to confer both nuclear protein import and export have

been identified For example the M9 domain of heterogeneous nuclear ribonucleoprotein

Al (hnRNP Al) is glycine and asparagines rich, and mediates both nuclear import and

export and is therefore termed a “shuttling” signal (Siomi and Dreyfuss, 1995) Its

nuclear import is mediated by a nuclear transport receptor called transportin (Pollard etal., 1996) Another example of a shuttling signal is the KNS domain of hnRNP K(Michael et al., 1997) (Figure 1.1) In general, analysis of these sequences is lessdissected compared to those via the classic NLS and NES

Examples of classical and non-classical nuclear localization signals (NLSs), leucine-rich

nuclear export signals (NESs), and two protein sequences that confer both nuclear import and export (shuttling signals) The protein containing each protein sequence is indicated All residues are given in single-letter code and crucial amino acids are in bold.

Nuclear Export Signal (NES)

HIV Rev L~PPLERLTL

PKI LALKLAGLDI

Shuttling Signals

hnRNPA1 (M9) NGSSNFGPMKGGNFGGRSSGPY hnRNPK (K) YDRRGRPGDRYDGMVGFSADETWD

SAIDTWSPSEWQMAY

The Nuclear Pore Complex (NPC) forms the pore across the nuclear envelope(NE) through which bidirectional nucleocytoplasmic transport occurs On closer inspection, NPCs are large protein assemblies that form aqueous channels at sites where the inner and outer nuclear membranes fuse (Figure 1.2) The number of NPCs per nucleus varies from ~1.9 x 10 in the budding yeast Saccharomyces cerevisiae (Rout and

Blobel, 1993) to ~5 x 10’ in the oocyte from Xenopus laevis (Cordes et al., 1995).

Overall, the NPC forms a barrel-like eight-fold symmetric framework embracing a

central pore; it is ~90 nm long and is narrowest (~30 nm) at the NPC’s midplane (Allen et al., 2000; Beck et al., 2004; Doye and Hurt, 1997; Fabre and Hurt, 1997; Pante and Aebi, 1996: Stoffler et al., 2003) The NPC can be subdivided into three sections: thecytoplasmic fibrils: including a cytoplasmic ring and eight ~50 nm long filaments

protrude from the ring into the cytosol; the nuclear basket: including a nuclear ring and a

basket structure attached to the ring; and the central core: composed of a radial

framework of eight spokes that is sandwiched between the cytoplasmic and the

nucleoplasmic ring (Stoffler et al., 2003)

At the molecular level, NPCs from both yeast and vertebrates are composedsimilarly of ~30 different proteins called nucleoporins (or Nups) (Cronshaw et al., 2002;Rout et al., 2000) Functionally, Nups can be divided into three major groups The firstgroup is a set of membrane proteins, called poms, which anchor the NPC into the NE.Members of the second group are mostly structural proteins These proteins form thecentral tube and provide a framework for the positioning of the third group of Nups

across both faces of the NPC This third group provides binding sites for transportfactors They are a related group of proteins, collectively called FG Nups because theycontain multiple copies of a Phe-Gly motif separated by hydrophilic linkers (Rout et al.,2003; Suntharalingam and Wente, 2003) With ~200 copies of FG Nups found in eachNPC, they comprise nearly half the mass of NPC (Rout et al., 2000).

The FG repeats are recognized by nuclear transport receptors and are believed tofacilitate their translocation across the NPC (Bayliss et al., 2000; Bayliss et al., 1999;

Fornerod et al., 1997b; Iovine and Wente, 1997; Marelli et al., 1998; Seedorf et al., 1999;

Shah and Forbes, 1998; Shah et al., 1998; Stochaj et al., 1998; Yaseen and Blobel, 1997).More recently, it was discovered that the linker regions between the FG repeats are alsocrucial to the affinity of the between nucleoporin and Kap95p by promoting simultaneousbinding of several FG-cores to Kap95p and hence influence the strength of binding (Liuand Stewart, 2005) The precise mechanism by which the cargo-receptor complex is

translocated through the NPC is still largely unknown But different translocation models

have been proposed: 1 affinity gradient model - transport complex driven by Brownian motion “steps” along FG Nups with increasing affinity for the receptor (Ben-Efraim and Gerace, 2001) 2 Brownian affinity gating model — cargos that can interact with NPCthrough receptors has a higher probability of entering and traversing the NPC thanproteins that don’t (Rout et al., 2000) 3 Selective phase model — FG repeats form ameshwork through weak hydrophobic interaction with each other and this meshworkforms a physical barrier only allow cargos that can incorporate into this meshwork to pass(Ribbeck and Gorlich, 2001; Ribbeck and Gorlich, 2002) 4 oily-spaghetti model — the

NPC is an open structure with flexible FG-containing nucleoporins coating the wall ofthis channel and providing binding sites for the passage of nuclear transport receptors.Transport receptors can push aside the loose nucleoporin chains during passage.Transient association with FG repeats and random motion would achieve translocation(Macara, 2001) Each model has its pros and cons None of them can provide acompletely satisfying molecular mechanism for nucleocytoplasmic transport But for all

of them, the ability of the transport receptors to interact with Nups plays an essential role

in translocation.

A subset of nucleoporins has been shown to be post-translationally modified(Gerace et al., 1982; Greber et al., 1990; Wozniak et al., 1989) In particular, somenucleoporins possess O-linked N-acetylglucosamine moieties (GlcNAc) (Davis andBlobel, 1987; Holt et al., 1987; Snow et al., 1987) Wheat-germ agglutinin (WGA), a

molecule that binds tightly to GlcNAc, can associate with the NPC and potently inhibit

nuclear transport (Dabauvalle et al., 1988; Finlay et al., | 1987) In addition,

nucleocytoplasmic transport can be arrested at 4°C (Pante and Aebi, 1996; Pante et al.,1997) It appears that there is a direct correlation between the temperature state of theNPCs and the existence of a plug-like material obstructing the central pore The plug-like material at the central pores probably represents cargo arrested in a transit state ratherthan a fixed and stationary component of the NPC (Stoffler et al., 2003)

Figure 1.2 Schematic representation of the Nuclear Pore Complex.

The main structural components of the NPC including the cytoplasmic ring, thecytoplasmic filaments, the central framework, the nuclear ring and the nuclear basket are

shown as seen from along the plane of the nuclear envelope.

The Nuclear Transport Receptors

Nuclear transport receptors are defined by their ability to interact directly withFG-containing nucleoporins, thereby facilitating the passage of cargo molecules throughthe NPC The largest class of nuclear transport receptors is the superfamily of importin-related proteins, sometimes referred to as the karyopherin family They are divided aseither importins or exportins depending on whether they are involved in nuclear import or

export (Macara, 2001) There are 14 members of this family in yeast and more than 20 in

higher eukaryotes (Fried and Kutay, 2003) Besides their ability to bind to nucleoporins,

nuclear transport receptors can form complexes with the GTP-bound state of the RanGTPase (Macara, 2001).

The first member of the karyopherin family to be identified was importin-B (alsocalled p97, karyopherin-b, Kap95, and PTAC 95) (Chi et al., 1995; Enenkel et al., 1995;Gorlich et al., 1995a; Radu et al., 1995) Importin-B translocates NLS-bearing proteins

using an adapter protein called importin-a (also called karyopherin-a, Srplp, p56, PFAC

38, and pendulin) (Adam and Gerace, 1991, Cortes et al., 1994; Cuomo et al., 1994;Gorlich et al., 1994; Kussel and Frasch, 1995; Moroianu et al., 1995; Weis et al., 1995).Importin-œ recognizes NLSs in cargo proteins, while importin-B interacts withnucleoporins and facilitates translocation of the trimetric NLS-importin-œ-importin-Bcomplex through the NPC (Gorlich et al., 1995a; Gorlich et al., 1995b; Imamoto et al.,1995) Once on the nuclear side of the NPC, RanGTP binds to importin-B and releasesimportin-œ-cargo complex Then the NLS-bearing cargo is displaced from importin-œ anexportin called CAS, the nuclear export receptor of importin-œ, and RanGTP (Kutay et

al., 1997) (Figure 1.3) There is only one importin-œ gene in the genome of the S.cerevisiae (Kussel and Frasch, 1995), but at least six importin-a isoforms in mammals(Malik et al., 1997).

Fewer exportins have been identified relative to importins The best-definednuclear export receptor is Crm1 (Fornerod et al., 1997a; Fornerod et al., 1997b; Gorlich

et al., 1997) Contrary to importins, Crm1 requires RanGTP in order to associate with

NES-bearing cargo (Fornerod et al., 1997a; Fukuda et al., 1997; Ossarehnazari et al.,

1997; Stade et al., 1997) Once the trimetric Crm1-NES-RanGTP complex reaches the

cytoplasm, RanBPi and RanGAP hydrolyze RanGTP and the complex is dissociated(Askjaer et al., 1999) (Figure 1.4) The study of Crm1 has been greatly aided by the use

of leptomycin B At nanomolar concentrations, leptomycin B has been shown to bindirreversibly to a cysteine residue in Crm1 which then prevents the binding of both NESand RanGTP (Kudo et al., 1999; Kudo et al., 1998; Neville and Rosbash, 1999).Leptomycin B inhibits specifically Crm1 but not other nuclear export receptors, such ascalreticulin (Holaska et al., 2001) and exportin-5 (Brownawell and Macara, 2002)

Other karyopherin family exportins have been described Exportin-t mediatesexport of transfer RNA (tRNA) (Hellmuth et al., 1998; Kutay et al., 1998) Exportin-4mediates export of eI[F5A (Lipowsky et al., 2000) Exportin-5 is an export receptor with

a broad spectrum of substrates It has been shown to mediate the nuclear export ofseveral RNA binding proteins and can also act as a minor export factor for transfer RNAs(tRNAs) (Bohnsack et al., 2002; Brownawell and Macara, 2002; Calado et al., 2002;Chen et al., 2004; Gwizdek et al., 2003) However, the major function of ExpŠ5 seems to

be the nuclear export of small structured RNAs such pre-miRNAs (Bohnsack et al.,2004; Lund et al., 2004; Yi et al., 2003), and the human Y1 and adenovirus VAl RNA(Gwizdek et al., 2001; Gwizdek et al., 2003; Rutjes et al., 2001) (Figure 1.4).

\

Figure 1.3 A model of Nuclear Import Pathway.

This model indicates that Ran drives net transport of import cargo against theirconcentration gradients by controlling the loading and unloading of cargo in acompartment specific manner Briefly cargo-importin œ-importin 6B complex isdissociated by RanGTP in the nucleus Importin œ and importin B recycles back tocytoplasm by CAS or by itself respectively

This model indicates that Ran drives net transport of export cargo against theirconcentration gradients by controlling the loading and unloading of cargo in acompartment specific manner Briefly cargo-Crm1/Exportin 5-RanGTP complex isdissociated by RanGTP hydrolysis via RanGAP and RanBPI in the cytoplasm.

The Ran GTPase system

The small GTPase Ran regulates most of the known karyopherin-mediatednucleocytoplasmic transport Ran is a highly abundant, ~25 kDa protein that is ~90% nuclear at steady state (Bischoff and Ponstingl, 1991) However, it constantly shuttlesbetween the nucleus and cytoplasm (Smith et al., 1998b) Like all GTPases, Ran cyclesbetween GTP- and GDP-bound states Unlike other small GTPases, Ran has only one

known guanine nucleotide exchange factor (GEF), RCC1 (Bischoff and Ponstingl, 1991),

and only one known GTPase-activating protein, RanGAP (Bischoff et al., 1994).

RanGEF RCCI functions by stimulating dissociation of Ran-bound nucleotide(either GDP or GTP) and stabilizes the nucleotide-free form of Ran (Amberg et al., 1993;Bischoff and Ponstingl, 1991; Kadowaki et al., 1993; Klebe et al., 1995a; Klebe et al.,1995b) RCCI is maintained exclusively in the nucleus by efficient nuclear import, aswell as strong and direct interaction with chromatin (Nemergut et al., 2001; Ohtsubo et al., 1989) (Figure 1.5) RCC1 is a mobile enzyme that it can cycle on and off the

chromosome (Cushman et al., 2004; Li et al., 2003; Moore et al., 2002)

RanGTP hydrolysis is stimulated by RanGAP, which increases the low intrinsic rate of RanGTP hydrolysis by five orders of magnitude (Bischoff et al., 1994; Klebe et al., 1995a) RanGAP localizes in the cytoplasm or the cytoplasmic face of the NPC(Hopper et al., 1990; Mahajan et al., 1997; Matunis et al., 1996; Saitoh et al., 1998).RanGTP is translocated to the cytoplasm by karyopherins The binding of karyopherinsand RanGAP to RanGTP is mutually exclusive (Bischoff and Gorlich, 1997; Bischoff etal., 2002; Villa Braslavsky et al., 2000) To make RanGTP accessible to RanGAP,

members of another family of Ran-binding proteins (RanBPs), including RanBP1 and RanBP2/Nup358, promotes dissociation of RanGTP from karyopherins (Bischoff et al.,

2002) (Figure 1.5)

The physical separation of RanGEF, RCC1, and RanGAP creates a steep RanGTPgradient across the nuclear envelope — a high level of RanGTP in the nucleus and a high

level of RanGDP in the cytoplasm (Figure 1.5) This gradient of RanGTP across the

nuclear envelope is believed to be pivotal for most forms of nuclear transport (Gorlich

and Kutay, 1999; Macara, 2001; Mattaj and Englmeier, 1998; Melchior and Gerace,

1998; Pemberton et al., 1998)

The function of Ran in nuclear transport is to regulate receptor-cargo interactions

in a compartment-specific manner, and therefore provides directionality for nucleartransport (Figure 1.4) Biochemically, RanGTP’s interactions with importins or exportins

lead to different consequences Importin-cargo complexes can only be assembled in the

absence of RanGTP; hence this is accomplished in the cytoplasm where RanGTP

concentration is low (Chi and Adam, 1997; Gorlich et al., 1996; Rexach and Blobel,

1995) Once the importin-cargo complexes reach the nucleus, where there is a highconcentration of RanGTP, the binding of RanGTP to the import receptor causesdisassembly of the importin-cargo complex, thereby unloading the cargo into the nucleus(Gorlich et al., 1996; Moore and Blobel, 1993) Formation of exportin-cargo complexonly occurs in the presence of RanGTP; hence this occurs in the nucleus where theconcentration of RanGTP is high (Fornerod et al., 1997a; Fukuda et al., 1997;Ossarehnazari et al., 1997; Stade et al., 1997) Once this complex reaches the cytoplasm

it encounters a high concentration of RanGAP The hydrolysis of RanGTP byRanGAP causes disassembly of the export complex, thereby releasing the exportsubstrate into the cytoplasm (Askjaer et al., 1999; Black et al., 2001; Black et al., 1999;Katahira et al., 1999; Kehlenbach et al., 1999) The return of Ran from cytoplasm tonucleus is mediated by another transport factor, NIT'F2 (Ribbeck et al., 1998; Smith et al.,1998a) (Figure 1.5).

Figure 1.5 The guanine nucleotide cycle of the Ran GTPase.

In the nucleus, RanGEF, RCC1, promotes the exchange of nucleotide to generateRanGTP In the cytoplasm, RanGAP stimulates hydrolysis of RanGTP to RanGDP withthe help of RanBP1 RanGTP binds to karyopherin in the nucleus and translocates withthem to the cytoplasm, where it is hydrolyzed to RanGDP Then RanGDP is recycledback into nucleus via NTF2.

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