May 8, 1987 - of many studies central to our understanding of how proteins .... C) are similar to signal sequences described for SV40 large-T ... the very carboxy terminus of nucleoplasmin not only has .... We can be sure that the fusion proteins enter nuclei ... Comparison of an aliquot of uninjected sample (lane S) to the.
The EMBO Journal vol.6 no.9 pp.2617-2625, 1987
The nuclear migration signal of Xenopus laevis nucleoplasmin
Thomas R.Burglin' and Eddy M.De Robertis' Biocenter of the University of Basel, CH 4056 Basel, Switzerland
'Present address: Department of Biological Chemistry, University of California, Los Angeles, CA 90024, USA Communicated by E.M.De Robertis
Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. Its ability to target to the nucleus when microinjected into the cytoplasm has been the subject of many studies central to our understanding of how proteins segregate into nuclei. Using a cDNA clone we constructed jgalactosidase-nucleoplasmin hybrids in modified bacterial expression vectors. The fusion proteins were expressed in Escherichia coli, purified and injected into the cytoplasm of X. laevis oocytes. The distribution of the fusion proteins between the cytoplasmic and nuclear compartments were analysed after incubation for various lengths of time. The results show that the signal sequence for nuclear transport is located close to the carboxy terminus of the protein. The signal sequence has been mapped to a small stretch of amino acids, containing a stretch of four lysines analogous to the SV40 large-T antigen signal. Key words: oocyte microinjection/nuclear transport/bacterial expression vector/g-galactosidase fusion proteins
Introduction Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes (Mills et al., 1980; Krohne and Franke, 1980). It is a protein with a mol. wt of ~-21 kd and forms a stable pentamer in vivo (Laskey et al., 1978; Earnshaw et al., 1980; Dingwall et al., 1987; Burglin et al., 1987). The protein has received much attention because of its ability to target rapidly to the nucleus after microinjection into the cytoplasm (Mills et al., 1980). Much of our present knowledge on nucleocytoplasmic transport of proteins comes from studies with Xenopus nucleoplasmin. It was the first protein for which a signal for nuclear transport was localized to a specific domain: Dingwail et al. (1982) cleaved nucleoplasmin proteolytically into a 'core' and a 'tail' fragment and only the 'tail' fragment was able to target to the nucleus. Feldherr et al. (1984) demonstrated that transport of nucleoplasmin occurs through the nuclear pore using gold particles coated with nucleoplasmin. Newmeyer et al. (1986) showed that ATP is required for its transport into oocyte nuclei and into nuclei reconstituted in vitro from egg extracts. In spite of these previous studies, the nature of the nuclear migration signal in nucleoplasmin was not known and is the subject of the present study. Since the initial proposal that nuclear proteins must contain in their mature molecular structure a signal that enables them to accumulate selectively in the nucleus (De Robertis et al., 1978), regions important for nuclear localization have been defined for several proteins. The proteins analysed were either yeast proteins (Hall et al., 1984; Silver et al., 1984; Moreland etal., 1985) C) IRL Press Limited, Oxford, England
or viral proteins (Kalderon et al., 1984a,b; Lanford and Butel, 1984; Fischer-Fantuzzi and Vesco, 1985; Davey et al., 1985; Krippl et al., 1985; Richter et al., 1985; Richardson et al., 1986; Wychowski et al., 1986). The SV40 large-T antigen signal has been analysed in detail by point mutations (Kalderon et al., 1984a,b; Lanford and Butel, 1984). Furthermore, it has been shown that synthetic peptides of this signal sequence which are cross-linked to non-nuclear proteins are able to target these proteins to the nucleus (Lanford et al., 1986; Goldfarb et al., 1986). So far no signal sequence has been defined for a non-viral protein in higher eukaryotes. Nucleoplasmin is not only a vertebrate non-viral protein, but has the advantage that its transport can be studied in the same cell in which it is normally synthesized, the Xenopus oocyte. As described previously, we had cloned a cDNA coding for nucleoplasmin (Burglin et al., 1987). Analysis of the sequence showed that although the cDNA was not complete, all the necessary information for nuclear targetting, i.e. the complete 'tail', was present in the clone (Burglin et al., 1987). In order to determine which sequences are important for transport, we constructed ,B-galactosidase-nucleoplasmin hybrids in modified bacterial expression vectors. The fusion proteins were expressed in Escherichia coli, purified and microinjected into the cytoplasm of oocytes. Analysis of the cytoplasmic and nuclear compartments after various times showed that the signal sequence for nuclear transport is located close to the carboxy terminus of the
protein.
Results Several putative signal sequences are present in the carboxy terminus of nucleoplasmin Dingwall et al. (1982) showed that the capacity for nuclear accumulation is located in a proteolytic 'tail' fragment of nucleoplasmin. Cloning of a cDNA for nucleoplasmin proved that this 'tail' is indeed the carboxy-terminal fragment (Burglin et al., 1987). As shown in Figure 1, a comparison of the carboxy terminus with other published signal sequences revealed several putative signal sequences in the last 50 amino acids. One element (box D) can be found which is very similar to the proposed signal sequence described for the yeast MATat2 protein (three hydrophobic amino acids containing a proline, flanked by basic amino acids, Hall et al., 1984), and a second element (box A) shows some similarity to this sequence, having an additional internal threonine. Two other amino acid stretches (boxes B and C) are similar to signal sequences described for SV40 large-T antigen (a stretch of five basic amino acids: Lys Lys Lys Arg Lys; Kalderon et al., 1984a,b; Lanford and Butel, 1984). Thus the very carboxy terminus of nucleoplasmin not only has homologies to histones (Buirglin et al., 1987), but also shows four homologies to nuclear migration signals (discussed by Dingwall et al., 1986, 1987). 2617
T.R.Burglin and E.M.De Robertis 1 40
1 30
Glu Glu Glu Asp Glu Gly Glu Glu Glu Glu Glu Glu Glu Glu Asp Pro Glu Ser Pro Pro
150 Lys Ala Val Lys
A
B
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180
Asp Lys Glu Asp Glu Ser Ser Glu Glu Asp Ser Pro Thr Lys Lys Gly Lys Gly Ala Gly
A
190D
Soc I
Arg Gly Arg Lys Pro Ala Ala
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Fig. 1. Amino acid sequence of the carboxy terminus of nucleoplasmin. Boxes A and D denote sequences which are similar to the proposed signal for the yeast MATcu2 protein (Hall et al., 1984) and boxes B and C indicate sequences which are similar to the SV40 large-T antigen signal (Kalderon et al., 1984a,b). Numbering is based on the assumption that only six amino acids are missing at the amino terminus of the cDNA clone used in this study (Burglin et al., 1987; Dingwall et al., 1987).
Technical approach for analysing the nuclear migration signal We had cloned nucleoplasmin Xgtl 1 as a fusion protein with 3galactosidase, which gave reasonably stable fusion proteins (Burglin et al., 1987). Furthermore, the ,B-galactosidase tetramer (465 kd) is much larger than the functional size of the nuclear pore for diffusion (Paine et al., 1975; Bonner, 1978). For example, bovine serum albumin (BSA) with a mol. wt of 68 kd hardly enters the oocyte nucleus (Bonner, 1978). Thus we chose to analyse the exact location of the nuclear migration signal by deletion analysis of f-galactosidase - nucleoplasmin fusion proteins. The vectors used for most constructs are shown in Figure 7. Since in many constructs the nucleoplasmin translation stop codon is deleted, we modified the vectors described by Ruther and Muller-Hill (1983) by cloning the translation stop codons and transcription stop signals from the vectors by Stanley and Luzio (1984) at the carboxy terminus of (-galactosidase (see Figure 7 in Materials and methods). These vectors (pTRB 0, pTRB 1, pTRB2) contain a short polylinker for each frame at the carboxy terminus of 03-galactosidase. The synthetic translation stop sequence provides stop codons in all three reading frames. The tandem transcription stop signals prevent readthrough. The resulting fusion proteins have wild-type ,Bgalactosidase activity, which facilitates purification and allows an easy assay for the hybrid proteins. However, the properties of the resulting fusion proteins strongly depend on the inserted sequences, since some fusion proteins have been found to be insoluble, or often degraded (C.V.E.Wright, personal communication; E.Frei, personal communication). The fusion proteins were expressed in E. coli and purified by affinity chromatography in a single-step procedure (Ullmann, 1984). The essentially pure fusion proteins were injected into the cytoplasm of oocytes and usually incubated overnight. Some fusion proteins were radiolabelled with 125I prior to injection. The oocytes were then manually separated into cytoplasmic and nuclear fractions. These fractions were then directly analysed for ,B-galactosidase activity or separated on SDS-polyacrylamide gels. Gels of radiolabelled fusion proteins were autoradiographed after drying, and gels of unlabelled fusion proteins were transferred to nitrocellulose and immuno-stained with anti-,B-galactosidase antibodies (Western blot). f-Galactosidase - nucleoplasmin fusion protein accumulates in the nucleus The cDNA described by Burglin et al. (1987) was cloned into pTRB 0 and the resulting plasmid was called pTRB 102. The fusion protein yield from this plasmid construct is higher than
2618
S
T
C N
S T C N
4.
-:-
pTRB 102
pTRB 0
Fig. 2. Western blot of pTRB 102 fusion protein and pTRB 0 3galactosidase after injection into oocytes and incubation overnight. Cytoplasms and nuclei were separated manually and the fractions were then centrifuged to remove yolk and lipids, electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose and stained with an affinity-purified anti-O3-galactosidase antibody (Western blot). Bound antibodies were visualized with [1251]protein A. Lane S is uninjected sample, corresponding to the amount of fusion protein injected into 10 oocytes, lane T is protein recovered from 10 total oocytes after overnight incubation, lane C is 10 cytoplasms and lane N is 10 nuclei. The fusion protein from pTRB 102 (arrow on left side) accumulates in the nucleus, whereas only a small trace of ,B-galactosidase from pTRB 0 (arrow on right side) is found in the nuclear fraction. The arrowhead on the left indicates the degradation product of approximately f-galactosidase size.
from Xgtl 1, and 50% of the fusion protein was recovered in the supernatant after sonication of the E. coli cells and centrifugation (see Materials and methods for details of purification). Several discrete degradation products can be seen, ranging from the 'full length' fusion protein to a degradation product of the size of 3-galactosidase (Figure 2, arrow and arrowhead in lane S). The longest fusion protein most likely represents the complete coding sequence, because its mobility on SDS gels (data not shown) displays the predicted shift corresponding to the expected apparent mol. wt of nucleoplasmin, as in the case of the Xgtl 1 fusion protein (Burglin et al., 1987; Dingwall et al., 1987). The lower mol. wt bands arise from carboxy-terminal degradation of the fusion protein, since all bands react well with an anti-
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The nuclear migration signal of nucleoplasmin
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Fig. 3. Analysis of the deletion constructs by Western blots. Mutant fusion proteins were injected into the cytoplasm of oocytes, incubated overnight, manually separated into cytoplasms and nuclei and analysed as in Figure 2. pTRB 102 and pTRB 108 were separated on 5% SDS-polyacrylamide gels, the others on 7.5% gels. The left side of the panel shows the results for the different constructs. Lane T represents 10 total oocytes, lane C, 10 cytoplasms, and lane N, 10 nuclei. Arrowheads on the left indicate the degradation product with a size similar to (3-galactosidase, arrowheads on the right indicate the fusion protein. In the middle part of the panel the different constructs are represented graphically to scale. The open frame on the left indicates (3-galactosidase; the long, narrow rectangle in the centre represents nucleoplasmin. The small horizontally hatched boxes indicate linker regions. The diagonally hatched box in nucleoplasmin indicates the polyglutamic acid stretch and the black boxes (A, B, C, D) indicate the four putative nuclear migration signals. A* stands for regenerated boxes (see Figure 8 for details). The right side of the panel gives a summary of the ability of the different clones to accumulate in the nucleus, represented by the + and signs. (*) indicates that the positive result for nuclear accumulation was confirmed with a re-injection experiment (see Figure 4 and text). -
against amino acids 3-92 of,-galactosidase (data not shown; Fowler and Zabin, 1983). About 10 ng of purified fusion protein was injected per oocyte, representing only a small fraction of the endogenous nucleoplasmin in the nucleus (250 ng/oocyte). Figure 2 shows that the pTRB 102 fusion protein accumulates in the nucleus after injection into the cytoplasm. About 70% of the 'full length' fusion protein (indicated by the arrow) is found in the nucleus (lane N) after incubation overnight (as determin-
serum
ed by densitometry of the autoradiographs). In several independent experiments with different protein preparations, between 50 and 75 % (1/4 in the cytoplasm, 3/4 in the nucleus) of the pTRB 102 fusion protein was found in the nucleus after overnight incubation (Figures 2 and 3, and data not shown). The nucleus is 8.3 smaller in volume than the cytoplasm (Bonner, 1978), which means that the protein is 8.3 -25 times more concentrated than in the cytoplasm. The degradation products of the fusion protein which have the -
2619
T.R.Biirglin and E.M.De Robertis Table I. ,B-Galactosidase activity in the nuclear and cytoplasmic fractions for pTRB 102 and pTRB 2. Compartment
% of total activity
pTRB 102
Cytoplasm Nucleus
31 69
pTRB 102
Cytoplasm Demembranated nucleus
46 54
Cytoplasm Nucleus
99.6 0.4
pTRB 2
Cytoplasmic to nuclear ratioa
1:18
1:9.5
1:.
30.1
For each experiment five oocytes were separated and assayed using ONPG according to Miller (1972). aAssuming a nuclear to cytoplasmic volume ratio of 8.3 (Bonner, 1978).
same length as ,B-galactosidase lack nucleoplasmin sequences and yet can accumulate in nuclei to some degree (see arrowheads for construct pTRB 102 in Figures 2 and 3, see next section for other constructs). The answer to this paradox lies in the fact that 03-galactosidase forms tetramers. Consequently, the microinjected samples will migrate into nuclei even if some of the chains are missing the carboxy-terminal nucleoplasmin fusion. The degradation occurs in E. coli, and there is no easy way of purifying the intact fusion protein without co-purifying some of the degradation products. We can be sure that the fusion proteins enter nuclei as tetramers, because only tetramers display enzymatic activity (Table I), and because co-injection of pTRB 102 protein together with the non-migrationg pTRB 103 protein, which can be distinguished on SDS gels (see below), showed that the tetramers do not dissociate and reassociate within the oocyte (data not
shown). Comparison of an aliquot of uninjected sample (lane S) to the total oocytes (lane T) or to the sum of the cytoplasmic (lane C) and the nuclear (lane N) fraction shows that the fusion protein, particularly the 'full length' product, is stable in oocytes. Some degradation products are sometimes slightly unstable, varying between different injection experiments (data not shown). Moreover, this comparison indicates that no substantial loss of protein occurs during the re-isolation from oocytes. Some variability is present (for example, panels 102 and 108 of Figure 3, see below), but as shown in the next section, there is a vast difference between migrating and non-migrating fusion proteins. In order to show that the (.-galactosidase - nucleoplasmin fusion protein actually enters nuclei (instead of attaching to nuclear envelope), we manually removed the nuclear envelope and measured the enzyme activity recovered from the nuclear gel that forms during isolation of nuclei in magnesium solutions (De Robertis et al., 1982). Table I shows that 54% of the 13-galactosidase activity was recovered from the nuclear gel of oocytes injected with the pTRB 102 fusion (corresponding to a 9.7-fold nuclear accumulation). This is a major proportion of the activity recovered from the intact nuclei (69%, corresponding to a nuclear accumulation of 18.5 times). The difference is easily explained by loss during the isolation, since even native nucleoplasmin leaks out of whole oocyte nuclei during isolation (Mills et al., 1980). Preliminary results from staining oocyte sections with anti-3galactosidase antibodies also show that the fusion protein is evenly spread throughout the nucleus (data not shown). In order to show that the nuclear accumulation is specific to the fusion protein, we prepared ,B-galactosidase from the pTRB 0 and pTRB 2 vectors. These proteins do not accumulate in the nucleus (Figure 2, Table I). However, long exposures of auto2620
radiographs show that some protein is present in the nuclear fraction. (3-Galactosidase activity assays also show that some activity can be detected in the nuclear fraction, being - 30 times less concentrated in the nucleus than if it were able to equilibrate uniformly throughout the cell (Table I). We also tested the behaviour of 3-galactosidase prepared from pTRB 0 when injected directly into the nucleus. About 50 ng of protein per oocyte was injected into nuclei of oocytes and after incubation overnight individual oocytes were analysed by Western blots as described above. 50-60% of the protein was found in the nuclear fraction, indicating that (3-galactosidase does not simply diffuse out of the nucleus into the cytoplasm. Although it is clear that most of the 3-galactosidase remains at the site of injection, we are unable to tell whether the protein in the cytoplasmic fraction passed through the nuclear pores, or whether some of the protein spilled into the cytoplasm during microinjection.
Mapping of the nuclear migration signal In order to define the location of the nuclear migration signal more precisely we took advantage of several restriction sites present in the carboxy terminus of nucleoplasmin which are indicated in Figure 1. The details of each construction are described in Materials and methods. The apparent mol. wt of the different fusion proteins was as predicted and the differences in mobility amongst themselves corresponded well with the difference in amino acids. Figure 3 shows the results of cytoplasmic injections of fusion protein prepared from the six constructs (which are referred to by their plasmid names below) as analysed by Western blots. pTRB 108 has the last four amino acids of nucleoplasmin replaced by vector sequences which therefore destroy the putative box D (Figure 1). It accumulates in the nucleus to the same extent as pTRB 102. pTRB 105 is missing the last 24 amino acids, and with them boxes C and D, but its ability to target to the nucleus is unaffected. In pTRB 103, the last 41 amino acids have been deleted, and with them boxes B, C and D. The last half of box A is destroyed but, due to the linker sequences, a sequence similar to box A is regenerated (Figure 8). This construct no longer accumulates in the nucleus, only entering the nucleus to the same extent as ,B-galactosidase . In pTRB 107 the last 35 amino acids are deleted, removing boxes B, C and D, but leaving box A intact. The construct behaves like pTRB 103 and does not accumulate in the nucleus. pTRB 106 has an internal deletion from amino acid 152 to 170, which removes part of box A and all of box B. This construct is not able to accumulate in the nucleus. However, it does seem to enter the nucleus slightly more than pTRB 103. These results indicate that there is an important sequence in the 11 amino acids from 161 to 172 (containing box B), which is necessary for nuclear migration. pTRB 100 has the last 42 amino acids joined to /3-galactosidase. This construct contains boxes B, C, D and, due to the linker region, also box A (see Figure 8 in Materials and methods). The majority of the fusion protein is degraded in E. coli (even on short induction periods), as shown by Western blots. Three bands can be recognized: a faint top band and two strong degradation products, the lower one having the same electrophoretic mobility as (3-galactosidase. After injection, the faint top band is able to accumulate in the nucleus, since it is about equally strong in the cytoplasm and the nucleus, indicating an 8-fold accumulation (Figure 3). The degradation products remain essentially in the cytoplasm. The results also show that the fusion proteins form oligomers
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Fig. 5. Rate of nuclear accumulation for the pTRB 102 and pTRB 105 fusion proteins. The fusion proteins were injected into the cytoplasm of oocytes and the nuclear and cytoplasmic fractions were analysed after 1.5, 6, 24 and 48 h by Western blots. Autoradiographs were scanned, the integral for the fusion protein (top band) was determined and from these data the percentage of fusion protein in the nucleus was calculated. (Open circle pTRB 102, solid circle pTRB 105). A value of 50% protein in the nucleus is equivalent to an 8.3-fold nuclear accumulation due to the differences in nuclear -cytoplasmic volume. The maximal value, - 80%, represents a 41-fold nuclear accumulation. Note the relatively slow uptake of the fusion proteins.
2
Fig. 4. Re-injection experiment. Panel A shows the design of the reinjection experiment. Radiolabelled pTRB 100 fusion protein is injected into oocytes and incubated overnight (o.n; 1). Nuclei are then isolated and their contents re-injected into new oocytes (2,3). After another incubation overnight, the nuclear and cytoplasmic fractions are analysed by gel electrophoresis (4). Panel B shows the experimental results. B1 is the autoradiograph of the first injection, each lane representing 10 total oocytes (T), 10 cytoplasms (C) and 10 nuclei (N). Nuclei of such oocytes were isolated, homogenized and the extract was injected into other oocytes. B2 shows the result of re-injecting the nuclear contents. Compare cytoplasm (C) and nucleus (N) of the first injection to cytoplasm (C) and nucleus (N) of the re-injection. The top band (arrow) is now preferentially accumulated in the nucleus. as j3-galactosidase does. All the nuclear-migrating fusion proteins (pTRB 102, 108, 105 and 100) are able to pull some of their degradation products into the nucleus. The results of the enzyme assay in Table I and the Western blots of Figures 2 and 3 show that the same proportion of pTRB 102 fusion protein accumulates in the nucleus in both cases. This is consistent with the notion that the fusion proteins enter the nucleus as tetramers and thus would pull degradation products along. The nonmigrating fusion proteins (pTRB 103, 107 and 106) do not display any degradation products in the nuclear fraction. In addition, since there is no reason to assume that pTRB 102 and pTRB 103, which have the same specific activity (data not shown), would oligomerize differently, we can exclude the possibility that the migrating fusion proteins enter the nucleus because they are mostly monomeric and enter the nucleus more readily than the tetrameric non-migrating fusions. A re-injection experiment demonstrates nuclear accumulation of pTRB 100 Since it is not possible to purify the faint top band of pTRB 100 biochemically in a native form, we devised an in vivo purification scheme in which the oocyte nucleus itself was used to enrich the pTRB 100 fusion protein mixture for non-degraded, nuclear fusion proteins (Figure 4, panel A). [1251]pTRB 100 protein was injected into the cytoplasm of oocytes and incubated overnight. Then the oocyte nuclei were isolated in phosphate-buffered saline (PBS) and transferred into an Eppendorf tube in a minimum volume (-0.5 IL/nucleus) using a siliconized 5 ILI micropipette. After collecting 20 nuclei, the tube was vortexed, the nuclear
envelopes and nucleoli removed by low-speed centrifugation and 50 nl aliquots of the supernatant were re-injected into a fresh batch of oocytes. After incubation overnight, the cytoplasmic and nuclear fractions were separated on an SDS -polyacrylamide gel and autoradiographed. Figure 4, panel B shows the result of this re-injection experiment. The first injection of the radiolabelled fusion protein gives a result similar to the Western blot of Figure 3 (Figure 4, panel Bi). When the nuclear content of such oocytes is re-injected, the ability of the faint top band to accumulate in the nucleus is displayed much better. In the re-injected sample of Figure 4, panel B2, the proportion of the total fusion protein able to migrate into the nucleus has much increased when compared with the first injection (panel Bl). This is due to the removal of part of the degraded proteins, which are left behind in the cytoplasm during the first nuclear purification step. In addition it is clear that the top band (arrowed) is the one that accumulates well in the nucleus, while the degradation products stay in the cytoplasm. From this we conclude that the last 45 amino acids (152-196) of nucleoplasmin are sufficient for accumulation in the oocyte nucleus. The deletion constructs indicate that a necessary sequence is located between amino acids 161 and 172. This stretch contains box B which is similar to the SV40 large-T antigen signal sequence. The last 24 amino acids do not enhance transport The polyoma virus large-T protein is known to have two transport signal sequences of different strength (Richardson et al., 1986). Although in nucleoplasmin the sequences around boxes C and D are neither necessary nor sufficient for nuclear migration, we thought they might possibly be required for the fast rate of nuclear accumulation which is observed for nucleoplasmin (Dingwall et al., 1982, 1986). Thus we injected oocytes with fusion proteins from pTRB 102 (whole cDNA clone) and pTRB 105 (missing the last 24 amino acids) and analysed the nuclear accumulation at different time points to test for a qualitative difference in their nuclear accumulation. Figure 5 shows graphically the results obtained by densitometry of autoradiographs of Western blots. There is no difference in the rate of accumulation for pTRB 102 and pTRB 105, thus there is no requirement for these sequences at all in nuclear targetting. The rate of accumulation, which has a half-time (time at which 2621
T.R.Biirglin and E.M.De Robertis 50% of the protein is found in the nucleus) of 15 h, is much slower than the 0.5 h observed for native nucleoplasmin (Dingwall et al., 1982, 1986). The accumulation seems to plateau only after 2 days. This slow uptake is most likely due to the large size of these tetrameric fusion proteins (550 kd). Diffusion in the cytoplasm is certainly not the limiting step, since radiolabelled pTRB 100 and pTRB 2 diffuse throughout the oocyte cytoplasm in < 1 h as analysed by sections (data not shown).
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Discussion E. coli-derived (3-galactosidase -nucleoplasmin fusions target to the nucleus Our results show that 3-galactosidase-nucleoplasmin fusion proteins purified from E. coli accumulate in the nucleus of X. laevis oocytes. These fusion proteins, which have a mol. wt of > 550 kd, are -8-25 times more concentrated in the nucleus after incubation overnight. The degradation products of the migrating constructs (pTRB 102, pTRB 108, pTRB 105) target less efficiently to the nucleus. One might expect that these degradation products should be present in equal amounts in the nucleus with the full-length fusion proteins, since the fusion proteins are at least tetrameric and one full length subunit should be sufficient to pull in associated degradation products. However, it might be possible that the degradation products and the intact fusion proteins are not mixed randomly in the tetramers. Furthermore, tetramers with only one tail may target slower to the nucleus. A similar phenomenon has been observed for nucleoplasmin pentamers that had been proteolytically digested so that some of the pentamers had only one or two 'tails' (Dingwall et al., 1982). It could be argued that the fusion proteins may be targetted to the nucleus because they associate with endogenous nucleoplasmin present in oocytes. This is most unlikely, since the mutations that abolish nuclear accumulation are all located in the carboxy-terminal 'tail' region, while the sequences involved in the formation of nucleoplasmin pentamers are located in the 'core' region (Dingwall et al., 1982). Non-migrating fusion proteins like pTRB 107 and pTRB 103 contain the same region of nucleoplasmin (amino acids 7-150, containing the almost complete amino-terminal 'core' and a part of the 'tail') as the migrating pTRB 105 (Figures 3 and 8). The fusion proteins accumulate much slower than native nucleoplasmin; - 20-30% of the best-migrating protein is still in the cytoplasm even after 2 days of incubation. It may be that the microinjected fusion proteins contain aggregates of tetramers, since i-galactosidase is known to form large aggregates (Fowler and Zabin, 1983). Furthermore, since our constructs also contain the nucleoplasmin 'core' region which forms pentamers, even larger aggregates could possibly be formed, although we do not have evidence for this. Such large aggregates might explain why not all of the migrating fusion proteins enter the nucleus and why the accumulation continues for long periods of time. The use of ,B-galactosidase as a carrier protein for nuclear migration studies has been questioned by the work of Kalderon et al. (1984b) who found that in somatic cultured cells some nuclear immunofluorescence could be detected even in the absence of a nuclear migration signal, although the presence of a SV40 large-T antigen signal increased nuclear localization. In our case, only small amounts of microinjected (3-galactosidase can be detected in manually isolated oocyte nuclei by Western blot analysis, as clearly shown in Figure 2 (pTRB 0, lane N). Similarly, the assays for 3-galactosidase enzyme activity in Table I show that the difference in nuclear accumulation for migrating 2622
6. Homologies to other nuclear migration sequences. Comparison of box B to other well-characterised nuclear migration signals of the SV40 large-T antigen, the two sequences of polyoma virus large-T protein and SV40 VP1. All these sequences contain a stretch of basic residues, mostly lysines, and a helix-breaking amino acid upstream.
fusion proteins and f-galactosidase is > 100-fold. Thus (3galactosidase is a valid tool for our nuclear transport studies in Xenopus oocytes. The difference with the findings of Kalderon et al. (1984b) might be of a technical nature; perhaps, if (3galactosidase has small degradation products these peptides might enter nuclei giving some nuclear immunofluorescence, while with our Western blots and enzymatic activity assays short peptides would remain undetected.
The nuclear migration signal maps to a short region in the car-
boxy terminus
The results obtained with deletion constructs indicate that the region from amino acid 152 to 172 is sufficient for nuclear accumulation, since the fusions pTRB 100 and pTRB 105 both accumulate in the nucleus. This region contains boxes A and B (see Figures 1 and 3). Furthermore, the region between amino acids 162 and 172, containing box B, is required for nuclear transport, because fusion pTRB 107, containing only box A, does not accumulate. The last 24 amino acids could have a very low affinity for the nucleus, as construct pTRB 106 (containing boxes C and D) did seem to enter the nucleus a little better than the non-migrating (pTRB 103) fusion protein, but at present our assays are not accurate enough to allow such a clear distinction between these constructs. However, sequences in those last 24 amino acids are apparently not required for nuclear accumulation. The region between amino acids 162 and 172, which is required for nuclear migration, contains the row of four lysines (box B) which is similar to the signal sequence for SV40 largeT antigen (Figure 6, Kalderon et al., 1984a,b; Langford and Butel, 1984). The nuclear migration signals of polyoma virus large-T protein (Richardson et al., 1986) and SV40 VP1 (Wychowski et al., 1986) have been well-mapped and they also contain stretches of basic residues, as shown in Figure 6. It is interesting to note that the Pro Thr Lys Lys Gly Lys sequence (box C), which is also quite similar to the SV40 large-T antigen signal, does not cause accumulation of fusion proteins in the nucleus. The sequences which are similar to that proposed for the yeast MATa2 protein (boxes A and D; Hall et al., 1984) are apparently not sufficient for nuclear targetting. Moreover, a sequence of this type also happens to be in the polylinker region of the pTRB 2 protein, which does not migrate. The structure of the nuclear migration signal apparently seems to centre around lysine residues. Possibly they fold into an oahelical secondary structure with a helix-breaker a few amino acids upstream (Langford and Butel, 1984, and Figure 6). Secondary structure prediction for box B in nucleoplasmin indicates that this region could adopt a turn-helix conformation, too (Burglin et al., 1987). This a-helix would probably have to be exposed on the surface of the protein, which is certainly feasible in the hydrophilic tail of nucleoplasmin.
The nuclear migration signal of nucleoplasnmin
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n
I
Therefore, although the region including box B is necessary for nuclear migration, it may not be sufficient. The mechanism of nuclear transport still remains to be elucidated (discussed by De Robertis, 1983; Dingwall, 1985). The precise role of the nuclear pores (Feldherr et al., 1983, 1984; Dingwall et al., 1986), of the ATP requirement (Newmeyer et al., 1986) and of intranuclear binding to non-diff-usible components (Feldherr and Ogbum, 1980; Bonner, 1978) in the transport process are still unknown. The clone used in this study contains all the necessary sequences for nuclear transport. Thus the signal sequence we defined here should be the same sequence that targets wild-type nucleoplasmin to the nucleus. Our experiments do not allow us to tell by which mechanism this signal sequence accomplishes nuclear accumulation. However, the fusion proteins generated in this study provide large proteins with enzymatic activity, which are targetted by a short signal sequence. Hopefulfly, these mutant proteins will provide probes to gain further insights into the mechanism of nuclear transport in oocytes.
4
A1
N.
EcoRI
-
Materials and methods
PTRB 0
Cys
Gin
Lys
Gly
TGT
CAA
AAA
GG-G
Cys GAT CCG3 TCG ACX TGC AGJC. C&A_ GCT TGC BamHI PstI SailI HindIII
Ser
Val
Asp
Pro
Ser
Thr
Cys
Ser
Gin
Ala
TGA
pTRB 1
Cys
Arg
Giy
TGT
CGG
GGA TCC. GTC GAC. CTG
oTRB
Asp
Leu
SailI
BamHI
Ser
Leu
Leu
Ilie
Asp**
CAG.CCA_AGC
TTG
CTG
ATT
GAT
Gin
Pro
TGA
HindIII
PstI
2
Cys
Gin
TGT
CAG
Ilie
Arg
G.GG ATC
QGT
Gly
Arg
Pro
CGA CdT
SailI
BamHI
Ala GCA
Ala
Lys
Ala
Asp
CTGCT HindIII
GAT
Leu
GiCC AA
PstI
TGA
Fig. 7. Expression vectors pTRB 0, pTRB and pTRB 2 which were used for constructing the (3-galactosidase-nucleoplasmin hybrids. The upper part shows a map of pTRB 0, pTRB and pTRB 2, the positions of the restriction sites are only approximately to scale. The total size of the 5600 bp. The size of the EcoRI fragment for pTRB plasmids is is 500 bp and for pTRB 0 and pTRB 2 530 bp. The bottom part shows the sequence in the
polylinker for
all three
reading
frames.
154
100
a-gal,-..
Cys
102
a-gal.
Gi-n Lys Gly Asp Pro Gin Phe Arg Asn
Gin
Giy
le Arq
Arg Pro Aia Ala 7
103
Aia
155 Ala Lys loeu Ala Asp
105
Asp
Giu Ala Lys Leu Ala Asp
106
155 175 Ala Ala Ala Giu Giu
107
161 Ala Ala Thr Lys Lys Ala Giy Gin Ser Leu Leu le Asp stop
108
Lys
stop
172
stop
192
Fig. 8. at
Pro Giu Leu Ala
Asp stop
Amino acid sequence of the different constructs in
the end of the construct. The sequence between
nucleoplasmin
shown for
pTRB
102 is the
107 and 108. Underlined amino acids
are
same
joining regions (3-galactosidase and
for
or
pTRB 103, 105, 106,
derived from linker
or vector
sequences.
In recent efforts to show whether box B alone is sufficient for
nuclear transport, seven or
eight
quences
were
tosidase
or
constructs
we
synthesized oligonucleotides coding region. These synthetic
amino acids from this
either cloned into the amino terminus of
the carboxy term-inus of pTRB migrated (data not shown). At
at
unable to tell the
reasons
for this failure.
for se-
fl-galac-
103. Neither of these the moment
Although
we
are
the fusion
proteins displayed the correct apparent mol. wt, a few impormight have been clipped off, we might have omitted some important flanking amino acids for the construction, the sequence might not be presented in the correct way or maybe some interaction with other regions close by is necessary. tant amino acids
Construction of a set of imzproved bacterial expression vectors T'he 03-galactosidase expression vectors pUR 290, pUR 291 and pUR 292 described by Ruither and Muiller-Hill (1983) were improved by cloning translation and transcription stop signals after the polylinker region. pEX 2, described by Stanley and Luzio (1984), containing synthetic translation steps in all three reading framies and tandem transcription stop signals, was digested with TthIll, 'filled in' with T4 DNA polymerase and then digested with PstI. The appropriate isolated fragment was cloned into pUR 291 that had been digested with HindIH, filled in, and digested with Pstl. This construct, called pTRB 1, was digested with EcoRI, filled in and digested with PstL. The restriction fragment, now containing the stops, was eluted out of an agarose gel after electrophoresis, and cloned into pUR 290 and pUR 292 that had previously been digested with HinduI, filled in and digested with Pstl. These constructs were called pTRB 0 and pTRB 2 (see Figure 7). Construction of (3-galactosidase-nucleoplasmin hybrids The deletion constructs were obtained by using different restriction fragments of the nucleoplasmin cDNA as described below. The amino acid sequences generated in the joining regions are shown in Figure 8. The M 13 constructs are intermnediates for the plasmiid constructs. pTRB 12. The cDNA described by Buirglin et al. (1987) was subcloned into the EcoRI site of pAT 153. The orientation of the clone is such that the 3' end points towards the Hindlil site in pAT 153. pTRB 1(X). pTRB 12 was digested with EcoRI, filled in with T4 DNA polymerase and digested with Pst. The digested fragments were separated on an agarose gel and the 200-bp band, containing the carboxy terminus of nucleoplasmin, was eluted from the gel. This fragment was cloned into pUR 292 that had been digested with HinduH, ffied in and then digested with Pst. MJ3mnp8 N4. pTRB 12 was digested with EcoRI and filled in with T4 DNA polymerase. This fragment was cloned into Ml13 mp8 that had been digested with SinaI. Both orientations were cloned and the junctions were sequenced. The clone contamning the 5' end of nucleoplasnmin towards the BamHl polylinker site of M 13 mp8 was called M13 mnp8 N4, the other orientation was called M13 mp8 N3. pTRB8 102. M13 mp8 N4 was digested with EcoRI, filled in and digested with BamHI. The isolated fragment was cloned into pTRB 0 that had been digested with SaiI, filled in and digested with BamHI. pTRB 103. pTRB 102 was digested with Pstl and re-ligated in diluted conditions. pTRB 105. pTRB 102 was digested with Sadl, filled in and then digested with BamHl. The isolated fragment was cloned into pTRB 0 that had been digested with Pstl, filled in and then digested with BamnHI. M13 mp8 N5. M13 mp8 N4 was digested with BamHl and Pst. The isolated fragment was cloned into M 13 mp8 that had been digested with BamHI and PstI. pTRB 106. pTRB12 was digested with Sadl and filled in. A 12-mer Pstl linker oligonucleotide (Serva) was ligated to the ends. It was then digested with Pst, passed over an Elutip column (Schleicher and Schuell) and finally digested with Hindlu and once more with PstI. The fragments were separated on an agarose gel and the appropriate band was isolated. This PstI-HindUll fragment was cloned into M13 mp8 N5 that had been digested with PstI and HindIll. The positive clones were identified by plaque hybridization (Maniatis et al., 1982) using the
2623
The nuclear migration signal of nucleoplasmin SacI-Hindml fragment from pTRB 12 as probe. Several positives were sequenced and the desired construct was selected. M13 mp8 Nll. M13 mp8 N5 was digested with PstI and HindIll. Two complementary synthetic oligonucleotides with PstI and HindIll sticky ends coding for Ala Thr Lys Lys Ala Gly Gln were cloned directly into this M13. After Ttrack sequencing, clones containing the oligo-insert were sequenced completely. pTRB 107. M13 mp8 N1I was digested with BarnHI and HindmI and the isolated fragment was cloned into pTRB 0 that had been digested with BamHI and HindIm. pTRB 108. pTRB 102 was digested with Pvull and BamHI. The isolated fragment was cloned into pTRB 0 that had been digested with HindIlI, filled in and digested wtih BamHI. Subcloning and sequencing For restriction maps and subcloning into pAT 153 (Twigg and Sherratt, 1980), pUR 290, pTRB 0, pTRB 1 and pTRB 2 standard agarose gel, restriction digest and ligation methods were used (Maniatis et al., 1982). The junctions of constructs and oligos were sequenced in M13 (Messing and Vieira, 1982) using the dideoxy method of Sanger et al. (1977). Preparation offusion proteins Constructs were transformed into Fl 1 'recA or BMH 71 - 18 (Ruther and MullerHill, 1983). Strains containing the constructs were maintained on minimal plates supplemented with 50 tg/ml ampicillin. The preparation of fusion proteins was adapted from Young and Davis (1983a,b). In short, 25 ml of L-broth containing 100 yg/ml ampicillin was inoculated with the desired construct and grown overnight at 37°C. The overnight culture was diluted - 1: 100 into either 25 ml of L-broth containing 100 ,ug/ml ampicillin for highly expressed proteins like (3galactosidase or 500 ml for other fusion proteins. The cultures were grown up to OD550 = 0.5 (2-3 h). Cultures were then induced by addition of IPTG (final concentration 0.5 mM) and incubated for another 2 h. The cells were centrifuged, the supernatant was completely removed and the pellet was resuspended in a 1/25 volume of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2 (buffer B), plus 1.6 M NaCl and 10 mM (3-mercaptoethanol. The cells were sonicated on ice and the sonicate was centrifuged at 12 000 r.p.m. in a Sorval rotor SS34 for 15 min at 4°C. The protein was then purified using the affinity matrix described by Uliman (1984). The supernatant of the large cultures was processed as follows. It was loaded on a 1 ml column of affinity matrix and washed as described by Ullman (1984). The fusion proteins were eluted with 100 mM sodium borate-NaOH (pH 10) and three-drop fractions were collected. The fractions were assayed for (3-galactosidase activity by testing 5 Fl of each fraction in 150 1l of buffer B with 5 Al of X-gal (20 mg/ml in dimethylformamide). The 1 ml sonication supernatants of small cultures were processed in a batch procedure as follows. The supernatant was diluted with 2 ml of buffer B plus 1.6 mM NaCl (HS buffer B) and incubated with 100-200 a1 of affinity matrix beads at room temperature for 1 h on a slow end-over-end rotator. The beds were centrifuged at 1000 r.p.m. in a table top centrifuge for 1 min and the supernatant was removed. The beads were washed three times with 3 ml HS buffer B and twice with 1/20 diluted buffer B. The fusion protein was then eluted twice with 200 1il of 100 mM sodium borate-NaOH (pH 10.0) each time. All fractions were immediately neutralized by adding 1/15 volume of 2 M TrisHCI (pH 7.0). The peak fractions were analysed by SDS-polyacrylamide gels. Fractions were stored at -20°C. Fractions chosen for injection were dialysed for 2-3 h against buffer B and after dialysis gelatin was added to a final concentration of 0.1 %. After purification over the affinity matrix, ,B-galactosidase eluted with a concentration of at least 5 mg/ml as determined by Bradford (1976). The large fusion proteins (pTRB 103 -pTRB 108) eluted with a relatively low concentration of -0.1-0.2 mg/mil. Subsequently all fusion protein injections were performed at a protein concentration of -0.1-0.2 mg/mil. Proteins were iodinated as described by Newmeyer et al. (1986). Injection of oocytes and manual separation Oocytes were obtained by surgery from adult female X. laevis. Stage S and 6 oocytes (Dumont, 1972) were manually separated for injection, kept in 1 x modified Barth's solution and microinjected as described by Gurdon (1976). About 50 nl of sample was injected into the cytoplasm of each oocyte. After incubation at 19°C overnight (consistently 20-24 h) the oocytes were manually separated into cytoplasm and nucleus in Jordan buffer [10 mM Hepes, 70 mM NH4Cl, 7 mM MgCl2, 0.1 mM EDTA, 2.5 mM dithiothreitol (DTT), 10% glycerol; pH adjusted to 7.4 with NaOH] in a small Petri dish which had been covered inside with parafilm so as to prevent sticking of the nucleus to the glass. Nuclei for the re-injection experiment were isolated in PBS (70 mM Na2HPO4, 30 mM NaH2PO4, 130 mM NaCl). Demembranated nuclei were prepared in glass Petri dishes: the nuclei were allowed to stick to the glass and by gently pushing the nuclei with a micropipette, the membrane was disrupted and the nuclear gel, which shrinks away from the membrane, could be recovered. Cytoplasmic
2624
or nuclear fractions from usually 10 oocytes were collected in small homogenizers containing 50 1l of 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCI2, homogenized, and the yolk and the lipids were then removed by centrifugation at 1000 g for 15 min at 4°C. ,3-Galactosidase activity assays with ONPG substrate were performed according to Miller (1972). Uninjected oocytes were used to adjust for background activity, which is very low in oocytes. Protein gels and Western blots SDS-polyacrylamide gel electrophoresis was performed according to Takacs (1979). 5% or 7.5% gels were used for ,B-galactosidase fusion proteins. Transfer of proteins to nitrocellulose (Schleicher and Schuell) was performed according to Towbin et al. (1979) (Western blots). For immunostaining of the blots the protocol of Fritz et al. (1984) was used. [125I]protein A was obtained from ICN. After immunostaining the nitrocellulose blots were stained with Amido black to check for efficiency of transfer. This is possible despite having blocked the filter by treatment with solution A (Zeller et al., 1983). During this study we noted that nitrocellulose filters can be re-stained with other antibodies, if the filter is still humid during the autoradiographic exposure. After exposure, the slightly humid filter is washed in 100-200 ml of 5% SDS, 50 mM Tris-HCl (pH 7.5) for 15-30 min, and is then washed three times with 100-200 ml of Tris-buffered saline, before submitting it to a fresh staining cycle.
Acknowledgements We are indebted to a number of colleagues for much help and advice during this project. We would like to thank lain Mattaj for his excellent technical advice and stimulating discussions, Don Newmeyer for stimulating discussions and for radiolabelling fusion proteins, Ken Cho, Johannes Wirz, Drs Fritz Melchers and Audree Fowler for gifts of (3-galactosidase antiserum, Drs B.Muller-Hill and K.Stanley for gifts of vectors and strains, Dr D.Glitz for providing the oligonucleotides. We specially thank Larry Tabata for his graphic work, and Christopher Wright, Ken Cho and Larry Zipursky for critical reading of the manuscript. This work was supported by grants of the Swiss National Science Fund, the Kanton Basel-Stadt, the Weingart Foundation, the W.M.Keck Foundation and the NIH.
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T.R.Burglin and E.M.De Robertis Laskey,R.A., Honda,B.M., Mills,A.D. and Finch,J.T. (1978) Nature, 275, 416-420. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY. Messing,J. and Vieira,J. (1982) Gene, 19, 269-276. Miller,J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, NY. Mills,A.D, Laskey,R.A., Black,P. and De Robertis,E.M (1980) J. Mol. Biol., 139, 561-568. Moreland,R.B., Nam,H.G., Hereford,L.M. and Fried,H.M. (1985) Proc. Natl. Acad. Sci. USA, 82, 6561-6565. Newmeyer,D.D., Lucocq,J.M., Burglin,T.R. and De Robertis,E.M.(1986) EMBO J., 5, 501-510. Paine,P.L., Moore,L.C. and Horowitz,S.B. (1975) Nature, 254, 109-114. Richardson,W.D., Roberts,B.L. and Smith,A.E. (1986) Cell, 44, 77-85. Richter,J.D., Young,P., Jones,N.C., Krippl,B., Rosenberg,M. and Ferguson,B. (1985) Proc. Natl. Acad. Sci. USA, 82, 8434-8438. Ruther,U. and Muller-Hill,B. (1983) EMBO J., 2, 1791-1794. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Silver,P.A., Keegan,L.P. and Ptashne,M. (1984) Proc. Natl. Acad. Sci. USA, 811, 5951-5955. Stanley,K.K. and Luzio,J.P. (1984) EMBO J., 3, 1429-1434. Takacs,B.J. (1979) In Lefkovits,I. and Pernis,B. (eds), Immunological Methods. Academic Press, NY, pp. 81-105. Towbin,H., Staehelin,T. and Gordon,J. (1979) Proc. Natl. Acad. Sci. USA, 76, 4350-4354. Twigg,A.J. and Sherratt,D. (1980) Nature, 283, 216-218. Ullmann,A. (1984) Gene, 29, 27-31. Wychowski,C., Benichou,D. and Girard,M. (1986) EMBO J., 5, 2569-2576. Young,R.A. and Davis,R.W. (1983a) Science, 222, 778-782. Young,R.A. and Davis,R.W. (1983b) Proc. Natl. Acad. Sci. USA, 80, 1194-1198.
Received on March 13, 1987; revised on May 8, 1987
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