The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors

Cell, Vol. 69, 939-950,

June

12, 1992, Copyright

0 1992 by Cell Press

The Two Steps of Nuclear Import, Targeting to the Nuclear Envelope and Translocation through the Nuclear Pore, Require Different Cytosolic Factors Mary Shannon Moore and Giinter Laboratory of Cell Biology Howard Hughes Medical Institute The Rockefeller University New York, New York 10021

Blobel

Summary We have isolated two cytosolic fractions from Xenopus oocytes that contain all of the activity necessary to support both steps of nuclear import in digitoninpermeabilized mammalian cells: binding at the nuclear envelope and translocation through the nuclear pore. The first cytosolic fraction (fraction A) interacts with an import-competent, but not a mutant, nuclear localization sequence-bearing conjugate and stimulates its accumulation at the nuclear envelope in an ATP-independent fashion. The second cytosolic fraction (fraction B) gives no discernible effect when added alone; but when added either together with fraction A, or after fraction A, stimulates the passage of the conjugate from the outer nuclear envelope to the interior of the nucleus in an ATP-dependent fashion. Introduction The passage of macromolecules between the nucleus and the cytoplasm occurs through openings in the nuclear envelope called nuclear pores (for review, see Dingwall and Laskey, 1986). These passageways through the double membrane of the nuclear envelope are formed by nuclear pore complexes, large (1.24 x 1O8 daltons) and geometrically complex structures that extend into both the cytoplasm and the nucleoplasm (Reichelt et al., 1990; Akey 1989). The nuclear pore acts as an aqueous channel allowing free diffusion of small macromolecules at a rate inversely proportional to their mass. Dextrans with a molecular weight of less than 20 kd diffuse very rapidly through nuclear pores, those of 40 kd more slowly, and those of 70 kd not at all (Paine et al., 1975). Both smaller (<70 kd) and much larger endogenous proteins accumulate inside the nucleus at rates much faster than would be predicted by their mass, but this import is an active process requiring ATP, and to be so imported, the protein must either contain a nuclear localization sequence (NLS) or if not, be bound to another protein that does (Dingwall et al., 1982; Zhao and Padmanabhan, 1988). The NLSs for a number of nuclear proteins have been functionally identified, and even though as a group they lack a strict consensus sequence, most contain a short stretch of basic amino acids (for review, see Garcia-Bustos et al., 1991). Many NLSs are similar to the one of the SV40 T antigen (PKKKRKV), which is one of the best characterized (Kalderon et al., 1984; Garcia-Bustos et al., 1991). Based on competition studies, proteins possessing these

T antigen-like sequences may belong to a general class of proteins that are imported through nuclear pores via the same mechanism (Michaud and Goldfarb, 1991, 1992). The addition of such a sequence to a normally nonnuclear protein can be sufficient to result in the targeting of that protein to the nucleus (Kalderon et al., 1984; Goldfarb et al., 1986; Lanford et al., 1986). The targeting efficiency of these sequences can be affected by modifications (such as phosphorylation) of the flanking sequences (Rihs and Peters, 1989; Rihs et al., 1991; Jans et al., 1991), by the presence of multiple copies within a protein and their distance from each other (Lanford et al., 1986; Roberts et al., 1987; Dworetzky et al., 1988; Robbins et al., 1991), and by the accessibility of the NLS to the import machinery (Roberts et al., 1987). Nuclear import in vivo is a highly specific and saturable process and therefore has been postulated to involve specific NLS receptors either as components of the nuclear pore complex or as soluble recognition factors (Goldfarb et al., 1986; Silver, 1991). Much of the work on nuclear import has focused on identifying the components of the import machinery that act as receptors for the T antigenlike NLSs. Affinity approaches have generated a number of potential candidates for these NLS receptors (Adam et al., 1989; Benditt et al., 1989; Yamasaki et al., 1989; Li and Thomas, 1989; Silver et al., 1989; Lee and Melese, 1989; Meier and Blobel, 1990; Adam and Gerace, 1991) from both mammalian cells and yeast, but to date there has been little consensus about the role of these different proteins in nuclear import (see Discussion). At the molecular level, the process of nuclear import is ill defined but can be divided into at least two steps, separable by their differing temperature and ATP requirements. In the absence of ATP, or in the cold, import substrates accumulate at or near the nuclear pore complex but are not transported through (Richardson et al., 1988; Newmeyer and Forbes, 1988). The addition of wheat germ agglutinin (WGA) has a similar effect, blocking translocation through the pore but not the binding of an import substrate to the cytoplasmic face of the nuclear pore complex (Finlay et al., 1987; Yonedaet al., 1987). Here we present evidence to show that these two stages of import (binding and translocation) can be separated in another way, by their requirements for distinct cytosolic factors. The possible relationship between these cytosolic factors and others previously implicated in nuclear import is discussed. Results The purpose of this study was to identify cytosolic factors required for protein import into the nucleus. To identify and purify these factors, we have used the in vitro nuclear import assay developed by Adam et al. (1990). In this system, tissue culture cells grown on coverslips are treated with a low concentration of digitonin, which permeabilizes the plasma membrane to the passage of macromolecules but leaves the nuclear envelope intact. A fluorescent im-

Cdl 940

Wild

HSA

Type

Peptide

Conjugate

128 Cys-Tyr-Thr-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val.COOH

0 Mutant

0 HSA

Figure

1. Peptide

Peptide

Conjugate 128

-Cys~Ty,-Thr-Pro-Pro-Lys-Thr-Lys-Arg-LyS-Val-COOH

Conjugates

Peptides containing the wild-type and mutant NLS of SV-40 T antigen were synthesized and coupled to rhodamine-labeled HSA. The amino terminal cysteine and tyrosine (in italics) are not part of the T antigen sequence.

port substrate is added, and nuclear import is monitored by the accumulation of this substrate within the nucleus. Adam et al. (1990) demonstrated that protein import into the nuclei of these permeabilized cells faithfully mimics that which occurs in vivo, requiring both ATP and a functional NLS, as well as being completely dependent on the addition of exogenous cytosol. We have used as an import substrate rhodaminelabeled human serum albumin (HSA) coupled to peptide containing the SV40 T antigen NLS Pro-Lys-Lys128-LysArg-Lys-Val (Figure 1) (Lanford et al., 1986; Newmeyer and Forbes, 1988). A mutant conjugate was also prepared in which a threonine was substituted for Lys128 within the targeting sequence (Figure 1); this mutation in the SV40 T antigen has been shown to result in the cytoplasmic localization of this protein in vivo (Kalderon et al., 1984). As shown in Figure 2, the wild-type conjugate was efficiently imported into the nuclei of permeabilized buffalo rat liver (BRL) cells in the presence (Figure 2 [2]) but not in

the absence (Figure 2 [l]) of cytosol. The mutant conjugate failed to enter the nucleus regardless of the presence of cytosol (Figure 2 [3] and Figure 2 [4]). The cytosol was obtained from Xenopus oocytes, and preliminary experiments indicated that cytosol from this source had a higher specific activity than cytosol prepared from either rat liver or rabbit erythrocytes (data not shown). Interestingly, once inside the nucleus, the wild-type conjugate always accumulated first in the nucleolus (Figure 2 121, uppermost nucleus), and this phenomenon has been observed by other investigators both in vivo (Breeuwer and Goldfarb, 1990) and in vitro (Meier and Blobel, 1990). It remains to be determined whether this localization of the conjugate is simply because of nonspecific accumulation within the nucleolus, or alternatively is a result of binding of the conjugate to specific sites that are more abundant in the nucleolus than the nucleoplasm. (Meier and Blobel, 1990, and references therein). Isolation of Fractions A and B The results shown in Figure2 simply confirmed what Adam et al. (1990) had already reported, namely that nuclear import in digitonin-permeabilized cells requires the addition of exogenous cytosol. Presumably, this addition is required to replace soluble factors necessary for nuclear import that are lost during permeabilization, and the next step was to fractionate the cytosol to locate these active components. Xenopus oocyte cytosol was passed over a DE-52 anion exchange column, the column was eluted with a salt gradient, and the fractions were assayed for nuclear import activity. While none of the column fractions supported nuclear import at levels seen with the starting material, one

Figure 2. Peptide in Nuclear Import

Conjugates

Are Functional

Digitonin-permeabilized BRL cells were incubated in the standard assay mixture containing either the wild-type ([I] and 121) or mutant ([S] and [4]) peptide conjugates. Coverslips were incubated for 15 min in either the presence (121 and 141) or absence ([I] and [2]) of Xenopus oocyte cytosol (IO mglml) prior to washing and fixation. ATP plus a regenerating system were present in all samples. Bar, 10 pm.

mut

;iylic

Factors

Required

for Nuclear

Import

Figure 3. The Effects of Xenopus Cytosol Fraction A in the Nuclear Import Assay

Wt

Digitonin-permeabilized BRL cells were incubated in the standard assay mixture containing either the wild-type ([I] and 121) or mutant ([3] and [4]) conjugate. Coverslips were incubated for 15 min in either the presence ([2] and [4]) or absence ([1] and [3]) of fraction A (1.3 mglml) prior to washing and fixation. ATP plus a regenerating system were included in all incubations. Bar, 15 pm.

mut

peak of activity was found in the fractions eluting at around 450 mM salt, which resulted in a striking visual change in the distribution of the fluorescent conjugate in the import assay (Figure 3). After incubation with these fractions, essentially all of the cytoplasmic background staining normally seen in the absence of cytosol was gone and was replaced by a bright rim of fluorescence around the nuclear envelope (Figure 3 [l] and Figure 3 [2]). This effect was seen only with the wild-type (Figure 3 [2]) and not with the mutant conjugate (Figure 3 [4]). These pooled DE-52 fractions (called fraction A) also appeared to stimulate a small amount of nuclear import, as noticed primarily by the increase in nucleolar staining. Increasing the concentration of fraction A, however, could not increase the amount of import beyond the level shown in Figure 3 (data not shown), and clearly, if fraction A was involved in nuclear import, another necessary activity had been lost during fractionation. In the hope of locating a synergistic activity, the DE-52 fractions were assayed again but this time in the presence of fraction A (data not shown). Such a stimulatory activity (called fraction B) was detected in the flow-through fractions of that column and was subsequently partially purified by different chromatographic steps (see Experimental Procedures). Fraction B (Figure 4 [l] and Figure 4 [2]), unlike fraction A (Figure 4 [3] and Figure 4 [4]), had no visible effect when added by itself in the import assay and gave a staining pattern indistinguishable from that of the buffer control (compare Figure 4 [I] and Figure 4 [2] with Figure 2 [l] and Figure 3 [l]). The simultaneous addition of fractions A and B, however, resulted in a level of nuclear import that appeared equal to (if not better than) that ob-

tained with unfractionated cytosol at saturation (Figure 4 IS]). This import-stimulating activity of fraction B was seen only in the presence (Figure 4 [5]) but not in the absence of ATP (Figure 4 IS]). In contrast, both the rim staining and the small amount of import obtained with fraction A did not require the addition of ATP (Figure 4 [3] and Figure 4 [4]) Quantitation of Relative Activities of Cytosolic Fractions A method of quantifying the nuclear fluorescence obtained in these import assays was developed using an image processing and analysis system. This system allows the quantitation of the mean nuclear fluorescence of any population of nuclei. It should be noted that this system of quantitation was designed to compare relative amounts of import between a large number of formaldehyde-fixed samples (i.e., for assaying column fractions) and was not designed (or calibrated) to measure absolute rates of import or levels of accumulation. The values obtained for mean nuclear fluorescence were quite reproducible from assay to assay for a given incubation condition and thus allowed a direct comparison of the relative activities of the cytosolic fractions. Shown in Table 1 are the results of one such assay. The addition of unfractionated cytosol(10 mglml) resulted in a large increase in nuclear import (samples 1 and 2) of the conjugate as measured by mean nuclear fluorescence. A titration experiment had previously shown that this protein concentration was saturating with respect to activity for this batch of cytosol (data not shown). Adding either a lower concentration of cytosol (sample 3) fraction A alone (sample 4) or fraction B alone (sample 5) did not result in

Cdl 942

+ATP

-ATP

I;‘-

Unlract,onated *

~Concenrrat~on

2

4 Concentration

Figure 5. The Activities spect to Each Other

of Fractions

;

of A (B at 2 mgiml)

-Concentration

0

Cytosol of B (A at 4 mg/ml)

6 (mglml)

A and 0 Are Saturable

with Rs

Digitonin-permeabilized BRL cells were incubated in the standard assay mixture (including the wild-type conjugate and ATP plus a regenerating system) for 15 min at room temperature prior to washing and fixation. The cytosolic fractions were included at the indicated concentrations. An average of 22 nuclei were scanned per point.

Figure

4. The Effects

of Fractions

A and B in the Nuclear

rating concentration (sample 2), the addition of either fraction A (sample 7) or B (sample 8) did not result in an increase in nuclear import. At a lower concentration of cytosol, however (sample 3), the addition of either fraction could stimulate import (samples 9 and 10) but only to a level of approximately 50% of that obtained with A and B together (sample S), or unfractionated cytosol at saturation (sample 2).

Import Assay

Digitonin-permeabilized BRL cells were incubated in the standard assay mixture (including the wild type conjugate) with either ATP plus a regenerating system ([I 1, [3], and [5]) or apyrase ([2], [4], and 161). Included in the incubation mixture were either fraction B (2.1 mglml; 111and [2]), fraction A (I .3 mglml; [31 and [4]), or both together ([S] and IS\). Incubation was for 15 min at room temperature prior to washing 2nd fixation. Bar, IO Km.

Fractions A and B Are Saturable with Respect to Each Other To determine whether either of the two fractions (A or 6) was a limiting factor in the import reaction, the experiment shown in Figure 5 was performed. In this experiment, either the concentration of A (open circles) or B (closed circles) was varied while keeping the concentration of the other fraction constant. The results indicated that not only were A and B both required for nuclear import, but that the activity of each fraction was saturable with respect to the

a measurable increase in the mean nuclear fluorescence relative to the buffer control. The addition of fractions A and B together (sample 6) resulted in a level of nuclear import slightly higher than that achieved with unfractionated cytosol at saturation (sample 2), indicating that all of the import activity present in the starting material could be accounted for in these two fractions. When unfractionated cytosol was present at a satu-

Table

1. Quantitation Unfractionated

1 2 3 4 5 6 7 8 9 10 A mmus scanned

of Nuclear Cytosot

Import Fraction

10 mglml 4 mglml

A

Fraction -

1.3 mglml

10 10 4 4

mglml mg/ml mglml mglml

1.3 mglml 1.3 mglml

0.5 mglml 0.5 mglml 0.5 mg/ml

1.3 mglml 0.5 mglml

B

Mean Nuclear 9 111 13 11 9 128 102 ‘22 57 63

Fluorescence

Percent

of Control

0% 100% 4% 2% 0% 1 17% 91% 91% 47% 53%

sign indicates that the addition was not made. Mean nuclear fluorescence is expressed in arbitrary units, and an average of 21 nuclei were to produce each mean value. The percent of control values are expressed relative to sample 1 (0%) and sample 2 (100%).

$&osolic

Table

Factors

Required

2. Summary

for Nuclear

of Purification

of Fractions

Protein Isolation

A and B

(mg)

Activity

(U)

Specific

Isolation

Fold Purification

Yield (o/o)

2460 240

19,680 33,720

8 141

18

100% 171%

2700 419 32

25,191 17,351 3,390

9 41 106

5 12

100% 75% 21%

of activitv

are as defined

in Experimental

Procedures.

other. Shown for comparison is a concentration curve of unfractionated cytosol alone (closed diamonds). This preparation of cytosol had a lower specific activity than the preparation used in Figure 4.

apparent during the purification of B. As shown, the purification steps for fractions A and B resulted in roughly similar increases in the specific activities (18 versus 12) but with very different yields (171% versus 21%). Regarding the purification scheme, it should be added that while the B activity could be recovered in the flow through from the DE-52 column during the purification of A, fraction A was irreversibly inactivated by the addition of ammonium sulfate during the isolation of B (data not shown).

Recovery of Activity During Purification of A and B When either the A or B fraction was included in the import reaction at saturation, the activity of the other fraction could be measured independently (Figure 5). This made possible the calculation of recovery and specific activities of each of the fractions during their isolation. Shown in Table 2 is a balance sheet for a representative partial purification of each of the two fractions. As can be seen, more A fraction activity (171%) was recovered from the DE-52 column than could be detected in the starting material, implying that some inhibitory factor was removed by this column. This inhibitory factor might be nuclear proteins, such as nucleoplasmin, released from the oocyte nuclei during the initial homogenization. Krohne and Franke (1980) showed that homogenization of isolated Xenopus oocyte nuclei results in a release of more than 90% of the nuclear protein, including all of the nucleoplasmin, into the high speed supernatant fraction. These nuclear proteins, if present, would presumably compete with the fluorescent conjugate for import in the assay. Whatever the nature of the inhibitory factor, it was not

3. Effects

of NEM Treatment

Unfractionated 1 2 3 4 5 6 7 8 9 10 11 12 13

(Ulmg)

of B

cytosol 25-50% (NH),SO, Q-Sepharose

Table

Activity

of A

cytosol DE-52

Units

Import

mock NEM NEM NEM

Cvtosol

on Activity

of Cytosolic

Fraction

A

Fractions Fraction

untreated untreated mock NEM mock NEM untreated untreated

Fraction A Is N-Ethylmaleimide Sensitive; B Is Not N-ethylmaleimide (NEM)-sensitive soluble factors from both Xenopus egg extract and bovine erythrocytes have been implicated in nuclear import (Newmeyer and Forbes, 1990; Adam and Gerace, 1991). Shown in Table 3 are the effects of NEM treatment on our cytosolic fractions. NEM pretreatment of unfractionated cytosol (sample 3) completely abolished its ability to support nuclear import (compare sample 3 with samples 1 and 2). The addition of untreated fraction A to this NEM-treated cytosol partially restored the import activity (sample 4) while the addition of fraction B (sample 5) had no effect. This result implied that the NEM-sensitive component present in unfractionated cytosol was likely to be present in fraction A, and this was shown to be the case. The mixture of either mock treated or untreated fractions A and B resulted in large

mock NEM untreated untreated mock NEM

E

Mean 9 75 13 48 15 18 10 9 15 139 16 142 118

Nuclear

Fluorescence

Percent

of Control

0% 100% 6% 59% 9% 14% 2% 0% 9% 197% 11% 202% 165%

A minus sign indicates that the addition was not made. Mock-treated, NEM-treated, and untreated cytosolic fractions were added at the following concentrations: unfractionated cytosol, 10 mg/ml; fraction A, 1.3 mglml; fraction B, 0.5 mglml. NEM treatment was carried out as described in Experimental Procedures. Mock refers to samples in which excess DTT was added prior to the addition of NEM. Mean nuclear fluorescence is expressed in arbitrary units, and an average of 17 nuclei were scanned to produce each mean value. The percent of control values are expressed relative to sample 1 (0%) and sample 2 (100%).

Cell 944

Mock A

NEM A

A+WGA

Figure 6. The Effects of Fraction A

of WGA and NEM Pretreatment

on the Activity

Digitonin-permeabilized BRL cells were incubated in the standard assay mixture (including the wild-type conjugate and ATP plus a regenerating system) for 15 min at room temperature prior to washing and fixation. (1) no additions; (2). mock treated fraction A (1.3 mglml); (3). NEM-treated fraction A (1.3 mglml). NEM and mock treatments were carried out as described in Experimental Procedures. In (4) WGA (Vector, 0.5 mglml) was present in addition to untreated fraction A (1.3 mglml). Bar, 10 urn.

amounts of nuclear import (samples 10 and 12). NEM pretreatment of the A fraction (sample 11) completely abolished the nuclear import obtained with the two fractions, while the same treatment of the B fraction had little or no effect (sample 13). As shown in Figure 6, NEM pretreatment of fraction A abolished not only its ability to support nuclear import but also its ability to stimulate binding of the conjugate to the nuclear envelope. The addition of NEM-treated fraction A (Figure 6 [3]) resulted in a staining pattern that appeared very similar to that obtained in the absence of fraction A (Figure 6 [l]). In these samples (Figure 6 [l] and Figure 6 [3]), the cytoplasmic background staining was quite noticeable, and the bright rim staining and the small amount of import obtained with untreated Aor mock-treatedA(Figure 6 [2]) was not seen. WGA inhibits nuclear import but does not inhibit binding of an import substrate to the nuclear pore complex (Finlay et al., 1987; Yoneda et al., 1987). This inhibition is thought to be due to an interaction of the lectin with a family of

nuclear pore complex proteins that contain O-linked N-acetylglucosamine (Davis and Blobel, 1986, 1987; Snow et al., 1987; Holt et al., 1987). Shown in Figure 6 (4) is a sample that received the simultaneous addition of fraction A and WGA. When added together with fraction A, WGA did not prevent the accumulation of the conjugate at the nuclear envelope. It did, however, block the small amount of import normally obtained with this fraction (Figure 6 [4]; compare with Figure 3 [2], Figure 4 [3], and Figure 6 [2]). Incubation with either fraction A alone or A and B together at 4% gave the same effect as WGA, resulting in rim staining without any import (data not shown). Fraction B Is Required for Passage into the Nucleus The results of the previous experiments indicated that the requirement for fraction A in the process of nuclear import preceded the requirement for fraction B. Figure 7 shows an experiment designed to determine whether conjugate prebound to the nuclear envelope by fraction A could be chased into the nucleus by the subsequent addition of fraction B. In this experiment, all coverslips were incubated for 10 min with fraction A plus conjugate and then were washed to remove unbound A fraction and conjugate. They were then incubated for 5 min with either ATP alone (Figure 7 [l]), fraction B plus ATP (Figure 7 [2]), or fraction B without ATP (Figure 7 [3]). The sample incubated with ATP alone appeared identical to samples fixed immediately after the first incubation (data not shown; compare Figure 7 [l] with Figure 3 121, Figure 4 [3], and Figure 4 [4]), demonstrating that even after removal of excess A fraction (and conjugate), the perinuclear binding of the conjugate was of sufficient affinity such that little if any was lost during the second incubation. In contrast, the addition of ATP plus fraction B (Figure 7 [2]) resulted in a redistribution of the conjugate associated with the nuclear periphery. After this incubation, the perinuclear rim was gone, and the interior of the nuclei appeared correspondingly brighter, an increase that was most apparent in the nucleolar staining. This increase in intranuclear fluorescence was not dramatic, nor could it have been, since the only conjugate available for import in the second incubation was that present in the perinuclear rim. The addition of fraction B without ATP (Figure 7 (31) resulted in a slight decrease in the amount of conjugate present in the nuclear rim. This loss could not be due to import, since there was not a corresponding increase in the intranuclear fluorescence and therefore was presumably due to dissociation. These results demonstrate that nuclear import in this in vitro system does not require the presence of additional A activity once the import substrate is bound at the nuclear envelope, and further show that the only other cytosolic activity that is required after the binding step can be supplied by fraction B. Whether ATP is required by the nuclear pore complex for transport, by fraction B, or by both cannot be determined at this time. D%ing the course of this work, Jans et al. (1991) showed that p34cdc*-mediated phosphorylation at Tlz4 inhibits nuclear import of the SV40 T antigen. To determine whether this phosphorylation site (present in our peptide conju-

;&osolic

Factors

Required

for Nuclear

1st Incubation:

Import

A + conjugate. then wash

2nd

Incubation

Buffer + ATP

Figure

7. Fractions

A and B Can Be Added Se-

Permeabilized BRL cells were incubated for 10 min at room temperature in the presence of the wild-type import conjugate and fraction A (1.3 mg/ml); neither ATP nor apyrase were included in this incubation. The co&slips were washed twice in 1 ml cold buffer A’, then incubated for 5 min at room temperature with either 1 mM ATP (1) fraction B (2.0 mglml) plus 1 mM ATP (2) or fraction B without ATP (3) prior to washing and fixation. Bar, 10 pm.

B + ATP

gates) was influencing our results, we repeated the experiments shown, substituting conjugates originally described by Meier and Blobel (1990). These conjugates, lacking the phosphorylation site, gave identical results (data not shown). Discussion We have isolated two cytosolic fractions (A and B) that contain all of the activity necessary to support import of HSA-NLS conjugates into the nuclei of digitonin-permeabilized cells. Fractions A and B appear to act in a sequential fashion, with fraction A involved in NLS recognition and targeting to the nuclear envelope and fraction B involved in the subsequent passage of the bound substrate into the interior of the nucleus. The recognition of the import substrate by fraction A was sequence specific, since it was abolished by a single amino acid substitution within the NLS. The accumulation of the import substrate at the nuclear envelope obtained with fraction A occurred in the absence of ATP, at 4’%, and in the presence of WGA. Any of these three conditions, in contrast, inhibited the second stage of import, where the B fraction was required. The activity of the A fraction was NEM sensitive, while that of the B fraction was not. The addition of the A fraction simultaneously with the

import substrate resulted in the accumulation of the substrate in a narrow band encircling the nucleus. This continuous rim of fluorescence does not resemble the clearly punctate pattern obtained when nuclear pore complex proteins are localized by immunofluorescence microscopy (Davis and Blobel, 1986) and therefore it seems unlikely that the nuclear pore complex can be the sole site of accumulation. Rim staining, rather than punctuate staining, is consistent, however, with the observations of Richardson et al. (1988) on the distribution of microinjected nucleoplasmin or nucleoplasmin-gold conjugates in ATPdepleted or chilled cells. They observed by electron microscopy that in these transport-inhibited cells, the gold conjugate accumulates not only at the cytoplasmic face of the pore complex but also on small fibers that radiate from the pore complex into the cytoplasm. These fibers are not visible on the isolated rat liver nuclei used in the in vitro system of Newmeyer and Forbes (1988). When tranport is blocked in their in vitro system, the import substrate accumulates only at the cytoplasmic face of the nuclear pore complex, and this localization is consistent with the punctuate labeling pattern they see by immunofluorescence microscopy. The precise location in our permeabilized cells where the conjugate binds in response to fraction A has to await electron microscopical analysis. One prediction based on the fluorescence pattern is that these

Cell 946

sites will involve the fibrils observed by Richardson et al. (1988). Whether the active component of the A fraction remains bound to the conjugate in this region or dissociates after the binding reaction is at present unknown. The simplest model for protein import into the nucleus involves the signal-mediated binding of a soluble receptor to a protein destined for import and a targeting of this complex to the nuclear envelope. Fraction A carries out both functions. In this respect, the active component of fraction A acts in a manner homologous to the signal recognition particle involved in protein transport into the endoplasmic reticulum (Walter and Blobel, 1980). In preliminary experiments, the relative molecular mass of the active component of the A fraction appears to be on the order of 200-250 kd as determined by both gel filtration chromatography and sucrose gradient centrifugation (data not shown). This relatively large size might indicate that the active component of fraction A, like the signal recognition particle, consists of a complex of proteins. An analysis of the 6 fraction, in contrast, by gel filtration chromatography indicated a relative molecular mass for the active component of around 80 kd (data not shown). In all previous studies of nuclear import, a requirement for the activity supplied by fraction B has gone undetected. This is because the activity of fraction B is required at a stage of import that takes place at, or very close to, the nuclear pore, and when import is blocked by energy depletion or cold or WGA, the distinction between the requirements of the pore complex itself versus the requirement for a distinct cytosolic factor cannot be made. The exact nature of the activity supplied by the B fraction is at present unknown, but there are several possibilities. Fraction B could be required for the opening of the nuclear pore complex to permit the passage of bound import substrate. Alternatively, it could modify in some way the import substrate bound at the nuclear envelope, thereby making it competent for translocation through the nuclear pore. Another possibility is that the active component of the B fraction functions as a motor protein that actively transports proteins through the pore complex. If so, its apparent molecular weight of 60 kd would make it smaller than any of the previously described motor proteins including kinesin, dynein, dynamin, or any of the myosins. With regard to the potential function of fraction B, we have been unable to demonstrate any affinity between B and the wild-type NLS on peptide affinity columns. In trial experiments, these affinity columns removed all of the A fraction activity from unfractionated cytosol (along with many proteins) but removed none of the B activity (data not shown). This finding may indicate that if fraction B acts on the import substrate, it does so without interacting with the targeting sequence directly and requires an intermediary (fraction A?) for its activity. One clue to the function of fraction B may lie in the small amount of import obtained with fraction A alone which, while not dependent on the addition of fraction B or ATP, was inhibited by WGA and by incubation at 4”C, implying that this small amount of import was mediated by the nuclear pore complex and was not simple diffusion. These results indicate that either a small amount of import can

occur independently of the B activityorthat a small amount of B is retained inside the cells after permeabilization. This small amount could either be bound at the pore complex or, if B shuttles between the cytoplasm and the nucleus, inside the nucleus. If this retained pool of B had access to ATP, it might be sufficient to catalyze a few rounds of internalization before being lost by dilution. What is the relationship between fractions A and B and factors previously implicated in nuclear import? Newmeyer and Forbes (1990) reported that NEM treatment of Xenopus egg cytosol abolishes its ability to support both nuclear import and signal-mediated binding of an import substrate to the nuclear pore complexes of isolated rat liver nuclei. Proteins precipitable by 40% ammonium sulfate from untreated cytosol are able to restore this activity to NEM-treated cytosol, and this salt fraction is called nuclear import factor-l (NIF-1). NIF-2 is another NEM-sensitive cytosolic factor that appears to stimulate NIF-1 and is inactivated by treatment with ammonium sulfate. Fraction A is also isolated from Xenopus tissue, is NEM-sensitive, and is involved in binding of the import substrate to the nuclear envelope, but its relationship to NIF-1 or NIF-2 is unclear for the following reasons. As mentioned previously, treatment of fraction A with ammonium sulfate destroyed all of its activity. Furthermore, fraction A (unlike NIF-1) did not require the addition of any exogenous cytosol to support nuclear envelope binding. Finally, NIF-1 was reported to resolve into two peaks of transport stimulating activity on gel filtration chromatography; one peak with a relative molecular mass of greater than 500 kd and another at around 50 kd, neither of which is consistent with the size of the active A component. These differences in the properties of fraction A and NIF-1 and NIF-2 might be a result of either their method of preparation or their tissue of origin. Fraction A was isolated from oocytes, cells that contain nuclei and actively engage in nuclear import. NIF-1 and NIF-2, in contrast, are obtained from eggs in which the nuclei are disassembled. The possibility exists that factors required for nuclear import exhibit different properties (modifications, associations with other components, etc.) in these two cell types. Other differences may lie in the assay system itself, namely in the use of digitoninpermeabilized cells as opposed to isolated nuclei. Isolated nuclei have to be “repaired” by the addition of egg extract before they are competent for import, and NIF-1 or NIF-2 might supply certain factors that are retained on the nuclei of permeabilized cells. A number of proteins have been tentatively identified and designated NLS-binding proteins on the basis of their ability to bind the SV40 T antigen and other NLSs (Adam et al., 1989; Benditt et al., 1989; Yamasaki et al., 1989; Li and Thomas, 1989; Silver et al., 1989; Lee and Melese, 1989; Meier and Blobel, 1990; Adam and Gerace, 1991). Of these, however, only the proteins purified .by Adam and Gerace (1991) have actually been shown to have any activity in a nuclear import assay. These ‘investigators ide%fified and purified a doublet of proteins (M, 54,00056,000) from bovine erythrocytes based on their ability to be chemically cross-linked with the SV40 T antigen NLS. These purified proteins exhibit no activity in the nuclear

Cytosolic 947

Factors

Required

for Nuclear

Import

import assay when added alone, but will stimulate several-fold the amount of import obtained with subsaturating concentrations of unfractionated cytosol, and this activity is NEM sensitive. Fraction A will also increase the activity of low amounts of cytosol (as will B), is NEM sensitive, and appears to interact with the targeting sequence. The most striking effect of fraction A, however, its ability to redirect the conjugate to the nuclear envelope, is not shared by the purified bovine NLS receptors. This difference might be explained by the rather trivial fact that our import conjugate contains a greater number of localization sequences than does theirs (mean of 10 versus 5), and an increase in the number of targeting sequences per molecule has been shown to result in more efficient accumulation at the nuclear envelope (Dworetzky et al., 1988). An alternative explanation, and the one that we favor, is that fraction A does contain the Xenopus functional equivalent of the bovine NLS receptors but as only one component of a larger complex. The bovine NLS receptors elute as monomers on gel filtration, and, as mentioned previously, the active component of the A fraction seems to be much larger than this. If the active component of the A fraction is a complex of more than one protein, this would explain both its larger size relative to the bovine NLS receptors and its additional targeting activity. These cytosolic factors (A and B) were isolated on the basis of their ability to support nuclear import of a conjugate containing the SV40 T antigen NLS. In experiments not shown, similar results were obtained with a peptide conjugate containing the NLS of human lamin A/C (SVTKKRKLE) (Loewinger and McKeon, 1988), which is similar but not identical to that of the T antigen. Whether fraction A has the capability to recognize most or all NLSs of the T antigen-like class, or whether there are instead additional receptors with different specificities remains to be determined. Targeting signals not of this class, such as the bipartite signal of the Ul snRNA, which is composed of both a 5’-trimethylguanosine cap as well as an unidentified element on the Sm proteins, will probably require a different receptor than that supplied by the A fraction (Mattaj and DeRobertis, 1985; Hamm et al., 1990; Fischer and Luhrmann, 1990; Fischer et al., 1991; Michaud and Goldfarb 1992). An interesting question will be whether the B fraction activity is required for all types of nuclear import, or is instead specific for only those import substrates targeted to the nuclear envelope by the A fraction. Purification of the active component of each of these cytosolic fractions, experiments that are in progress, should permit a more detailed dissection of the import pathway. Experimental

Procedures

Buffers Buffer A: 20 mM HEPES-KOH (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol (DTT). Buffer A*: buffer A containing leupeptin. aprotonin, and pepstatin at 1 wglml each. Buffer B: 20 mM HEPES-KOH (pH 7.3), 2 mM magnesium acetate, 2 mM DTT. Buffer C: buffer B containing 10% glycerol. The pH of all buffers was adjusted at room temperature. Preparation of Import Conjugate Peptides were synthesized at the Rockefeller

University

biopolymer

facility on a model 430A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA.). To prepare theconjugates, 6 mg of HSA(Calbiochem)wasdissolved in 8 ml of 0.1 M sodium carbonate (pH 9.0), 50 mM NaCI. This solution was mixed with 600 fd of a 1 mglml solution (in dimethyl sulfoxide) of tetramethylrhodamine isothiocyanate (TRITC, Sigma #T-5646) and rotated end over end for 3.5 hr at room temperature in the dark. The solution was concentrated to 1 ml by centrifugation in a Centricon(Amicon), and unbound TRITC was removed by passage over a 10 ml G-25 column equilibrated in 50 mM sodium borate (pH 7.6). The fractions containing TRITC-HSA were pooled and concentrated to 500 ~1 as before. Two milligrams of sulfo-SMCC (Pierce) was added and incubated for 45 min at 37°C with occasional shaking. Excess crosslinking reagent was removed by passage over a G-25 column equilibrated in 0.1 M Tris (pH 7.0). The sulfo-SMCC-activated HSA-TRITC was split into two halves and to each half was added 250 pl of a 6 mgl ml solution of either wild-type or mutant peptide dissolved in 0.1 M Tris (pH 7.0). The mixture was incubated overnight at 4°C in the dark. Ten microliters of 0.1 M P-mercaptoethanol was added to each tube and incubated for 20 min at room temperature. The resulting conjugates were dialyzed extensively in the dark against 20 mM HEPES-KOH (pH 7.3), 100 mM potassium acetate, adjustecl to 2 mglml in bufferA, frozen in liquid nitrogen, and stored at -20°C. For short-term use, an aliquot was thawed, diluted 1:5 in buffer A’, microfuged to remove aggregates, and held at 4OC (in the dark) for a period of several weeks. From the shift in mobility on SDS-polyacrylamide gel electrophoresis, the conjugation resulted in a mean incorporation of ten peptides per albumin molecule for both the wild-type and mutant peptides.

Nuclear Import Assay We are using the digitonin-permeabilized cell method for studying nuclear importessentiallyasdescribed byAdam etal. (1990); however, we have made a number of technical modifications. Their method, including our modifications, is as follows. BRL cells were grown on 12 mM glass coverslips in individual wells of a 24-well plate in Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum, 20 mM HEPES, penicillin (100 U/ml), and streptomycin (100 fig/ml). The cells were passaged onto the coverslips from l-4 days in advance of use at a density such that on the day of use they were subconfluent. For permeabilization, the tissue culture plate was placed on ice, the wells were aspirated, and 1 ml of cold buffer A’ was added. The wells were aspirated, and 1 ml of cold buffer A’ containing 35 pglml digitonin (Gallard-Schlesinger Industries, Inc.) was added. The digitonin was diluted into buffer A* immediately before use from a 20 mglml stock in dimethyl sulfoxide. The plate was incubated on ice for 5 min, and then the wells were aspirated, and 1 ml of cold buffer A” was added. The coverslip containing the permeabilized cells was removed from the well and pressed briefly on its edge against filter paper to wick off excess moisture. The coverslip was placed cell side down on top of 20 pl of incubation mixture on Parafilm. Incubation was for 15 min at room temperature, and the reaction was terminated by the addition of 250 ~1 of cold buffer A’ to the sample. The coverslip was transferred back to the original plate on ice into 1 ml of cold buffer A’. The well was aspirated, and 1 ml of cold fixative consisting of 3% paraformaldehyde (w/v) in buffer A without DTT was added and incubated for 15 min on ice. The coverslips were removed from the fixative, blotted as above to remove excess moisture, and mounted on a drop of 10% phosphate-buffered saline and 90% glycerol containing 1 mglml phenylenediamine (Aldrich). The edges of the coverslip were sealed with nail polish. For a standard assay reaction, the following reagents were mixed in a 500 VI microfuge tube on ice: 1 pl of 20 mglml bovine serum albumin (Sigma #A-7030), 1 pl of 20 mM ATP (Sigma #A-5394), 1 pl of 100 mM phosphocreatine (Sigma #P-6915), and 1 ~1 of 400 U/ml creatine phosphokinase (Sigma #C-3755). Each of these stock solutions was prepared in bufferA’, stored at -20°C, and thawed immediately before use. One microliter of either the wild-type or mutant conjugate (400 pglml in buffer A’) was added to give a final working concentration of 20 pg/ml. Xenopus oocyte cytosol or the partially purified fractions were added at the indicated concentrations, and the total volume was adjusted to 20 VI by the addition of buffer A’. For

Cell 948

ATP depletion experiments, 1 ~1 of 2000 U/n.; ,pyrase (Sigma#A-6410) was substituted for ATP and the regeneratl ; system. Fluorescence Microscopy and Quantitation Photography The samples were observed and photographed on a Zeiss Axiophot microscope equipped with a 63 x II .4 N. A. objective. The fluorescence micrographs were obtained with T-MAX 400 film developed at 1600 ASA. All of the panels within each given figure were from the same experiment and were exposed and printed for the same length of time. Quantitation of Fluorescence Nuclear fluorescence was quantitated using the Image-l/AT image processing and analysis system (Universal Imaging Corporation, West Chester, Pennsylvania) composed of software and hardware linked to a 25 MHz computer. AVideoscope-intensified CCD camera, consisting of a Hamamatsu microscope video camera (C2400) and a model KS-1381 Videoscope Image Intensifier (Videoscope International, LTD., Herndon, Virginia), was mounted on a Zeiss Axiophot microscope, and the samples were observed through a 63 x Il.4 N. A. objective. Sixty-four frames of a live image were summed, and the resulting image was displayed on a color monitor (Sony PVM 1342Q). Using the mouse, a square was centered within each nucleus, and the mean nuclear fluorescence in that region was obtained. Usually between 10 and 30 nuclei were scanned per coverslip. The settings for the camera gain and black level, the intensifier gain and black level, and the computer analog gain and black level were empirically chosen to generate arbitrary values in the range of 5-15 for nuclei that have not imported substrate and in the range of 100-150 for those nuclei that exhibit maximal import (within the standard conditions of the assay) out of a possible scale of O-255. These values were quite reproducible from assay to assay, and this made possible the calculation of standard units of activity. One unit of activity is defined as the mean nuclear fluorescence obtained by the addition of cytosol minus the mean nuclear fluorescence of the buffer only control. The resulting value is divided by ten. This formula gives a specific activity (Ulmg) for unfractionated cytosol of approximately 1 .O. One unit of A fraction activity is defined as the mean nuclear fluorescence obtained by the addition of A (with fraction B at saturation) minus the value obtained by B alone, and this value was divided by 10. Units of B activity are the same, except that the A fraction is added at saturation, and the A alone value is subtracted. Isolation of Cytosolic Fractions Preparation of Cytosol All procedures were performed at 4% unless otherwise stated. Mature Xenopus laevis females (Nasco #LM535WC) were sacrificed by carbon dioxide overdose. The ovaries were removed and placed in cold Dulbecco’s phosphate-buffered saline on ice. When all were collected, they were rinsed with multiple changes of cold Dulbecco’s phosphate-buffered saline, blotted briefly on a paper towel, and transferred to an equal volume of cold buffer 8. Leupeptin, pepstatin, aprotonin (1 pglml each), and phenylmethylsulfonyl fluoride (1 mM) were then added. The oocytes were disrupted by three 5 s bursts with a Polytron homogenizer (3.5 cm probe) at power setting 3. The homogenate was centrifuged for 20 min in a Sorvall GSA rotor at 10,000 rpm (16,319 x g). The supernatant was removed (avoiding the fat layer on top) and filtered through ten layers of cheesecloth. The filtered supernatant was centrifuged for 2 hr in a Beckman Ti 50.2 rotor at 40,000rpm.Thesupernatantwasremoved,avoidingasmuchaspossible the loose yellow aggregate that remained unpelleted near the bottom of the tube. For unfractionated cytosol, this supernatant was dialyzed overnight against multiple changes of buffer A and centrifuged in a Beckman Ti 50.2 rotor for 1 hr at 40,000 rpm (146,000 x g). The supernatant was collected, and aprotonin, pepstatin, and leupeptin were added to a final concentration of 1 pglml each. Aliqouts were frozen in liquid nitrogen and stored at -80%. Isolation of Fraction A The high speed supernatant from the cytosol preparation (prior to dialysis) was used as the starting material for the isolation of fraction A. The following is the procedure used for the cylosol obtained from the oocytes of 21 frogs. The cytosol was adjusted to 150 mM potassium acetate by the dropwise addition (with stirring) of buffer B containing 1 .O M potassiuim acetate. This solution was loaded at a flow rate of

100 mllhr on a 100 ml DE-52 (Whatman) column (2.5 x 20.4 cm) equilibrated in buffer B containing 150 mM potassium acetate. The column was washed with 400 ml of column buffer and eluted with a 500 ml linear gradient of 150-750 mM potassium acetate in buffer B. Fractions (10 ml) were collected, and 5 ~1 of each fraction was assayed for activity (in the presence of fraction B). The active fractions, which eluted between 400 and 500 mM potassium acetate, were pooled and dialyzed overnight against multiple changes of buffer A. The solution was centrifuged for 1 hr in a Beckman Ti 50.2 rotor at 40,000 rpm to remove insoluble material, aliquoted, frozen in liquid nitrogen, and stored at -80%. Fraction B activity could be detected in the flow-through fractions of this DE-52 column (after concentration). To obtain a purer and more concentrated preparation, the following procedure was used to isolate the B activity. kolation of Fraction B The high speed supernatant from the cytosol preparation (prior to dialysis) was used as the starting material for the isolation of fraction B. The following is the procedure used for the cytosol obtained from the oocytes of 20 frogs. Solid ammonium sulfate was added to the cytosol to a final concentration of 25% (13.4 g per 100 ml) and allowed to stir on ice for 30 min. The solution was centrifuged in a Sorvall GSA rotor for 10 min at 8000 rpm (10,444 x g), and the pellet was discarded. Solid ammonium sulfate was added to the supernatant to a final concentration of 50% (14.6 g per 100 ml) and was allowed to stir on ice for 1 hr. The solution was centrifuged in a Sorvall GSA rotor for 15 min at 10,000 rpm. The pellet was resuspended in 60 ml of buffer C and dialyzed overnight against multiple changes of the same buffer. The sample was centrifuged in a Beckman Ti 50.2 rotor for 1 hr at 40,000 rpm. The supernatant was applied at a flow rate of 60 mllhr lo a 70 ml Cl-Sepharose (Pharmacia) column (2.6 x 13.2 cm) equilibrated in buffer C. The column was washed with 70 ml of buffer C, then with 200 ml of buffer C containing 150 mM potassium acetate and eluted with a 375 ml linear gradient of 150-500 mM potassium acetate in buffer C. Fractions (6 ml) were collected, and 5 @I of each was assayed for activity (in the presence of fraction A). The active fractions, eluting between 200-300 mM potassium acetate, were pooled. Two different methods of concentrating these fractions were used in different preparations. In one method, solid ammonium sulfate was added to a concentration of 60% (36.1 g per 100 ml). The solution was stirred for 30 min on ice and centrifuged in a Sorvall SS-34 rotor for 15 min at 10,000 rpm. The pellet was resuspended in 1 .O ml of buffer A, dialyzed overnight against multiple changes of buffer A, and centrifuged for 30 min at 54,000 rpm (104,000 x g) in a TLA-100.2 rotor. In an alternate method, the pooled O-Sepharose fractions were concentrated by centrifugation in a Centriprep-10 (Amicon) to a protein concentration of around 15 mglml, then were dialyzed and centrifuged as above. These two methods gave similar results. Aliquots were frozen in liquid nitrogen and stored at -80%. NEM Treatment of Fractions One volume of cytosol or of each partially purified fraction was diluted with 3 vol of buffer A’ without DTT to reduce the DTT concentration to 0.5 mM. The samples were then concentrated back to their original volumes by centrifugation in a Centricon(Amicon). A 1 .O M stock solution of NEM (Sigma) was prepared in dimethyl sulfoxide immediately before use, and subsequent dilutions were made in buffer A’ without DTT. NEM was added to the cytosolic fractions at a final concentration of 5 mM and incubated for 10 min on ice. The reaction was terminated by the addition of DTT to a final concentration of 10 mM. For mock treated fractions, excess DTT was added prior to the addition of NEM. Untreated fractions were neither treated with NEM-DlT nor diluted and reconcentrated. Miscellaneous Protein values were obtained with the Bio-Rad bovine serum albumin as the standard.

protein

assay

using

We thank Thomas Meier for his generous gift of peptide conjugates and for sharing his expertise regarding nuclear import assays. We would also like to thank Christopher Nicchitta and Patrick Casey for

tr9osolic

Factors

Required

for Nuclear

Import

many helpful discussions regarding protein purification and Thomas Meier and Christopher Nicchittafor acritical reading of the manuscript. M. S. M. was supported by National Institutes of Health fellowship 1 F326M13758502. This paper is dedicated to George Palade. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

9, 1992; revised

April 14, 1992.

Adam, S. A., and Gerace, L. (1991). Cytosolic proteins bind nuclear location signals are receptors for nuclear 837-847.

that specifically import, Cell 66,

Adam, S. A., Lobl, T. A., Mitchell, M. A., and Gerace, L. (1989). Identification of specific binding proteins for a nuclear localization sequence. Nature 337, 276-279. Adam, S. A., Sterne Marr, Ft., and Gerace, in permeabilized mammalian cells requires tors. J. Cell Biol. 7 7 7, 807-816. Akey, C. W. (1989). Interactions complex revealed by cryo-electron 970.

L. (1990). Nuclear import soluble cytoplasmic fac-

and structure of the nuclear pore microscopy. J. Cell Biol. 709,955-

Benditt, J. O., Meyer, C., Fasold, H., Barnard, F. C., and Riedel, N. (1989). Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling. Proc. Natl. Acad. Sci. USA 86, 9327-9331. M., and Goldfarb, D. S. (1990). Hl and other small nucleophilic

Facilitated nuclear transport proteins. Cell 60,999-1008.

Davis, L. I., and Blobel, G. (1986). Identification and characterization of a nuclear pore complex protein. Cell 45, 699-709. Davis, L. I., and Blobel, G. (1987). Nuclear pore complex contains a family of glycoproteins that includes ~62: glycosylation through a previously unidentified cellular pathway. Proc. Nat. Acad. Sci. USA 84, 7552-7556. Dingwall, C., and Laskey, R. A. (1986). Protein nucleus. Annu. Rev. Cell Biol. 2, 367-390.

import

into the cell

Dingwall, C., Sharnick, S. V., and Laskey, R. A. (1982). A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30, 449-458. Dworetzky, S. I., Lanford, R. E., and Feldherr, C. M. (1988). The effects of variations in the number and sequence of targeting signals on nuclear uptake. J. Cell Biol. 707. 1279-1287. Finlay, D. R., Newmeyer, D. D., Price, T. M., and Forbes, D. J. (1987). Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J. Cell Biol. 704, 189-200. Fischer, U., and Luhrmann, the m,G cap in the transport 786-790.

R. (1990). An essential signaling role for of Ul snRNP to the nucleus. Science 249,

Fischer, U., Darzynkiewicz, E., Tahara, S. M., Dathan, N. A., Luhrmann, R., and Mattaj, I. W. (1991). Diversity in the signals required for nuclear accumulation of U snRNP’s and variety in the pathways of nuclear transport. J. Cell Biol. 173, 705-714. Garcia-Bustos, J., Heitman, J., and Hall, M. N. (1991). localization. Biochim. Biophys. Acta 1077, 83-101. Goldfarb, Synthetic 644.

Krohne, G., and Franke, W. W. (1980). Immunological identification and localization of the predominant nuclear protein of the amphibian oocyte nucleus. Proc. Natl. Acad. Sci. USA 77, 1034-1038. Lanford, R. E., and Butel, J. S. (1984). Construction tion of an SV40 mutant defective in nuclear transport 37, 801-813. Lanford, R. E., Kanda, P., and Kennedy, nuclear transport with a synthetic peptide antigen signal. Cell 46, 575-582.

References

Breeuwer, of histone

Kalderon, D., Roberts, 9. L., Richardson, W. D., and Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509.

Nuclear

protein

D. S., Gariepy, J., Schoolnik, G., and Kornberg, R. D. (1986). peptides as nuclear localization signals. Nature 322, 641-

and characterizaof T antigen. Cell

R. C. (1986). Induction of homologous to the SV40 T

Lee, W.-C., and Melese, T. (1989). Identification and characterization of a nuclear localization binding protein in yeast. Proc. Natl. Acad. Sci. USA 86, 8808-8812. Li, R., and Thomas, J. 0. (1989). interacts with nuclear localization

Identification of a human protein that signals. J. Cell Biol. 109,2623-2632.

Loewinger, L., and McKeon, F. (1988). Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm. EMBO J. 7, 2301-2309. Mattaj, snRNA 118.

I. W., and DeRobertis, E. M. (1985). Nuclear segregation of U2 requires the binding of specific snRNP proteins. Cell 40, 11 l-

Meier, U. T., and Blobel, G. (1990). A nuclear localization protein in the nucleolus. J. Cell Biol. 777, 2235-2245.

signal binding

Michaud, N., and Goldfarb, D. (1991). Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J. Cell Biol. 7 72, 215-223. Michaud, N., and Goldfarb, D. (1992). Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways. J. Cell Biol. 776, 851-861. Newmeyer, D. D., and Forbes, D. J. (1988). Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52, 641-653. Newmeyer, D. D., and Forbes, D. J. (1990). An N-ethylmaleimidesensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J. Cell Biol. 770, 547-557. Paine, P. L., Moore, L. C., and Horowitz, permeability. Nature 254, 109-l 14.

S. (1975).

Nuclear

envelope

Reichelt, R., Holzenburg, A., Buhle, E. L., Jarnik, M., Engel, A., and Aebi, U. (1990). Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J. Cell Biol. 770, 883-894. Richardson, W. D.. Mills, A. D.. Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1988). Nuclear protein migration involvestwosteps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52, 655-664. Ribs, H.-P., and Peters, R. (1989). Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the simian virus 40 T-antigen. EMBO J. 8, 1479-1484. Ribs, H.-P., Jans, D. A., Fan, H., and Peters, R. (1991). The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of SV40 T-antigen. EMBO J. 70, 633-639. Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615-623.

Hamm, J., Darzynkiewicz, E., Tahara, S. M., and Mattaj, I, W. (1990). The trimethylguanosine cap structure of Ul snRNA is a component of a bippartite targeting sinal. Cell 62, 569-577.

Roberts, 9. L., Richardson, W. D., and Smith, A. E. (1987). The effect of protein context on nuclear location signal function. Cell 50, 465475.

Holt, G. D., Snow, C. M., Senior, R. S., Haltiwanger, R. S.. and Gerace, L. (1987). Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Biol. 704, 1157-l 164.

Silver, P., Sadler, I., and Osborne, M. (1989). Yeast proteinsthat recog nize nuclear localization sequences. J. Cell Biol. 109, 983-989.

Jans, D. A., Ackermann, M. J., Bischoff, J. R.. Beach, D. H., and Peters, R. (1991). ~34~“~ -mediated phosphorylation at TjZ4 inhibits nuclear import of SV-40 T antigen proteins. J. Cell Biol. 7 751203-l 212.

Snow, C. M., Senior, A., and Gerace, L. (1987). Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 704, 1143-1156.

Silver, P. A. (1991).

How proteins

enter the nucleus.

Cell 64,489-497.

Cell 950

Walter, P., and Blobel. ciated protein complex endoplasmic reticulum.

G. (1980). Purification of a membrane-assorequired for protein translocation across the Proc. Natl. Acad. Sci USA 77, 7112-7116.

Yamasaki. L., Kanda, P., and Lanford, R. E. (1969). Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Mol. Cell. Biol. 9, 3028-3036. Yoneda, Y., Imamoto-Sonobe, N., Yamaizumi, M., and Uchida, T. (1987). Reversible inhibition of protein import into the nucleus bywheat germ agglutinin injected into cultured cells. Exp. Cell Res. 173, 586595. Zhao, L., and Padmanabhan, Ft. (1988). Nuclear rus DNA polymerase is facilitated by interaction tein. Cell 55, 1005-1015.

transport of adenoviwith preterminal pro-

. “.,