Nuclear location signal-mediated protein transport

Biochimica et Biophysica Acta, 1008 (1989) 263-280 263 Elsevier

BBAEXP 91980

Review

Nuclear location signal-mediated protein transport

Bruce Roberts Integrated Genetics, Framingham, MA (U.S.A.)

(Received 22 May 1989)

Key words: Nuclear location signal; Signal sequence; Nuclear protein localization; Nuclear pore; Active transport

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

II. Structure of the nuclear envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

IlL Compartmentalization of nuclear proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

IV. Identification of nuclear location signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 A. SV40 large-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 B. Yeast a 2 mating factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 C. Papovavirus capsid proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 D. Human iamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 E. Yeast histone H2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 F. Adenovirus EIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

V. Proteins with more than one nuclear signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 A. Polyomavirus large-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 B. Human c.myc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 C. Rat glucocorticoid receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

VI. Complex nuclear location determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27i A. Nucieoplasmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 B. N l / l q 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 C. Yeast GALA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

VII. Structural features of nuclear signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

VIII. Features of sigr~-mediated nuclear protein import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 A. Route of signal-mediated import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 B. Effect of nuclear signal number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 C. Unidirectionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 D. Energy dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 E. Multi-step nature of signal-mediated nuclear protein import . . . . . . . . . . . . . . . . . . . . . . . . . 275

IX. Mechanism of nuclear signal-mediated protein uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 A. Temperature dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 B. Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 C. Competition and saturability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

Abbreviations: BG, ~-8alactosidase; BSA, bovine serum albumin; DNA, deoxyribonucleic acid; GK, galactokinase; HSA, human serum albumin; PK, pyruvate kinase; WGA, wheat-germ agglutinin.

Correspondence: B. Roberts, Integrated Genetics, 1 Mountain Road, Framingham, MA 01701, U.S.A.

0167-4781/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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X. Identification of cellular factors involved in nuclear protein uptake . . . . . . . . . . . . . . . . . . . . . . . 277 A. Inhibition of nuclear protein import by antibodies and WGA . . . . . . . . . . . . . . . . . . . . . . . . 277 B. Identification of putative nuclear signal receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 C. Other proteins potentially involved in nucl~z protein uptake . . . . . . . . . . . . . . . . . . . . . . . . 278

XI. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

!. Introduction II. Structure of the nuclear envelope

Nuclear protein transport has been the subject of considerable attexition as of late. In part, this is due to the desire of researchers to contribute towards our knowledge of how proteins are sorted within eukaryotic cells. However, an understanding of the mechanisms by which individual viral or cellular proteins are targeted to the cell nucleus allows one to devise means to perturb their subcellular distributions. By studying the biologi- cal properties of cytoplasmic variants of otherwise nuclear proteins, insights into complex processes such as cellular transformation [1-4] and viral capsid assembly [5,6] have been gained. Hence, an understanding of nuclear protein transport has provided the basis for a variety of other studies.

Progress has been made in the area of the identification and characterization of amino-acid sequences which can confer a nuclear location. Paradoxically, these studies have raised more questions than they have answered. In spite of a wealth of data, no concensus sequence has emerged for a nuclear location signal.

Progress has also been made in the elucidation of factors which influence nuclear protein import. It is apparent that nuclear signal-mediated protein transport is most likely a muti-step process consisting of a recognition event which precedes a translocation event. Nev- ertheless, the mechanism of signal-mediated translocation of proteins into the nucleus is still unknown.

Finally, progress has been made in the identification of nuclear envelope-associated proteins which may bind to nuclear signals. However, it has yet to be shown that any of these proteins are directly involved in the recognition and translocation of nuclear proteins.

In the present review, we examine what is known about the structure and function of amino-acid sequences which can confer a nuclear location. Further- more, we review recent studies designed to identify the cellular components involved in the recognition and selective nuclear uptake of nuclear proteins° To begin with, I present a brief overview of the structure of the nuclear envelope, since it constitutes the intracellular barrier which must be traversed by nuclear proteins.

The nucleus consists of an internal aqueous compartment, the nucleoplasm, bounded by the nuclear envelope [7]. The nuclear envelope consists of two lipid bilayers, calle0 the inner and outer nuclear membranes, which are separated by a perinuclear space. The nuclear envelope is studded with gromlet-like structures called nuclear pores. At the site of nuclear pores, the inner and outer nuclear membranes may be continuous; however, this has not been firmly established.

Nuclear pores are cylindrical channels, approx. 1100 A in diameter, which conta~ annuli which face towards the nucleoplasm and the cytoplasm [8]. Each annulus is composed of eight 100-250 A particles which are arranged in a symmetrical, octagonal pattern [7]. The center of each channel appears to taper towards a central plug, 300-350 A in diameter [9]. Radial spokes eminate from this central plug and extend towards each of the eight particles.

Our understanding of the structure of the nuclear envelope is very limited. Only within the last few years have researchers made significant advances in the characterization of nuclear envelope-associated proteins. It is also apparent that our knowledge of nuclear structure and nuclear protein import stems from the examination of a limited number of eukaryotic systems, in consider- ing questions of how nuclear proteins traverse the nuclear envelope, we must be aware of the extraor- dinary diversity of nuclear envelope architecture in vari- ous eukaryotic systems [7].

HI. Compartmentalization of nuclear proteins

Following their introduction into the cytoplasm, macromolecules enter the nucleus at a rate which is inversely proportional to their size. Thus the nuclear envelope behaves like a molecular sieve consisting of aqueous diffusion channels, 90 A in diameter (reviewed in Refs. 10, 11). While the diffusion of dextrans and proteins into the nucleus has been studied in detail, the efflux of macromolecules from the nucleus has not received much attention. Small non-nuclear proteins 43

kDa can diffuse out of the nucleus [12]; however, we do not know the effective diameter of the diffusion channels which perm/t protein efflux from the nucleus.

The nucleus contains its own distinct set of cellular proteins which range in size from 10 ~o 200 kDa (reviewed in Ref. 10). Thus, the import of cellular proteins into the nucleus must be selective. The fact that the nuclear envelope can behave like a molecular sieve imposes some degree of selectivity on nuclear protein import. However, tiffs in itself can not account for the net accumulation of nuclear proteins which are small enough tb diffuse into the nucleus. One model of nuclear protein import holds that proteins enter the nucleus by passive diffusion and are retained there due to association with non-diffusable elements (DNA, protein). This is the so-called 'diffuse and bind' model of nuclear protein transport (reviewed in Ref. 10). However, this model is not sufficient to account for the rapid nuclear accumulation of proteins which are too large to pas- sively diffuse into the nucleus.

The nuclear signal-mediated transport model con- tends that nuclear pr¢,teins contain inherent information in the form of a signal sequence which allows them to be selectively translocated into the nucleus [13]. This model is analogous to the signal hypothesis which has been proposed to account for the selective entry of proteins into the lumen of the endoplasmic ret icuhm [14]. As with the 'diffuse and bind' model of nuclear protein accumulation~ the nuclear-signal-mediated transport model mus~ account for the net accumulation of proteins within the nucleus. In this case, it is not necessary to invoke a nuclear retention process, since transported proteins are too big to freely d i f f~e c~ut of the nucleus. However, one must concede that the signal-mediated transport process must be uni-direc- tional. The nuclear signal can not allow a protein to exit the nucleus as ~eadily as it gained entrance.

In the following section, we examine the evidence in favour of the existence of nuclear location signals.

IV. Identlficafion of nuclear location signals

Nucleoplasmin is the most abundant nuclear protein of Xeng~us o,'¢~yte~s. Through its interaction with his- tones H2A and H2B, nucleoplasmin plays a role in nucleosome assembly. It is a pentameric protein with an apparent mass of 165 kDa. Each subunit consists of a highly charged carboxy-terminal tail and a globular amino-terminal domain. Under mild proteolytic conditions, the charged tails can be removed from the pentameric structure leaving a residual core fragment (110 kDa) [15]. Intact nucleoplasmin, as well as tail frag- ments, can rapidly enter the nucleus following introduction into the cytoplasmic compartment of Xenopus oocytes. However, the core fragment is excluded from the nucleus. A single tail fragment attached to the

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pentameric core is sufficient for nuclear localization. Following its introduction directly into the nucleus, the core fragment is retained. This suggests thai the tail fragment of nucleoplasmin is necessary for nuclear entry but not nuclear retention and the carboxy-terminal fragment of nucleoplasmin constitutes a nuclear location don-~ain. This study provided the first concrete evidence for the existence of nuclear location signals.

Two approaches have been adopted to identify nuclear location signal sequences. In the 'subtractive' approach, typified by the study above [15], a putative nuclear location signal is either deleted or mutated to demonstrate that it is necessary for the nuclear localization of a given protein. A second strategy, the 'additive' approach, involves the transposition of a putative nuclear signal onto a non-nuclear protein or macro- molecule to demonstrate that it is sufficient for nuclear localization. Neither one of these approaches is sufficient in itself to categorically prove that a given amino- acid sequence is a bona fide nuclear location signal. As we shall see, there are examples of amino-acid sequences which are necessary to target a given nuclear protein to the nucleus but which are not sufficient to target otherwise cytoplasmic proteins to the nucleus. The converse is also true. In reviewing the literature, we will highlight those instances in which both the additi,,e and subtractive approaches have been utilized in concert to identify nuclear location signals.

IV.A. SV40 large-T

The 92 kDa large-T antigen of SV40 virus is a nuclear, multifunctional protein capable of transfor- ming rodent cells and it is necessary for viral replication in monkey kidney cells. A number of biochemical properties have been ascribed to large-T, including an ability to bind to viral as well as cellular DNA sequences [16].

To determine whether the DNA binding activity of SV40 large-T is required for either transformation of rodent cells or viral replication, a mutagenic study of T antigen was conducted [1]. In particular, the study focused on a striking sequel~ce of five contiguous basic amino acids (Table I). In examining the subcellular distributions of the mu*.~! T antigens, a surprising observation was made. A single point mutation (dl0) resulting in the substitution of threonine for lysine at the codon 128 position of the largr-T gene completely abolishes the ability of the encoded protein to localize to the nucleus. Alteration of any one the basic residues flanking iysine 128 impairs but does not abolish the ability of large-T variants to accumulate in the nucleus. A series of deletion mutants were constructed to delineate the boundaries of the region of large-T which is required for nuclear localization. It was concluded that a sequence of only eight amino acids, Pro Lys Lys(128)

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TABLE 1

Nuclear proteins with single nuclear location signals

Signal sequences which are either necessary ( + ) or not necessary ( - ) for nuclear localization are indicated. Signal sequences which are either sufficient ( + ) or insufficient ( - ) for the nuclear localization of either pyruvate kinase (PK), immunoglobulin (lgG), bovine serum albumin (BSA), fl-galactosidase (BG), polio vinous VPI (POL) ot galactokinase (GK) are indicated. In cases where no data are availa- ble, a (.9) appears.

Nuclear Number Signal Signal Sequence of protein of resi- neces- sufficient putative

dues sary signal

Ref.

128 I

SV40 708 + + (PK) P P K K K R K V 19 large-T + (BSA) 54

+(IgG) 12

3 I

Yeast ,~ 2 210 ? +(BG) N K i P I K D 23

320 I

SV40 VP2 352 + + (PK) P N K K K R K L 25 + (POL) 6

5 I

SV40 VPl 362 ? - (PK) M A P T K R K G 25 + (POL) 27

? ?

17 !

A P K K P K E P

o,17 Human I lamin A 664 + ? SVTKKRKL 30

Yeast 31 I H2B 131 .9 + (BG) G K K R S K A 31

285 Adenovims I E1A 289/243 - + ( G K ) SCKRPRP 32

Lys Arg Lys Val Glu, is necessary for nuclear localization of SV40 large-T antigen.

Simultaneously, the genetic lesion within an SV40 large-T antigen (p~'.a-cT) encoded by an SV40-adeno hybrid virus was characterized [2]. It had been shown previously, that this large-T mutant localizes to the cytoplasm of virus-infected cells [17]. Interestingly, the authors discovered that a single point mutation resulting in the conversion of lysine-128 to asparagine is responsible for the aberrant subcellular distribution of the para-cT mutant. This report confirmed the critical role played by lysine 128 in the nuclear localization of large-T. In a subsequent study, it was reported that the para-cT mutant is structurally indistinguishable from wild-type large-T as judged by peptide mapping studies [18]. Thus, the failure of the para-cT mutant to accumulate in the nucleus can not be attributed to a gross structural change in the protein.

Subsequently, the subceilular distributions of truncation and deletion mutants of wild-type large-T were examined [19]. It was determined that the nuclear localization of full-length large-T is solely dependent on the presence of the wild-type nuclear location signal. The fact that the signal is sufficient for nuclear localization when appended to the amino-termirms of a cytoplasmic variant of large-T suggested that the sequence Pro Lys Lys Lys Arg Lys Vat Glu can act as an indept;~:lent element specifying nuclear location.

To test this hypothesis directly, chimaeric genes were constructed such that a small DNA sequence encoding the putative nuclear location signal was fused to genes encoding either E. coli fl-galactosidase or chicken muscle pyruvate kinase [19]. These proteins were selected because they are theoretically large eaough to be excluded from eukaryotic nuclei. Surprisingly, fl-galactosidase is not excluded from the nuclei of monkey kidney cells [19]. Nevertheless, the putative nuclear location signal of SV40 large-T can suffice to bring about the complete nuclear localization of fl-galactosidase. Pyruvate kinase, unlike fl-galactosidase, is completely excluded from the nuclei of monkey kidney cells [19]. However, the wild- t3"pe nuclear location signal can bring about the complete nuclear localization of pyruvate kinase. Substitu- tion of threonine for lysine-128 diminishes the ability of the signal to target either fl-galactosidase or pyruvate kinase to the nucleus. Thus, the nuclear location signal of SV40 large-T is sufficient to target otherwise cytoplasmic proteins to the nucleus. Furthermore, it was determined that a short sequence of just eight amino acids, Pro Lys Lys Lys Arg Lys Val Glu, is sufficient for the nuclear Iocalizatio~ of pyruvate kinase [19]. This minimal SV40 large-T sequence is identical to the minimal sequence which is necessary for the nuclear localization of SV40 iarge-T itself [1].

The biochemical properties of the cytoplasmic mutant of SV40 large-T (dl0) have been characterized [20]. The dl0 mutant is indistinguishable from wild-type large-T in its ability to undergo oligomerization, associate with the nuclear cellular antigen p53 and bind to DNA in vitro [20]. Thus, the failure of the dl0 mutant to localize to the nucleus can not be attributed to a failure to bind to p53, DNA or iiself. This suggests that the sole determinant of nuclear localization of SV40 large-T is the single nuclear location signal.

The DNA binding properties of a variety of point and deletion mutants of SV40 large-T in vitro have been examined [21]. These studies have indicated that the nuclear location signal does not comprise part of the DNA binding domain of large-T. Furthermore, it is apparent that a variety of large-T mutants which are difficient in DNA binding [21], at least in vitro, can nevertheless accumulate in the nucleus [22]. This suggests that DNA binding is not required for the u~clear localization or retention of SV40 large-T.

In spite of the fact that SV40 large-T is a complex multifunctional protein, the mechanism by which it accumulates in the nucleus is elegant in its simplicity. Large-T contains a single nuclear location signal consisting of just eight amino acids. The nuclear signal is necessary for the nuclear localization of large-T and it is sufficient for the nuclear localization of otherwise cytoplasmic proteins. The nuclear localization of large-T exhibits a high degree of specificity. A single amino-acid substitution within the signal can abolish its abilty to target large-T as well as cytoplasmic proteins to the nucleus.

A study of the nuclear localization of large-T is potentially complicated by the fact that it can bind to the nuclear cellular antigen p53 and DNA. However, carefully controlled experiments have shown that neither DNA binding nor p53 binding is required for the nuclear localization of iarge-T. Furthermore, neither DNA binding nor p53 binding is sufficient for the nuclear localization of large-T mutants containing defective nuclear location signals.

Thus the nuclear localization of SV40 large-T repre- sents a classic example of signal-mediated transport. The studies described above exemplify how the additive and subtractive approaches can be utilized to identify a nuclear signal.

IV.B. Yeast a 2 mating factor

Coincident with the identification of the SV40 large-T nuclear location signal, a putative nuclear location signal was also identified in yeast a 2 mating factor [23]. Yeast a 2 is a nuclear DNA binding protein consisting of 210 amino acids. The subceilular distributions of ot 2:fl-galactosidase fusion proteins in yeast have been examined by indirect immunofluorescence and subcellu- lax fractionation techniques [23]. The authors concluded that while fl-galactosidase itself is excluded from yeast nuclei, the amino-terminal 13 residues of yeast a 2 are sufficient to enhance the nuclear accumulation of /~- galactosidase. The authors were unable to conclude whether the amino-terminal 13 residues of a 2 con- stit~.e a signal specifying selective nuclear entry or a domain which enhances the nuclear retention of //- galactosidase. However, they faw.~z~'ed the former possibility because at the level of resolution employed in this study, fl-galactosidase appeared to be excluded from yeast nuclei. The authors highlighted a sequence motif within the amino-terminal 13 residues of yeast a 2 which consists of two basic residues separated by two hydrophobic amino acids and one proline residue (Ta- ble I). The authors pointed out that this sequence motif also appears in other small, nuclear DNA binding proteins and for this reason they proposed that it constitutes the putative nuclear location signal sequence of yeast a 2 [23]. In this report the authors did not

267

investigate whether this putative signal sequence is also necessary for the nuclear localization of yeast 2 itself.

The identification of a putative nuclear location signal in yeast a 2 immediately raises two questions. Firstly, yeast a 2 is theoritically small enough to diffuse into the nucleus and be retained there by DNA bindiag. Hence, the 'diffuse and bind" model can potentially account for its nuclear accumulation. The discovery of a putative nuclear location signal in yeast a 2 suggests that while not strictly necessary, a nuclear location signal may enhance the rate of nuclear entry of small nuclear proteins.

Secondly, a comparison of the SV40 large-T and yeast a 2 nuclear signals does not reveal any obvious structural similarity other than the presence of basic amino acids. This raises the interesting possibility that there may be different kinds of nuclear location signal sequences.

Since the discovery of the SV40 large-T and yeast a 2 nuclear signals, a variety of putative nuclear Iocatioa signals have been identified within cellular as well as viral proteins. In each case, investigators have en- deavoured to relate the newly discovered signal sequences to one of the two signals described above.

IV. C. Papovavirus capsid proteins

The late region of the SV40 genome encodes three capsid proteins called VP1, VP2 and VP3. The carboxy-terminal 234 residues of VP2 and VP3 are identical and the two proteins differ only in the fact that the amino-terminal portion of VP2 is 118 residues longer. The VP2 and VP3 capsid proteins of simian virus 40 contain a basic amino sequence, homologous to the SV40 large-T nuclear location signal, within the shared carboxy-terminal portions of the proteins (Table I) [24]. The sequence encompassing residues 312-325 of VP2 is sufficient to target the cytoplasmic pratein pyruvate kinase to the nucleus of microinjected cells [25]. To evaluate the role of this sequence in the nuclear localization of SV40 VP3 alone, the subcellular distributions of carboxy-terminal truncation mutants in trans- fected TC7 cells have been examined [5]. The authors reported that the carboxy-tetminal 35 residues of VP3 are required for nuclear localization. While the 35-residue carboxy-terminal fragment of VP3 does contain the putative nuclear location signal identified previously [24,25], the authors could v.ot discount the possibility that additional sequences witthin this region might contribute towards the nuclear Ilocalization of VP3 [5].

The nuclear localization of SV40 VP2/polio virus VP1 fusion proteins in virus;-infected cells is dependent on the presence of residues 317-323 of SV40 VP2 [6]. A single amino-acid substitution at the lysine-128 equivalent position of the putatiwe nuclear signal (lysine-320 converted to either threonine or asparagine) abolishes

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the ability of this sequence to enhance nuclear accumulation. This further substantiates the important role played by this basic region in the nuclear localization of SV40 VP2/VP3.

Like VP2/VP3, SV40 VP1 appears to contain its own nuclear localization determinant. An SV40 large T-like nuclear location signal is preser~t within the amino terminal eight residues of SV40 VP1 (Table I) [24]. Subsequently, it was shown that the amino-terminal 11 residues of SV40 VP1 are not sufficient to target pyruvate kinase to the nucleus of microinjected Veto cells [25]. However, the authors reported that the amino- terminal region of 42 residues of VPl, which encom- passes a second basic sequence, is able to target pyru- rate kinase to the nucleus in some but not all microinjected cells. The authors concluded that either the amino-terminal 42 residues of SV40 VP1 contain an inefficient nuclear location signal or this region is able to target pyruvate kinase to the nucleus by some means other than signal-facilitated transport. The amino-terminal 94 residues of SV40 VP1 are sufficient to target polio virus VP1 to the nucleus in virus-infected C'~ 1 cells [26]. The authors subsequently showed that the amino-terminal 11 residues of SV40 VP1 are sufficient to target polio virus VP1 to the nucleus of virus-infected cells [27]. This serves to illustrate that, while a particular basic amino-acid sequences may function as a nuclear location signal in the context of one protein (polio virus VP1) in virus-infected cells [27], it may fail to do so in the context of another cytoplasmic protein (pyruvate kinase) in microinjected cells [25]. Thus, both SV40 capsid proteins VP1 and VP2/VP3 appear to contain basic amino-acid sequences which can function as nuclear location determinants. It has been pointed out that a single nucleic acid sequence can be translated in two different reading frames to encode both the SV40 VP1 and VP2/VP3 putative nuclear signals [6,24].

The putative nuclear location signal sequence of SV40 VP2/VP3 is conserved in the VP2/VP3 capsid proteins of mouse and hamster polyomaviruses [25]. By contrast, the basic sequence contained within the amino-terminal 11 residues of SV40 VP1 is conserved in mouse but not in hamster polyomavirus VP1 [25]. This suggests that additional sequences within VP1 may suffice for nuclear localization.

IV.D. Human lamin A

The lamina is composed of three lamin proteins called A (70 kDa), B (67 kDa) and C (60 kDa) (reviewed in Ref. 28). Lamins A and C are both structurally and immunologically releated. Recent eDNA clon- ing studies have revealed that the basis for this similarity lies in the fact that the amino-terminal 566 residues of human lamins A and C are identical [29]. A putative

nuclear location signal sequence, homologous to that of SV40 large-T, lies between residues 417 and 422 of human lamins A and C [29]. Loewinger and McKeon [30] sought to identify the nuclear location signal of human lamin A. Initially, carboxy-terminal truncation mutants were generated and used to demonstrate that a region between amino acids 407 and 444 is required for the nuclear localization of human lamin A in CHO cells. Mutation of the lysine-128 equivalent residue of the putative signal sequence of lamin A (lysine-417) to either threonine or isoleucine severely impairs the ability of lamin A to localize to the nucleus. It is not known whether the putative signal sequence of human lamin A (Table I) is also sufficient for the nuclear localization of otherwise cytoplasmic proteins. The putative signal of human lamin A is conserved in the lamin proteins of a variety of eukaryotes [30].

IV.E. Yeast histone H2B

The amino-terminal 33 residues of yeast histone H2B are sufficient to enhance the nuclear accumulation of ~-galactosidase in yeast as judged by indirect immunofluorescence [31]. Deletion of residues 2-22 within this 33-residue segment does not impair the ability of the H2B-derived sequence to enhance the nuclear accumulation of ~-galactosidase. This suggests that residues 23-33 of H2B are sufficient for nuclear localization (Table I). A basic amino-acid sequence, homologous to the SV40 large-T prototypic signal, is contained within this region of H2B. Mutation of the lysine-128 equivalent residue of the H2B sequence (lysine-31) abolishes its ability to enhance the nuclear uptake of ~-galactosidase [31].

The subcellular distributions of H2B:/t-galactosidase fusion proteins in yeast cells have also been examined by immunoelectron microscopy [31]. It was found that ~-galactosidase is not excluded from yeast nuclei. This observation raised the possibility that the sequence between residues 23-33 of H2B may simply enhance the nuclear retention of p-galactosidase. The ability of /~-galaetosidase fusion proteins to bind to cellular DNA in vitro was investigated. It was reported that the amino-terminal 33 residues of H2B does not confer a DNA binding activity on /t-galactosidase. Taken together, the data suggest that the basic sequence of H2B does not simply enhance the nuclear retention of ~-galactosidase molecules that enter yeast nuclei of their own accord. Rather, residues 23-33 of H2B constitute the putative nuclear location signal of H2B. It is not known whether this sequence is also necessary for the nuclear uptake of H2B itself. The SV40 large-T-like signal sequence of yeast H2B is conserved in the H2B proteins of a variety of eukaryotic species [31].

IV.F. Adenovirus EIA

Transcription of the E1 A gene of adenovirus gives rise to 13 S and 12 S mRNAs. The 13 S RNA encodes a nuclear protein of 289 amino acids, while the 12 S RN/~ encodes a nuclear protein of 243 amino acids. Both proteins contain a basic amino-acid sequence proximal to their carboxy terminii (Table I). Carboxy4er~:ninal truncation mutants of the EIA protein are impaired in their ability to localize to the nucleus [32]. This suggests that, while not absolutely required, the carboxy-terminal basic sequence enhances the nuclear accumulation of E1A proteins. This basic sequence has been shown to be sufficient for the nuclear localization of the otherwise cytoplasmic protein E. coli galactokinase in microinjected CV1 cells [32]. Since galactokinase is retained when directly introduced into the nucleus, this suggests that the carboxy-terminal basic sequence of the E1A proteins specifies selective nuclear uptake and not nuclear retention. This basic an~no-acid sequence has been conserved in the E1A proteins of adenovirus types 2, 5, 7 and 12; hence, it may fulfil a common function in all E1A proteins [33]. Interestingly, the nuclear location signal of EIA is homologous to one of the nuclear signals of polyomavirus large-T (next section).

V. Proteins with move than one nuclear signal

V.A. Polyomavirus large-T

The large-T antigen of polyomavirus is a nuclear DNA-binding protein consisting of 785 amino acids. Like SV40 large-T, polyomavirus large-T is a multifunctional protein which has the capacity to immortal- ize rodent cells as well as support viral replication in permissive cells [16]. Richardson et al. [34] identified three basic amino-acid sequences within polyomavirus large-T which exhibit some homology to the SV40 large- T prototypic signal sequence. The authors examined the subcellular distributions of a variety of polyomavirus large-T:pyruvate kinase fusion proteins in microinjected monkey kidney cells. The amino4erminal 295 residues of polyomavirus large-T are sufficient for the nuclear localization of pyruvate kinase. A hybrid consisting of the amino-terminal 243 residues of polyomavirus large-T and consequently lacking the sequence which is the direct counterpart of the SV40 large-T signal, is localized to both the nuclear and cytoplasmic compartments. This suggests that additional information within the amino-terminal 243 residues of polyomavirus large-T contributes towards its nuclear localization. A pyruvate kinase fusion protein containing the amino-terminal 194 residues of large-T is confined to the cytoplasm. Thus, two regions within polyomavirus large-T appear to be responsible for its nuclear accumulation.

269

TABLE II

Proteins with two nuclear location signals

Nuclear signals which are either necessary ( + ) or not necessary ( - ) for nuclear localization are indicated. Nuclear signals which are either sufficient ( + ) or insufficient ( - ) for the nuclear localization of either pyruvate kinase (PK) or fl-galactosidase (BG) are indicated.

Nuclear N u m b e r Signal Signal Sequence of Ref. protein of resi- neces- suffi- signal

dues sary cient

Polyoma 282 I

large-T 785 + - ( P K ) P P K K A R E D 34

192 t

+ + ( P K ) V S R K R P R P 34

Human 323 I

c-myc 439 + + ( P K ) P A A K R V K L 36

367 I

+ ( P K ) R Q R R N E L K R S F 36

513 Glucocort icoid I receptor ( r a t ) 795 + + ( B G ) R K T K K K I K

Estrogen 257 receptor I ( h u m a n ) 595 '; ? I R K D R R G

39

40

Deletion mutants of polyomavirus large-T were constructed to delineate the boundaries of the two nuclear location domains. An ll-amino-acid-long basic sequence including lysine-282 is necessary for nuclear localization of polyomavirus large-T, as is a second sequence consisting of 9 amino acids includ;ng lysine- !92 (Table II). Polyomavirus large-T muraT, its which contain single deletions which impinge on either of these two regions are impaired in their ability to localize to the nucleus, while a double deletion mutant lacking both sequences is cytoplasmic [34].

These observations serve to illustrate that nuclear protein localization phenomena can be more complex than was originally found for SV40 large-T. In the case of polyomavirus large-T, two basic an~no acid sequences contribute towards the nuclear localization of the protein. Richardson et al. [34] suggested that the two sequences function independently to bring about the nuclear localization of polyomavirus large-T. This proposal is supported by the observation that one of the two sequences can suffice to target pyruvate kinase to the nucleus.

As with SV40 large-T, a study of the nuclear localization of polyomavirus large-T is potentially complicated by the fact that it exhibits an affinity for cellular as well as viral DNA. However, a mutant of polyomavirus large-T which contains a deletion within the putative

270

DNA binding domain and consequently is unable to bind to DNA in vitro can nevertheless localize ~o the nucleus [35]. This suggests that DNA binding is not required for the nuclear translocation and retention of polyomavirus large-T.

V.B. Human c-myc

The 439-amino-acid-long nuclear DNA binding protein encoded by the human c-myc proto-oncogene also appears to contain two nuclear location signals. Stone et ai. [4] examined the subcellular distributions of mutant c-myc proteins in COS cells. They reported that the amino-acid sequence between residues 320 and 368, while not absolutely required, can enhance the nuclear accumulation of c-myc. The authors were unable to conclude whether a nuclear location signal is contained within the sequence between residues 320 and 368 or whether this region is required for the proper presentation of another signal located elsewhere within the structul;= ~f c-myc.

To discriminate between these two possibilities, Dang and Lee [36] sought to identify the minimal sequence or sequences required for the nuclear l~calization of c-myc. The authors reported that a carboxy-terminal arginine- rich hexapeptide within c-myc is not required for nuclear localization [36]. Smith et al. [24] had previously speculated that this highly basic sequence is an unlikely nuclear location signal. This contention was based on the observation that variants of the SV40 large-T signal which are rich in lysine residues are more efficacious than signal variants rich in arginine residues. Dang and Lee [36] also reported that, within the large nuclear location domain defined earlier [4], deletion of residues 320-335 alone is sufficient to impair the nuclear localization of c-myc. By examining the subcellular distributions of c-myc:pyruvate kinase fusion proteins, the authors were able to identify a sequence (M1) between residues 320 and 328 of c-myc which is sufficient for nuclear localiTation (Table II). However, the authors also made the unexpected discovery that an additional sequence (M2) encompassing residues 364-374 is partially able to target pyruvate kinase to the nucleus (Table II). Thus, two regions of the c-myc protein appear to contain amino-acid sequences which are sufficient (to different degrees) for nuclear localization.

To examine the kinetics of nuclear accumulation facilitated by these two signal sequences, synthetic peptides homologous to the sequences of M1 and M2 were fused to human serum albumin (HSA) and the subcenular distributions of the peptide: HSA conjugates in microinjected ce~, ~vere examined. As had been found with the pyruvate kinase fusion proteins, the M1 signal is a potent nuclear location signal, able to bring about rapid nuclear loealiTation of HSA. How- ever, the M2 sequence is a much weaker signal, able to

only partially localize HSA to the nucleus. Dang and Lee [36] concluded that the c-myc protein, like polyomavirus larg~.-T, contains two nuclear location signals. One of the ~wo signals (the M1 signal) appears to be the principal nuclear localization determinant of c-myc. The second M2 signal most likely does not participate in the nuclear local~.ation of wild-type c-myc, since deletion of this sequence does not impair the ability of c-myc to localize to the nucleus and the M2 signal only appears to function when the principal M1 signal is deleted. Thus, unlike the two signals of polyomavirus large-T, which are both required for nuclear localization, the two signals of c-myc differ dramatically in their ability to target c-myc to the nucleus. Both the M1 and M2 sequences are conserved in the c-myc proteins of a variety of eukaryotes [36].

V. C. Rat glb~cocorticoid receptor

The rat ~,,lucocorticoid receptor is a nuclear DNA binding prollein consisting of 795 amino acids. In vitro mutagenic studies have defined the boundaries of the DNA and hormone binding domains of the glucocorti- cold receptor [37,38]. In the absence of steroid, the receptor resides in the cytoplasm. However, in its presence, the receptor undergoes a conformational change coincident with its translocation into the nucleus. Thus, the glucocorticoid receptor constitutes an example of a protein which contains a masked or hidden nuclear location signal. Picard and Yamamoto [39] examined the subcellular distributions of mutants of the rat glucocorticoid receptor to identify the nuclear localization determinants of the protein. The authors concluded that the rat glucocorticoid receptor, like polyomavirus large-T [34] and human c-myc [36], contains two nuclear location signals. One sequence, lying between the hinge region connecting the DNA and hormone binding domains of the receptor, bears a striking resemblance to the SV40 large-T prototypic signal (Table II). This sequence is sufficient to target E. coli ~8-galactosidase or a truncation mutant of the glucocorticoid receptor, lacking the steroid binding domain, to the nucleus in the absence of hormone. However, a second nuclear localization domain, which coincides with the steroid binding domain, is required for nuclear localization of the intact receptor. When the two nuclear location determinants are juxtaposed in the context of the entire receptor, the signals function only in the presence of the hormone. This suggests that the activity of the SV40 large-T-like signal is modulated by a determinant within, the steroid binding domain. Hormone binding is !:nown to induce a conformational change in the receptor and this may be sufficient to ummask tl~e SV40 large-T-like signal. These observations suggest a novel mechanism whereby the nucleocytoplasmic distribution of the glucocorticoid receptor is regulated in a hormonally responsive manner.

Similar basic amino-acid sequences, homologous to the SV40 large-T-like nuclear signal of the glucocorticoid receptor, are located within a ' hinge' region linking the DNA and steroid binding domains of other nuclear hormone receptors (Table II). Kumar et al. [40] reported that the 'hinge' region and the D N A binding domain are required for tight nuclear association of the estrogen receptor. In these studies, the authors did not attempt to discriminate between sequences which specify selective nuclear entry as opposed to nuclear retention. By analogy to the glucoeorticoid receptor, the DNA binding domain of the estrogen receptor may be required for nuclear retention while the 'hinge' region may contain the respective nuclear location signal of the estrogen receptor. Unlike the glucocorticoid receptor, however, nuclear localization of the estrogen receptor aoes not appear to be steroid hormone-dependent [41].

VL Complex nuclear location determinants

VI.A. Nucleoplasmin

The highly charged carboxy-terminus of nudeo- plasmin is rich in basic amino-acid sequences [42,43]. Unlike the situation with SV40 large-T, where a single tract of basic residues immediately draws one's attention, nucleoplasmin contains four basic amino-acid sequences. These nuclear location signal candidates, called A, B, C and D (in order from amino- to carboxy- terminus), can be grouped on the basis of a resemblance to either the SV40 large-T signal (B and C) or the yeast a 2 signal (A and D) [44].

.Burglin and De Robertis [45] reported that the carboxy-terminal 196 residues of nuclcoplasmin, when fused to E. coli /~-galactosidase, are sufficierr~ to enhance nuclear accumulation in Xenopus oocytes. The authors reported that two of the four candidate sequences (C and D) can be deleted without impairing the ability of nuclcoplasmin to enhance the rmclear accumulation of ,8-galactosidase. However, a mutant which lacks three of the candidate sequences (B, C and D) is

271

cytoplasmically disposed. This suggested indirectly that signal candidate B alone can suffice to enhance the nuclear accumulation of fl-galactosidase.

Subsequently, Dingwall et al. [46] sought to identify the minimal sequence of nucleoplasmin which is sufficient for nuclear localization. The authors discovered that residues 149 to 176 of nucleoplasmin can target pyruvate kinase to the nuclei of microinjected cells (Table IIl). This result is in agreement with the observation that candidate sequences A and B together are sufficient for nuclear localization [45]. However, when the authors evaluated the efficacy of each candidate sequence individually, it was found that neither sequence A nor B alone was sufficient for nuclear localization. Thus, both sequence elements appear to work in concert to bring about nuclear localization.

The minimal nucleoplasmin nuclear location signal is twice as long as either the SV40 large-T or yeast a 2 mating factor signals [46]. Secondly, the nucleoplasmin signal is more complex than the SV40 large-T signal. While Bal 31 deletion analysis reveals that the minimal nucleoplasmin nuclear location signal potentially consists of 14 amino acids, this sequence fails to target pyruvate kinase to the nucleus when tested directly [46]. The addition of a single basic amino-acid residue to either the amino- or carboxy-terminal ends of the minimal sequence restores its ability to confer a nuclear location.

VI.B. NI / N2

The N1 /N2 polypeptides of Xenopus laevis ooeytes are abundant nuclear proteins of 105 and 110 kDa, respectively. These proteins participate in chromatin assembly through their interaction with histories H3 and H4. Recently, it has been shown that a single mRNA species codes for both proteins [47]. The N1 and N2 proteins appear to differ only in the degree to which they are phosphorylated. The sequence of the cDNA reveals a basic amino-acid tract between residues 531 and 537 which is strikingly similar to the SV40 large-T

TABLE III

Proteins with complex nuclear location determinants

Nuclear signals which are necessary (+) for nuclear localization are indicated. Nuclear signals which are sufficient (+) for the nuclear localization of either pyruvate kinase (PK) or jS-galactosidase (BG) are indicated.

Nuclear Number of Signal Signal Sequence of Ref. protein resiaues necessary sufficient signal

155 168 I I

Nucleoplasmin 200 + + ( P K ) AVKRPAATKKAGQAKKKKLD 46 +(BG) 45

534 549 I I

N1/l ' , l '2 589 + ? L V R K K R K T E E E S P L K D K D A K K S K Q 48

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prototypic signal sequence (Table Ill). Kleinschmidt and Seiter [48] constructed a series of deletion mtd point mutants of the N1 protein to delineate the boundaries of the nuclear location signal(s). A deletion mutant lacking the sequence encompassing residues 531-537 is localized to the cytoplasm. However, truncation mutants lacking a second basic sequence encompassing residues 544-554, while retaining the SV40 large-T like sequence at residues 531-537, are also unable to localize to the nucleus.

Single amino-acid substitutions within either of the basic tracts encompassing residues 531-537 or 544-554 impair the nuclear localization of N1 [48]. In particular, the mutation of either lysines 534 or 549 to threonine completely abolishes nuclear localization. Thus N1 appears to contain two critical lysine residues, each equivalent to lysine-128 of the SV40 large-T nuclear location signal, which are absolutely required for nuclear signal function. The authors concluded that either the two basic amino-acid tracts centered or~ lysines-534 and -549 constitute tandemly-linked, interdependent nuclear location signals or a nuclear location domain encompassing residues 531-554 of N1 is required for nuclear location. If the second interpretation is correct, then the complex nuclear location domain of N1 is similar to that of the functionally related nuclear protein, nucleoplasmin [46]. However, there is no obvious sequence similarity between the nuclear location signal domains of nucleoplasmin and N1 (Table III).

VI.C Yeast GAL4

The protein product of the yeast GALA gene is a 99 kDa nuclear DNA binding protein which functions as a transcriptional activator. The amino-terminal 74 residues of the GAlA protein are sufficient for nuclear accumulation of fl-galactosidase [49] and for DNA binding to specific yeast gene sequences [50]. This suggests that either nuclear location and DNA binding domains map to the same region of the GALA protein or that DNA binding alone is sufficient for the nuclear accumulation of fl-galactosidase in yeast. Silver et al. [51] reported that a fusion protein consisting of the DNA binding domain of the LexA protein of E. coil fused to/~-galactosidase does not accumulate in yeast nuclei. Furthermore, substitution of the LexA DNA binding domain for the ami~o-terminal 74 residues of GALA protein abolishes the nuclear localization of GAL4 in yeast [51]. Together, these data suggest that DNA binding alone is not sufficient for nuclear localization of/3-galactosidase in yeast cells and that a nuclear location determinant is present in the amino-terminal 74 residues of GALA protein.

Amino-acid substitutions introduced between residues 6 and 53 impair the ability of the amino-terminal 74 residues of GALA protein to enhance the nuclear

accumulation of fl-galactosidase [52]. This suggests that either the entire amino-terminal 74 residues of GALA protein constitute a nuclear location domain or that there are multiple nuclear location signals within this region of GAL4 protein. Analogous point mutations in the context of GALA protein do not affect nuclear localization. It has been suggested that sequences located between residues 75 and 881 of GAL4 protein may force the amino-terminal domain to adopt a conformation which will permit nuclear localization [52]. Interest- ingly, mutants of the GAL4 protein which are unable to localize to the nucleus are overexpressed relative to wild-type GALA protein. This phenomenon may be analogous to the observation that cytoplasmic variants of SV40 large-T are overexpressed due to the fact that they are unable to autoregulate their own synthesis [20,21].

Nelson and Silver [53] sought to determine the minimal sequence of GALA protein which is sufficient for the nuclear localization of otherwise cytoplasmic proteins. They reported that the amino-terminal 74 but not 61 residues of GALA protein are required for the nuclear localization of fl-galactosidase in yeast cells. However, the amino-terminal 29 residues of GALA are sufficient to target invertase to the nucleus. It is not known whether there are multiple nuclear signals within the amino-terminal 74 residues of GAIA protein and whether one of these signals alone is sufficient for the nuclear localization of invertase but not/~-galactosidase in yeast. The efficacy of the nuclear determinant within the amino-terminal 29 residues of GALA may be affected adversely by its fusion to /~-galactosidase sequences. Residues 30-74 of GAL4 protein may insulate the GALA nuclear determinant from these negative influences.

VH. Structural features of nuclear signals

To date, no X-ray crystallographic or physical-chemical characterization of any nuclear location signal has been performed. In the absence of such information, one can only speculate as to the structure of nuclear location signals. It is apparent that the strict mainte- nance of an exact primary structure is not required for nuclear location signal function. This suggests that nuclear location signals may adopt a common secondary structure or motif. This notion is supported by the finding that anti-nuclear location signal peptide antibodies recognize a variety of nuclear proteins [54].

In attempting to predict the structure of nuclear signals, one must take into account the fact that they are highly charged due to the presence of contiguous basic amino acids. One might imagine that these basic residues would be positioned as far apart as possible to minimize charge repulsion effects. A Chou and Fasman secondary structure prediction of the SV40 large-T sig-

nal sequence suggests that it might form an a-helical structure [2]. Circular dichroism studies have indicated that polylysine can form an a-helical structure only at high pH when the e-amino groups of the lysine residues are uncharged [55]. At physiological pH, polylysine is more likely to adopt a random coil configuration to minimize charge repulsion. It would seem unlikely, therefore, that the SV40 large-T nuclear signal, which also contains contiguous basic residues, can adopt an a-helical conformation at physiological pH conditions unless acidic cellular factors stabilize the helical structure by neutralizing the basic charges of the lysine residues. In the absence of such hypothetical cellular neutralizing factors, the SV40 large-T nuclear signal most likely adopts a random coil conformation.

Why is it, then, that lysine-128 is so crucial? It is conceivable that a particular spatial distribution of basic residues within the nuclear signal is essential for function. Since lysines-128 and -131 of the SV40 iarge-T nuclear location signal appear to be the most sensitive residues to mutation (albeit to different degrees) and the substitution of arginine for lysine-131 does not impair signal function [24], the arrangement lysine-X- X - l y s i n e / a r ~ i n i n e (where X can be any residue) may be important. In an extended-coil or a-helical structure, this arrangement could place the positively charged side-chains of both essential basic residues on the same side of the signal peptide. The sequence motif lysine-X- X-'ysine/arginine occurs twice in the SV40 large-T signal. The fact that conversion of lysine-128 to any one of seven other amino acids abolishes nuclear transport of large-T [3,24] suggests that the array lysine(127)-X- X-arginine(130) is not sufficient for nuclear transport and that lysine-127 can not substitute for lysine-128. Perhaps, this may be due to its proximity to proiin¢-i26 which may act as a reference point for the 'reading' of the signal in the context of SV40 large-T.

The motif lysine-X-X-lysine/arginine appears in all the nuclear location signal sequences described to date (Table IV) except for those of yeast a 2, SV40 VP1 and the sequence centered on arginine-367 of human c-myc. Since the first lysine of the motif corresponds to lysine- 128 of the SV40 large-T signal, the corresponding lysine residues of other nuclear location signals should play a critical role in signal function. This has been verified for Xenopus oocyte N1 [48], human lamin A [30], yeast H2B [31] and SV40 VP2/VP3 [6]. Therefore, the niotif lysine-X-X-lysine/arginine may be the minimal feature of some nuclear signals. Many nuclear signals are flanked by proline residues or amino acids with bulky side-chains (such as threonine, serine or valine) (Table IV). These flanking residues may serve to insulate the nuclear signal from the effects of surrounding sequences and enhance the presentation of the signal on the surface of proteins.

A sequence motif consisting of two lysine residues

273

TABLE IV

Identification of a sequence motif in the nuclear location signals of a variety of proteins

Nuclear protein Signal sequence

SV40 large-T

SV40 VP2

Lamin A

Histone H2B

Adenovirus EIA

Polyomavirus large-T

Human c-myc

Nucleoplasmin

NI

Glucocorticoid

receptor (rat)

Concensus sequence

128

PKKKRK

320

NKKKRK

41"/

VTKKRK

31

GKKRSK

285

SCKRPR

281

PPKKAR

192

SRKRPR

323 I

AAKRVK

167 I

QAKKKK

549 I

DAKKSK

533 I

VRKKRK

514 I

TKKKIK

XXKKXK R R

separa'.ed by two hydrophobic amino acids and a proline residue has been highlighted in yeast a 2 [23]. A sequence motif consisting of two basic residues separated by four hydrophobic amino acids appears twice in the nucleoplasmin signal [46]. However, there is no experimental evidence to support the contention that these sequence motifs are important for nuclear locafi- zation.

VIII. Features of signal-medlated nuclear protein import

VIII.A. Route of signal-mediated import

Feldherr et al. [56] reported that nucleoplasmin- coated gold particles up to 200 ,A in diameter can rapidly accumulate in the nucleus following their microinjection into the cytoplasmic compartment of Xenopus oocytes. On the basis of the distribution of the gold

274

particles adhering to the nuclear envelope, it was concluded that transport of nucleoplasmin-coated gold particles occurs via nuclear pores. Gold particles coated with the nucleoplasmin core fragment (lacking the carboxy-terminal transport domain) do not accumulate in the nucleus. Nuclear translocation mediated by the SV40 large-T signal also appears to occur via nuclear pores. A synthetic peptide, homologous to the sequence of the SV40 large-T signal, can target albumin-coated gold particles to the nucleus via nuclear pores [57,58].

Apart from nuclear pores, no other cellular components have been shown to participate in the translocation of proteins into the nucleus. In particular, there does not appear to be a requirement for a soluble cytosolic factor analogous to a signal recognition par- ticle [14]. Rather, it would appear that nuclear signal recognition occurs at the level of the nuclear pore [57,59]. Indeed, microinjection studies have shown that prior to translocation into the nucleus, nuclear proteins exhibit a perinuclear distribution around the nuclear envelope [6,59,60]. Nucleoplasmin-coated gold particles associate with fibrils attached to the cytoplasmic side of nuclear pore complexes [59]. This association may account for the perinuclear accumulation of nuclear protein~ prior to their nuclear uptake.

At least one nuclear protein, VP1, binds to the cytoskeleton in the cytoplasm prior to its transport to the nucleus [61]. If this is a general phenemenon, what role might this play in the nuclear accumulation of proteins? It has been proposed that cellular factors may enhance the concentration of nuclear proteins on the cytoplasmic surface of the nuclear envelope in prepara- tion for their translocation into the nucleus [62]. Hence nuclear proteins could 'ride' along cytoskeletal fibers which radiate from the outer nuclear membrane. Ding- wall and Laskey [10] have estimated that a reduction of the three-dimensional random migration of nuclear proteins to two dimensions would result in a only 2-fold enhancement of the rate of cytoplasmic migration of nuclear proteins to the nuclear periphery. They have suggested that, if guiding of nuclear proteins to the nuclear periphery does occur, it most likely contributes very little towards the overall rapid rate of signal-mediated nuclear protein uptake.

Chemical treatments known to disrupt the actin, tubulin and intermediate filament networks of eukaryotic cells do not appear to abolish the nuclear accumulation of at least some nuclear proteins [63]. Furthermore, in vitro studies suggest that an ex- tranuclear cytoskeleton is not required for nuclear signal-mediated uptake [64]. By contrast, Lin et al. [61] have reported that the nuclear uptake of SV40 capsid proteins in virus-infected cells is diminished during latter stages of i,.tfection when the integrity of the cytoskeleton is compromised. Whether the nuclear envelope-associated fibrils described above form part of a

larger intraceiiuiar network which plays a role in vivo in the concentration of nuclear proteins at the cytoplasmic sui'face of the nuclear envelope has yet to be determined

VIII.B. Effect of nuclear signal number

The rate of signal-mediated protein transport appears to be directly proportional to the number of functional nuclear signals within a protein. Thus, Ding- wall et al. [15] reported that nuclear uptake rate in- creases with the number of tail domains attached to the nucleoplasmin core fragment. Subsequently, Roberts et al. [65] demonstrated that inefficient nuclear location signals can co-operate to bring about the nuclear localization of pyruvate kinase. Two copies of the sequence Arg-Lys-Arg-Lys in any one of three different spatial combinations is more effective than a single copy of the defective signal in targeting pyruvate kinase to the nuclei of microinjected cells. Furthermore, three copies of the signal are more effective than two. This suggests that the signals function independently of one another. Dworetzky et al. [58] have reported that the rate of nuclear accumulation of BSA is directly proportional to the number of synthetic peptides, homologous to the sequence of the SV40 large-T signal, which are cross-linked to it. An interpretation of this study [58] is, however, complicated by the fact that heterogeneous popu- lations of molecules containing an average of either 5, 8 or 11 synthetic peptides cross-linked to BSA were used for measurements of nuclear protein uptake. Fischer- Fantuzzi and Vesco [60] reported that the rate of nuclear uptake of a deletion mutant of SV40 large-T is dependent on the number of tandemly linked copies of the SV40 large-T signal which are fused to its carboxy- terminus. Thus, multiple copies of the SV40 large-T signal appear to enhance nuclear accumulation whether they are closely linked together [60] or separated from one another [65].

Two models can account for these observations. In the first model, a cellular factor can only recognize one copy of a signal sequence within a protein at a time. The likelihood that the factor will recognize a given nuclear protein will depend on the number of signal sequences, exposed on the surface of the protein, which the receptor can potentially interact with following random association [65]. In a second model, multiple signal recognition factors can recognize a given nuclear protein simultaneously [58]. The rate of nuclear uptake of a protein is directly proportional to the number of factors which can bind to it and hence the number of nuclear signals exposed on its surface. Dingwall et al. [46] have proposed that the complex nuclear location signal of nucleoplasmin may consist of two interdependent nuclear signals. Simultaneous recognition of both nuclear signals by two cellular factors may be necessary

for the nuclear uptake of nucleoplasmin. Tiffs would imply that the two recognition factors must be in close proximity to one another to interact with the nucleoplasmin signal. Elucidation of wh/ch of these two models best describes the mechanism of nuclear signal recognition must await the identification of the cellular recognition factors and the generation of reconstituted systems to study signal-mediated nuclear protein uptake.

VIII.C Unidirectionality

With the exception of a handful of proteins which are able to shuttle back and forth across the nuclear envelope [66], signal mediated-nuclear protein uptake in rive appears to be a unidirectional process. In the case of nucleoplasmin, it has been shown lhat the core fragment is retained following its direct introduction into the nucleus [15]. It is not known whether other nuclear proteins can be retained within the nucleus when their nuclear signals have been removed. While it is difficult to measure nuclear protein efflux in rive, this can readily be detected in vitro. Isolated rat liver nuclei which have been allowed to accumulate either nucleoplasmin [67] or SV40 large-T [64] have been recovered and incubated for extended periods of time to ascertain the amount of nuclear protein which leaks out. On the basis of these studies it appears that the nuclear import of nucleoplasmin and SV40 large-T is unidirectional in vitro.

The nuclear location signals described to date appear to facilitate translocation across the nuclear envelope only in the direction from the cytoplasm to the nucleus. The signal modulation theory proposes that nuclear signals become inoperative once a nuclear protein is within the nucleoplasm [68]. The fact that proteins purified from the nucleus retain the ab~ty to rapidly re-enter the nucleus following their introduction into the cytoplasm argues against ~.his possibility [13,69,70]. Alternatively, the nucleoplasmic face of nuclear pores may be unable to respond to the signal. If this is the case, this implies that nuclear pores are asymmetric. Recent immunological studies have shown preferential binding of monoclonal antibodies to the nucleoplasmic [71] side of nuclear envelopes. Thus nuclear pores may indeed be asymmetric and cellular factors which recognize the nuclear signal may reside only on the cytoplasmic side of nuclear pores.

VIII.D. Energy dependence

The nuclear accumulation of radiolabelled nucleoplasmin in Xenopus oocytes can be abolished by prior injection of either apyrase or hexokinase plus glucose [72]. Both treatments drastically reduce intracellular ATP pools. Nucleoplasmin is retained within Xenopus oocyte nuclei when endogenous ATP pools are depleted

275

after nuclear uptake has been allowed to occur. The nuclear accumulation of nucleoplasn'fin in monkey kidney cells can be abolished by prior treatment of cells with deoxyg!ucose and sodium azide [59]. This suggests that nuclear translocation but not retention of nucleop- lasmJn in rive requires an energy source, possibly ATE It has yet to be shown that this energy-dependent phenomenon is reversible in rive or that nuclear localization mediated by other nuclear signals is also energy- dependent in vivo.

Nuclear import but not retention of nucleoplasmin in vitro can be abolished by treatment with either hexokinase or apyrase [67]. This effect can be reversed by addition of an ATP-regenerating system [57]. Nuclear import but not retention of SV40 large-T in vitro into isolated rat liver nuclei is abolished in the presence of apyrase [64]. It is not known whether this effect is reversible.

Taken together, the data suggest that the nuclear translocation of nucleoplasmin both in vitro and in rive and of SV40 large-T in vitro is energy-dependent. Only in one case has it been shown that this effect is reversible. It has yet to be shown that ATP is a direct co-factor for nuclear import and that ATP depletion does not have adverse effects on nuclear envelope structure, rendering them less permeable.

VIII.E. Multi-step nature of signal-mediated nuclear protein import

Evidence from recent microinjection studies suggests that nuclear protein translocation occurs via a two-step process. The nuclear accumulation of nucleoplasmin in monkey cells is characterized by a distinct perinuclear accumulation of the protein within minutes of its introduction into the cytoplasm [59]. No such accumulation of the nucleoplasmin core fragment is observed. Nuclear but not pednuclear accumulation can be abolished by chilling microinjected cells on ice or treating them with deoxyglucose plus sodium azide ir. the absence of glucose. Thus nuclear import of nucleoplasmin appears to consist of at least two steps; a temperature- and energy-independent binding to the nuclear envelope followed by a temperature- and energy-dependent translocation into the nucleus. This conclusion is supported by the observation that nucleoplasmin-coated gold particles exhibit a perinuclear accumulation in Xenopus oocytes, minutes following their introduction into the cytoplasm, while gold particles coated with the nucleoplasmin core fragment do not show a marked preference for adhering to the outer nuclear membrane [59]. Taken together, these observations suggest that the import process can be subdivided into a recognition event, which occurs at the level of nuclear pores, prior to a translocation event. Only the latter step appears to be energy- and temperature-dependent. Similarly, New.-

276

meyer and Forbes [57] reported that the nuclear import of an SV40 large-T signal peptide:human serum albumin conjugate into isolated nuclei can be separated into signal recognition and translocafion steps.

IX. Mechanism of nuclear signal-mediated protein uptake

While the discovery of nuclear location signals pro- vides a means to account for the selective nature of nuclear protein import, this does not reveal the mechanism of nuclear signal-mediated translocation of proteins into the nucleus. Two possible mechanisms exist. In the active transport model, proteins containing nuclear location signals are recognized and actively carried across the nuclear envelope. In the facilitated diffusion model, the nuclear location signal triggers a conformational change in the nuclear envelope such that the passive diffusion of nuclear signal-bearing proteins is no longer restricted.

To gain insight as to the mechanism of action of nuclear signals, researchers have examined factors which influence nuclear signal-mediated import of proteins both in vitro and in vivo. In the following section we review those factors and highlight observations which potentially allow us to discriminate between active transport and facilitated diffusion processes.

iX.A. Temperature dependence

Dingwall et al. [15] reported that the import of nucleoplasmin into Xenopus oocyte nuclei is reduced 10-fold when import studies are conducted at 4°C instead of 25°C. More recently, Richardson et al. [59] showed that the nuclear import of nucleoplasmin into microinjected monkey cells can be blocked by maintaining the microinjected cells on ice. This effect is reversible, since nuclear import of nucleoplasmin resumes once the temperature is shifted to 37 ° C.

The nuclear import of nucleoplasmin into synthetic nuclei [72] or isolated rat liver nuclei [67] can be abolished by maintaining samples at 4 ° C. Similarly, the nuclear uptake of SV40 large-T into isolated rat liver nuclei in vitro is abolished at 4°C [64]. It is not known whether the nuclear import of large-T in vivo is also temperature-dependent. Taken together this suggests that nuclear localization mediated by either the nucleoplasmin or SV40 large-T signals is temperature dependent. It has been argued that chilling to 4°C should abolish nuclear localization if it occurs via an active transport mechanism [73]. However, if nuclear import occurs via facilitated diffusion, the rate of nuclear import of proteins at 40C should be 90~ of that at 37°C [73]. The observations presented above would seem to suggest that nuclear localization of nucleo-

plasmin and SV40 large-T occurs via an active transport process.

IX.B. Kinetics

Dingwall et al. [15] reported that radiolabelled nucleoplasmin accumulates in the nuclei of microin- jetted Xenopus oocytes within 60 rain of its introduction into the cytoplasmic compartment. Richardson et al. [59] reported that the maximal nuclear accumulation of nucleoplasmin in Veto cells is more rapid, occurring within 20 min of its introduction into the cytoplasm. Differences in cytoplasmic viscosity may account for different rates of nuclear uptake of nucleoplasmin in Xenopus oocytes versus monkey cells. Schulz and Peters [68] used the technique of fluorescence microphotolysis to quantitate the nuclear uptake of nucleoplasmin in rat hepatoma cells. They reported that the half-time for nuclear uptake is 5 rain at 37 o C. The nuclear import of nucleoplasmin in vitro is also extremely rapid. New- meyer et al. [67] reported that the maximal level of uptake of fluorescently labelled nucleoplasmin into isolated nuclei occurs within 30-45 rains.

Similarly, the nuclear uptake of SV40 large-T in vivo, as determined by pulse-chase labelling experiments, is rapid. Schickedanz et al. [74] showed that SV40 large-T is translocated into the nucleus within 15-30 min of its synthesis in the cytoplasm of monkey cells. Proteins, covalently coupled to peptides homologous to the SV40 large-T signal sequence, are rapidly targeted to the nucleus following their introduction into the cytoplasm of either Xenopus oocytes [54] or monkey cells [12]. Similarly, Markland et al. [64] reported a half-time of 5 min for the maximalrate of uptake of SV40 large-T into isolated rat liver nuclei in vitro.

Rates of nuclear import mediated by either the SV40 large-T or nucleoplasmin signals greatly exceed what would be expected for a processes dependent on facilitated diffusion [44]. These data support the contention that nuclear import mediated by the nucleoplasmin and SV40 large-T signals occurs via an active transport mechanism.

IX. C. Competition and saturability

The active transport model of nuclear protein import requires that signal recognition must be a prerequisite for nuclear import. If one accepts that there must be a finite number of cellular signal receptors which are involved in this process, it should be possible to saturate the uptake process by introducing an excess of a transport competent protein. Furthermore, an excess of a synthetic peptide homologous to the transport signal should be an effective competitive inhibitor of import.

The nuclear uptake of BSA conjugated to a synthetic peptide homologous to the SV40 large-T signal exhibits

saturability in Xenopus oocytes [54]. The estimated maximum rate of uptake is 6.4- 109 molecules per cell per min. Tiffs is the only report in which saturation of nuclear protein import has been demonstrated. The same study reported that a synthetic peptide homologous to the SV40 large-T signal can also act as a competitive inhibitor of the nuclear uptake of an SV40 large-T signal peptide:bovine serum albumin conjugate, but it does not influence the maximal level of nuclear accumulation [54].

Taken together, the above data argue in favour of an active transport mechanism of signal-mediated protein import. However, the data are not unequivocal. The elucidation of the precise mechanism of signal-mediated protein import must await the identification of the cellular factors which permit the selective entry of nuclear proteins. The generation of reconstituted systems may allow researchers to determine the mechanism of nuclear protein import in the same way as this approach has enabled researchers to understand selective entry of proteins into the lumen of the endoplasmic reticulum.

X. Identification of cellular factors involved in nuclear protein uplake

X.A. Inhibition of nuclear protein import by antibodies and WGA

Finlay et at. [75] reported that WGA binds to the nuclear pores of isolated rat liver nuclei and blocks the nuclear uptake of nucleoplasmin. In an effort to identify the factor(s) responsible for this effect, they conducted a Western blot of nuclear envelope proteins using radiolabelled WGA and detected a 63-65 kDa protein. This may be analogous to the 62 kDa N- acetylglucosamine-containing nucleoporin identified previously [76]. However, this study did not determine whether WGA binding to nuclear envelopes nonselec- tively blocks nuclear uptake of proteins due to occlusion of pores.

WGA can also reversibly block the nuclear import of nucleoplasmin and a large-T signal peptide: BSA conjugate in vivo [77]. The effect of WGA was judged to be specific, since the binding of WGA to nuclear envelopes in vivo does not block the efflux of RNA or the passive diffusion of small low-molecular mass dextrans into the nucleus. WGA also blocks the nuclear uptake of a va~ety of nuclear proteins in Xenopus oocytes [78]. The ,:~fect has been shown to be specific, since the passive diffusion of low-molecular-mass dextrans into the nucleus is not affected by WGA. Neither of these studies has, however, identified a protein or proteins which fail to permit nuclear protein uptake when bound to WGA.

277

A monoclonal antibody (RL1) which recognizes two N-acetylglucosanfine-containing proteins of 180 and 63 kDa can block the uptake of nucleoplasm/n in Xenopus oocytcs [79]. Monoclonal antibodies that recognize other N-acetylglucosamin¢ bearing nucleoporins do not block uptake. The monoclonal RL1 also inh/bits the export of 5 S ribosomal RNA and tRNA; however, it does not preturb the passive diffusion of low-molecular-mass dextrans into the nucleus. The authors suggested that, since the monoclonal antibody used in their studies is substantially smaller than WGA, it is unlikely that the observed inhibition can be accounted for by steric hinderance. This report implicates two N-acetylglucosamine containing nucleoporins in nuclear protein uptake. The smaller of these two proteins may be equivalent to the 62 kDa nucleoporin described previously [76]. Similarly, a monoclonal antibody which recognizes a 68 kDa nuclear envelope-associated polypeptide of Xenopus oocytes can significantly inhibit the nuclear uptake of a variety of nuclear proteins in Xenopus oocytes [80].

X.B. Identification of putative nuclear signal receptors

A novel approach towards identifying a putative nuclear signal receptor is to raise an antibody to a synthetic peptide which might resemble the nuclear signal binding pocket of the receptor. Yoneda et al. [81] speculated that the binding pocket of the nuclear signal receptor might contain numerous acidic residues which could interact in a complementary fashion with the basic residues of a nuclear signal sequence. An antibody was raised to the synthetic peptide Asp-Asp-Asp-Glu- Asp because it was reasoned that this sequence might interact favourably with the SV40 large-T signal sequence. The anti-peptide antibody was found to bind to nuclear envelopes; however, it was not determined whether the antibody binds specifically to nuclear pores. As a consequence of binding to the nuclear envelope, the anti-peptide antibody can block the uptake of nucleoplasmin and an SV40 large-T signal peptide: BSA conjugate in a variety of tissue culture cells. The authors reported that, on a Western blot, the antibody recognizes protein species of 69 and 59 kDa, while the antibody immunoprecipitates proteins of 65, 54, 50, 43 and 34 kDa. It is not known whether these molecular mass species are related to one another, whether these proteins are associated with nuclear pores and whether they resemble any of the N-acetylglucosamine-bearing nucleoporins which have been identified by others [76,82,83,84]. Finally, it is not known whether the binding of this anti-peptide antibody to the nuclear envelope blocks the passive diffusion of low-molecular- mass dextrans into the nucleus and, most importantly, whether any of the proteins detected on Western blots actually bind to nuclear location signal peptides.

278

Adam et al. [85] reported that rat liver nuclear envelope proteins of 60 and 70 kDa specifically bind to a radiolabelled synthetic peptide homologous to the sequence of the SV40 large-T nuclear location signal. Interestingly, these proteins do not bind to a synthetic peptide homologous to the sequence of a defective SV40 large-T nuclear location signal. The authors demonstrated competition between labelled and unlabelled peptide for binding to these proteins and showed that the binding event is energy-independent. Furthermore, the binding of the synthetic peptide to the 60 and 70 kDa proteins demonstrates saturability. These observations are reminiscent of those reported for the binding of nucleoplasmin and SV40 large-T to nuclear envelopes prior to their translocation in the nucleus [57,59]. A variety of synthetic peptides, homologous to the sequences of SV40 large-T nuclear location signal variants, were tested for their ability to bind to the 60 and 70 kDa proteins. There is a direct correlation between the ability of these synthetic peptides to interact with the signal binding proteins and the ability of the sequences to function as nuclear location signals in the context of SV40 large-T [3,24]. Interestingly, none of the nuclear signal binding proteins identified in this study appears to be immunologically related to the N-acetylglucosamine-containing nucleoporins which have been described [76,82,83,84]. It has yet to be established that the 60 and 70 kDa proteins are in fact nuclear pore-associated proteins and that they participate in nuclear protein import.

X.C. Other proteins potentially involved in nuclear protein uptake

ATP depletion blocks nuclear signal-mediated protein import both in vitro [64,67] and in vivo [59,72]. A nuclear pore-associated ATPase may provide the energy for the translocation event. Photoaffinity labelling experiments have identified the presence of a 40-46 kDa ATPase in rat-liver nuclear envelopes [86,87] and a conserved 188 kDa ATPase/dATPase in the nuclear envelopes of a variety of different eukaryotes [88,89]. The 40-46 kDa ATPase has been implicated in RNA export from the nucleus, because its activity can be stimulated by either synthetic poly(A) or the poly(A) segments of natural mRNAs [87].

The 188 kDa ATPase/dATPase has been shown to be both structurally and immunologically related to myosin heavy chain [89]. Indirect immunofluorescence studies have localized the 188 kDa protein to the nuclear envelopes of a variety of eukaryotic cells. Berrios and Fisher [89] have noted that the eight annular subunits located on both the nucleoplasmic and cytoplasmic faces of nuclear pores [8] are nearly identical in size to the head of a myosin molecule. Furthermore, the myosin tail is sufficiently long and flexible to span the nuclear

envelope and form the lumenal walls of nuclear pores. It has been proposed that bidirectional transport of macromolecules through nuclear pores could be accom- plished by a peristaltic wave of contractions generated by ATP hydrolysis in the myosin heads at the margins of the nuclear pore complex [89].

The majority of nuclear signal bearing proteins described to date are larger than the 90 A diffusion channel of nuclear pores. It would seem likely that nuclear pores must dilate to allow the passage of nuclear proteins. Indeed, it has been shown that nucleoplasmin- coated gold particles of 200 A [58] and SV40 large-T signal peptide : "mununoglobulin conjugates of a theoret- ical size of 150 A [12] can traverse the nuclear envelope. Thus, a nuclear signal-responsive contractile machinery may regulate the size of the channel within nuclear pores. Evidence in support of a role for contractile-like proteins in nuclear upteke ¢~mes from the work of Schlinder and Jiang [90]. The authors reported that anti-myosin antibodies can block the accumulation of fluorescently labelled dextrans in isolated nuclei. Whether the myosin-like ATPase described by Berrios and Fisher [89] plays a role in nuclear signal-mediated uptake of proteins is yet to be established. Future studies may reveal how nuclear signal recognition regu- lates dilation of nuclear pores. In vitro studies suggest that nuclear signal-mediated pore dilation does not simply involve the enlargement of a diffusion channel, since non-nuclear proteins do not adventiously co- migrate with nuclear proteins into the nucleus, even when present in vast excess [64]. Thus, a 'transit' protein within the nuclear pore may selectively carry nuclear proteins through the dilated nuclear pore. Future studies will be required ~o test these mechanisms of nuclear protein translocation.

XI. Future prospects

Are there different classes of nuclear pores or do all nuclear pores have the capacity to recognize the bewildering number of different nuclear location signal sequences described to date? Evidence from electron microscopy studies suggests that individual nuclear pores have the capacity to translocate proteins containing different nuclear location signals [58]. Gold particles coated with either nucleoplasmin or an SV40 large-T synthetic peptide:BSA conjugate are able to pass through the same nuclear pore.

Additional evidence may come from the study of novel mutants of nuclear proteins which are ineffi- ciently transported to the nucleus. Knipe and Smith [91] reported that a temperature-sensitive mutant of the ICP4 protein of herpes simplex virus is not able to localize to the nucleus at the non-permissive temperature. In virally infected cells expressing the m~Jtant ICP4 protein at the non-permissive temperature, the

nuclear localization of at least two other viral proteins, ICP0 and ICPS, is impa/red~ The nuclear uptake of another viral protein, ICP27, is, however, unaffected. In the absence of the mutant ICP4 protein, both ICP0 and ICP8 are able to localize to the nucleus. This suggests that the mutant ICP4 protein blocks the nuclear localization of ICP0 and ICP8 but not ICP27. ICP4 can bind to ICP0 hence cytoplasmic retention of ICP0 by the mutant ICP4 may account for some of these observations. However, there is no evidence that ICP4 binds to ICP8. Hence, the mutant ICP4 may block the nuclear uptake o~f other proteins by obstructing the nuclear protein translocation machinery within nuclear pores. The fact that the ICP27 protein retains the ability to localize to the nucleus in the presence of the mutant ICP4 suggests that there may be functionally different classes of nuclear pores.

A frame-shift mutant of SV40 large-T appears to block the nuclear uptake of an unrelated nuclear protein, adenovirus fibre [92]. The expression of the frame- shift mutant lead to lethal consequences for the ceil. Introduction of a mutation into the wild-type nuclear location signal sequence of the frame-shift mutant abolishes its ability to block the nuclear uptake of adenovirus fibre. This suggests that the large-T mutant blocks the nuclear uptake of cellular factors which are essential for cell viability. One possib!e explanation for these observations is that there is only one type of nuclear pore and the frame shift mutant blocks the nuclear uptake of a variety of nuclear proteins. Alterna- tively, the SV40 large-T mutant may only block nuclear uptake through a subset of nuclear pores which are the usual route of nuclear transport for a crucial cellular factor. The data described above do not allow us to discern whether there are different classes of nuclear pores. However, the novel transport-blocking antigens of herpes simplex and SV40 viruses may prove to be useful in the identification of the cellular factors which mediate signal-dependent nuclear protein uptake.

Acknowledgements

The author would like to thank Dr. William Mark- land and Dr. Alan Smith for critical reading of the manuscript.

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Nuclear location signal-mediated protein transport