The analysis of the transcriptional activator PrnA reveals a tripartite nuclear localisation sequence 1

doi:10.1006/jmbi.2000.3666 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 298, 585±596

The Analysis of the Transcriptional Activator PrnA Reveals a Tripartite Nuclear Localisation Sequence Anna Pokorska, Christine Drevet and Claudio Scazzocchio* Institut de GeÂneÂtique et Microbiologie, UMR CNRS C8621, Universite Paris-Sud BaÃtiment 409, Centre Universitaire d'Orsay F-91405, Orsay Cedex France

Nuclear localisation signals (NLSs) have been classi®ed as either monoor bipartite. Genetic analysis and GFP fusions show that the NLS of a Zn-binuclear cluster transcriptional activator of Aspergillus nidulans (PrnA) is tripartite. This NLS comprises two amino-terminal basic sequences and the ®rst basic sequence of the Zn-cluster. Neither the two amino-terminal basic sequences nor the paradigmatic nucleoplasmin bipartite NLS drive our construction to the nucleus. Cryosensitive mutations in the second basic sequence are suppressed by mutations that restore the basicity of the domain. The integrity of the Zn-cluster is not necessary for nuclear localisation. A tandem repetition of the two basic amino-terminal sequences results in a strong NLS. Complete nuclear localisation is observed when the whole DNA-binding domain, including the putative dimerisation element, is included in the construction. At variance with what is seen with tandem NLSs, all ¯uorescence here is intranuclear. This suggests that retention and nuclear entry are functionally different. With the whole PrnA protein, we observe localisation, retention and also a striking sub-localisation within the nucleus. Nuclear localisation and sub-localisation are constitutive (not dependent on proline induction). In contrast with what has been observed by others in A. nidulans, none of our constructions are delocalised during mitosis. This is the ®rst analysis of the NLS of a Zn-binuclear cluster protein and the ®rst characterisation of a tripartite NLS. # 2000 Academic Press

*Corresponding author

Keywords: tripartite NLS; transcriptional activator; nuclear localisation; green ¯uorescent protein; Aspergillus nidulans

Introduction Eukaryotic transcriptional regulators obviously play a role in the nucleus, where they activate or repress gene expression. Transport across the nuclear pore has been studied in some detail (for reviews see Melchior & Gerace, 1995; Hurt, 1996; Fabre & Hurt, 1997). The ®rst step in this process is the recognition of the protein to be transported by a speci®c transporter called either a-importin (GoÈrlich et al., 1994), karyopherin a (Moroianu et al., 1995) or SrpIp in Saccharomyces cerevisiae (Yano et al., 1992). A gene coding for a protein highly Present address: A. Pokorska, Division of Neurobiology, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Abbreviations used: NLS, nuclear localisation signal; DAP1, 4,6-diamino-2-phenylindole; GFP, green ¯uorescent protein. E-mail address of the corresponding author: [email protected] 0022-2836/00/040585±12 $35.00/0

similar to karyopherin a has recently been characterised in Aspergillus nidulans (A. P., J. Doonan & C. S., unpublished results). To be recognised by karyopherin a, the target protein has to include a so-called nuclear localisation signal (NLS). There is no clear consensus for the sequence recognised by karyopherin a, besides the fact that it must include a stretch of basic amino acid residues. Broadly, NLSs have been classi®ed in two groups, monopartite and bipartite NLSs. The ®rst category is exempli®ed by the sequence found in the simian virus 40 (SV40) large T antigen (Kalderon et al., 1984). The second category, exempli®ed by the nucleoplasmin NLS, is a bipartite sequence comprising two clusters of basic amino acid residues separated by a spacer (Robbins et al., 1991). The length of the spacer can vary considerably in different bipartite NLSs, but oscillates around ten residues, while the precise sequence of the spacer seems to be irrelevant to nuclear transport (Nath & Nayak, 1990; Robbins et al., 1991). Not only the precise sequence organisation of NLSs is quite vari# 2000 Academic Press

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The Tripartite NLS of the Proline Pathway Activator PrnA

able, their localisation can be amino or carboxyterminal, or even map elsewhere in the protein (Matheny et al., 1994). Bipartite NLSs are well characterised in higher eukaryotes (Schmidt-Zachmann et al., 1987; Moll et al., 1991; Addison et al., 1990; Picard & Yamamoto, 1987; Schreiber et al., 1992; Slingerland et al., 1993; Rubtsov et al., 1997). No systematic work on the nature of NLSs has been carried out in fungi. This contrasts with the substantial body of work carried out on transcription factors or repressors, such as Gal4p, Put3p, Gcn4p, Mig1p of Saccharomyces cerevisiae, AreA, CreA, AlcR, NirA, UaY of A. nidulans or QUT1-F, NIT2 and NIT4 of Neurospora crassa and the homeodomain mating type factors of the yeasts and the basidiomycetes (for reviews see Schjerling & Holmberg, 1996; Todd & Andrianopoulos, 1997). Even for the paradigmatic Gal4p protein, all that is known is that the ®rst 74 amino-terminal residues are suf®cient to ensure nuclear localisation (Silver et al., 1984). A carboxy-terminal putative bipartite nuclear localisation sequence from the StuA protein of A. nidulans has been shown to be suf®cient to drive nuclear localisation of the green ¯uorescent protein (Suelmann et al., 1997). Nuclear transport can be an essential step in transcriptional regulation. Different mechanisms, including reversible phosphorylation of sequences adjacent to the NLS, unmasking of the NLS or release from an inhibitory protein can result in inducible nuclear localisation (for a review, see Vandromme et al., 1996). In S. cerevisiae, Mig1p is only localised in the nucleus under carbon catabolite repressing growth conditions (DeVit et al., 1997). The NUC1 transcription factor of N. crassa is localised in the nucleus only under conditions of phosphate limitation (Peleg et al., 1996). We have previously described the domain organisation of the PrnA transcriptional regulator of A. nidulans (Cazelle et al., 1998). This protein mediates induction proline and is essential for the expression of the four structural genes of the prn gene cluster, coding for all the activities involved

in the utilisation of proline as the sole nitrogen and carbon source. This protein is thus isofunctional to, and presents some similarities with, the Put3p protein of S. cerevisiae, even if it binds to quite different DNA sequences (Siddiqui & Brandriss, 1989; Swaminathan et al., 1997; D. Gomez, B. Cubero & C. S., unpublished results). We suggested previously that the ®rst exon of the prnA gene, comprising 28 residues, contained the NLS of the PrnA protein (Cazelle et al., 1998). We show here that this domain is indeed necessary but not suf®cient for nuclear localisation and here we present for the ®rst time evidence that a nuclear localisation signal is tripartite.

Results Characterisation of cold-sensitive mutations mapping in the putative NLS of PrnA The peptidic sequence of the PrnA regulator revealed a region in the N terminus of the protein containing two basic amino acid residue clusters (Cazelle et al., 1998). It seemed reasonable to assume that these two clusters constituted the NLS, as no other clusters of basic residues are found elsewhere in the protein sequence, with the exception of the DNA-binding domain (see below). Moreover, a mutation resulting in a cryosensitive phenotype (Arst et al., 1981), prnA29 (Arg23Leu) is located in the second basic cluster (Cazelle et al., 1998). A second mutation with an identical phenotype (prnA121) had been located by classical genetic analysis in the same region (Arst et al., 1981; Sharma & Arst, 1985). Sequencing of this mutation has shown it to occur in the same codon and to result in an Arg23Pro change (Figure 1). We have con®rmed that these mutations are indeed responsible for the inability to utilise proline as a nitrogen source at 25  C by transforming each mutant strain with overlapping PCR fragments, as described by Cazelle et al. (1998), and recovering strains with a prnA‡ phenotype.

Figure 1. Sequence changes in the prnA29 and prnA121 mutants and in their revertants. Basic amino acid residues are in bold, mutated amino acid residues are underlined. The ‡, ÿ and ‡/ÿ signs indicate the growth at 25  C of different strains as shown in Figure 2, on proline as the sole nitrogen source. The number of sequenced revertants carrying the three different reversion changes is shown. Besides these suppressor mutations, 15 true revertants of prnA29 and three of prnA121 were found. Numbers above the peptide sequence indicate the position of the ®rst and last residue shown in the sequence of PrnA.

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The Tripartite NLS of the Proline Pathway Activator PrnA

Reversion of the prnA29 and prnA121 mutations We hoped to identify the gene coding for the protein interacting with the putative PrnA NLS by isolating extragenic suppressors of the two cryosensitive mutations. We also hoped that isolation of intragenic suppressors would contribute to the determination of which sequences are acceptable as NLSs by the transport machinery of the cell. In spite of a very thorough search we failed to obtain extragenic suppressor mutations, either using UV light or 4-nitroquinoline-1-oxide as mutagens. The search included the methodology detailed by Goldman & Morris (1995). A total of 1020 revertants were screened by this method. To include in our search dominant suppressors which could be also recessive lethals, we repeated, with no success, the search in a diploid strain homozygous for the prnA29 allele. We did, however, obtain intragenic suppressors. The results are summarised in Figure 1 and shown in Figure 2. All intragenic suppressors restore the overall number of basic residues in the region of the putative NLS. prnA29 is suppressed by Glu(21)Lys, while prnA121 is suppressed by Asn(22)Lys. Both these suppressors restore wildtype growth. Glu(20)Lys suppresses partially both prnA121 and prnA29. We also obtained true revertants of both cryosensitive mutations. The fact that we obtained each suppressor mutation and true reversions several times suggests, but does not prove, that we have saturated the system and thus, that Asn(22)Lys does not suppress Arg(23)Leu, and Glu(21)Lys does not suppress Arg(23)Pro. It

Figure 2. Growth of strains carrying the prnA29 and prnA121 mutations and their revertants on proline as the sole nitrogen source. A prnA‡ strain and the prnA404 total loss of function mutant are included as controls. (a) Growth at 25  C; (b) growth at 37  C. The position and relevant genotype of each strain are indicated under the Petri dishes.

should be mentioned that the three suppressor mutations obtained are all the mutations to a basic amino acid residue that are possible as a result of a one base-pair change in the region between the two basic clusters. Mapping of the PrnA NLS by GFP fusions In order to map precisely the NLS of PrnA we made use of a reporter protein, the green ¯uorescent protein (GFP). We have transformed an A. nidulans wild-type strain with plasmid pAN521-GFP (gift from Corinne ClaveÂ, Bordeaux), containing the strong constitutive A. nidulans gpd promoter and the SGFP-TYG version of the GFP. This construction was shown to result in strong overall ¯uorescence of A. nidulans hyphae and conidia. The GFP localises uniformly in the cytoplasm and nuclei of the cell, but not in vacuoles and mitochondria. Its expression is clearly visible in the hyphae as well as in the conidia. This is consistent with the observation of others (Suelmann et al., 1997; FernaÂndez-Abalos et al., 1998). Temperature affects neither the expression nor localisation of the GFP: the same pattern is seen at 25  C and 37  C. While this work was in progress, and in line with our ®ndings (see below), it has been reported that, while the GFP stains the nuclei in the absence of any NLS, preferential localisation could be observed clearly if a putative NLS was included in the construction (Suelmann et al., 1997; FernaÂndezAbalos et al., 1998). We then proceeded to map the PrnA NLS by fusing to the amino terminus of the GFP coding region a number of sequences from the aminoterminal region of the open reading frame of the prnA gene. The large number of constructions analysed made it unpractical to introduce them in vectors that would allow insertions of all of them in the same locus of the genome. Thus, all constructions were introduced in A. nidulans by cotransformation with a plasmid carrying an argB‡ gene and only transformants containing single copy integrations of the plasmid carrying the GFP constructions were used for further work. In order to offset any artefacts due to differential expression resulting from position effects, at least three different single copy transformants were analysed for each construction with identical results in all cases. Four clusters of basic amino acid residues are present in the amino terminus of PrnA, two in the ®rst exon, comprising the putative NLS and two within the DNA-binding domain. The basic clusters are underlined in Figure 3 and are numbered 1, 2, 3 and 4 from the amino terminus of the protein. Constructions carrying different combinations of the basic clusters were checked for their ability to drive nuclear entry. The different constructions were checked after growth at 25  C and 37  C. Only the constructions where nuclear entry is cryosensitive are shown at both temperatures in Figure 4.

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The Tripartite NLS of the Proline Pathway Activator PrnA

Figure 3. Amino acid sequence of the N-terminal fragment of PrnA used for the GFP fusion protein constructions. Clusters of basic amino acid residues are underlined and are numbered 1 to 4 from the N terminus of the PrnA protein. The Zn-binuclear cluster DNA-binding domain is overlined. The putative dimerisation domain is framed. The ability of different constructions to locate the GFP into the nucleus is indicated as follows; (ÿ), no nuclear localisation; (‡, ‡ ‡ , ‡ ‡ ‡), increasing intensities of ¯uorescence in the nucleus (see Figures 4 and 5). The presence of the putative dimerization element is indicated by dim. Of the total number of transformants obtained with each construction and showing GFP ¯uorescence, we indicate the number which was shown to carry single-copy integration events by Southern blot analysis. The sequence coding for the bipartite nucleoplasmin NLS was introduced between PrnA residues so as to substitute exactly the PrnA tripartite NLS.

Constructions containing clusters 1 and 2 failed to localise the GFP into the nucleus (Figure 4(b)). So did constructions containing clusters 2, 3 and 4 (Figure 4(c)). Constructions containing clusters 1, 2, 3 and 4 and 1, 2 and 3 localised the GFP into the nucleus, weakly but clearly. This is shown in Figures 4 and 5. Making use of a construction containing clusters 1, 2, 3 and 4 with the prnA121 Arg(23)Pro substitution in cluster 2 showed the role of the latter. It was satisfying to ®nd that this construction resulted in ability to transport the GFP into the nucleus at 37  C but not at 25  C (Figure 4(d)-(f)). These studies show that clusters 1, 2 and 3 are necessary and suf®cient for a weak (see below), but clearly seen, nuclear localisation of the GFP protein. The role of cluster 3 was also investigated by site-directed mutagenesis. We have substituted Arg34 and Arg35 with two glycine residues. In Figure 5(d), it is shown that this mutation results in complete loss of nuclear localisation. Identical results were obtained at 25  C and 37  C (shown only at 25  C in Figure 5).

In order to determine whether other NLSs could drive our construction to the nucleus we deleted the basic clusters 1, 2 and 3 and inserted in their place the well-studied nucleoplasmin bipartite NLS. The construction is shown in Figure 3 and detailed in Materials and Methods. No nuclear localisation was observed in ®ve different singlecopy transformants at either 25  C or 37  C, the germlings being identical to the control with the GFP alone (not shown as identical to Figures 4(a) and 6(a)). Even when this construction was present in as many as four copies per nucleus, no nuclear localisation could be observed. We have also constructed a hybrid gene comprising basic clusters 1, 2, 3 and 4 and the GFP as described above but driven by the weak, physiological, prnA promoter, rather than by the strong constitutive gpd promoter. While very weak expression of the GFP is seen, the localisation into the nucleus is evident (data not shown). It should be noted that the integrity of the binuclear zinc cluster is not necessary for nuclear localis-

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The Tripartite NLS of the Proline Pathway Activator PrnA

sequences (data not shown). Thus, no other sequences in PrnA are able to act as a NLS. Multimers of the 1, 2 clusters are artificial strong NLSs We have made constructions containing the 1 and 2 basic clusters twice and three times in tandem. These constructions result in an extremely strong nuclear localisation signal. We have constructed a trimer containing in each repeated unit the prnA121 Arg(23)Pro mutation. This mutation results in very clear cryosensitivity of the nuclear localisation of this very strong NLS (Figure 5(e)-(h)). Sequences carboxy-terminal to the NLS and to the Zn-binuclear cluster improve drastically nuclear retention

Figure 4. Subcellular localisation of different GFP fusion proteins. (a) Localization of the GFP protein without any PrnA sequence added. (b) and (c) GFP fusion with PrnA sequence 1-29 and 18-74 (see Figure 3) comprising the 1 and 2 and 2, 3 and 4 basic amino acid residue clusters, respectively. (d) GFP fusion with PrnA sequence 1-74 comprising the 1, 2, 3 and 4 basic amino acid residue clusters. (e) and (f) As (d) but carrying the prnA121 mutation (Arg23Pro) in cluster 2 at 25  C and 37  C, respectively. (g) GFP fusion with PrnA sequence 1-27 (clusters 1-2) repeated three times in tandem.

ation (but see below). The construction containing the 1, 2, 3 basic clusters obviously does not contain an intact Zn cluster. This was further investigated by introducing in 1, 2, 3, 4 construction the prnA1 mutation, Pro(45)Leu (Cazelle et al., 1998). This mutation affects a proline residue universally conserved in all binuclear Zn clusters, and it is supposed to affect the formation of the Zn2‡-cysteine complex (Johnston, 1987). A construct carrying this mutation drives the GFP reporter into the nucleus with the same ef®ciency as a wild-type construction (Figure 5(c)). A construction carrying the whole PrnA protein with the exception of the ®rst two exons showed the same pattern of subcellular localisation as the GFP control carrying no extraneous protein

We constructed a longer fusion protein, including up to residue 130. The results are shown in Figures 6(b) and 8(b). We obtain with this fusion excellent nuclear localisation of the order of that obtained with the triplicate tandem 1, 2. What is particularly interesting is that while the apparent intensity of ¯uorescence in strains carrying the duplicate and triplicate tandems is very strong, this is seen on the background of a ¯uorescent cytoplasm. The 1-130 construction shows a similar apparent intensity of ¯uorescence, but the cytoplasmic background is virtually nil (Figure 6(b)). It may be important that this construction includes a putative prnA dimerisation element (see Discussion). Localisation of the whole PrnA protein We made two constructions with the GFP fused to the carboxy terminus of the whole prnA open reading frame. One construction was driven by the physiological prnA promoter, the second one by the strong constitutive gpd promoter. A prnA404 null mutation strain was used as a recipient. The ®rst observation is that full complementation of the prnA404 mutation is seen. The GFP-PrnA fusion allows the prnA404 strain to grow as well as the wild-type on proline as the sole nitrogen source (not shown). Observation of several transformants containing single copies of these constructions showed a very clear nuclear localisation of the GFP (Figures 6(c), (d) and 7). The two constructions behave identically, except that the ¯uorescence is much stronger in the construction containing the gpd promoter (Figure 7). The cytoplasmic background is as low as in the construction including only the putative dimerisation element. The surprising observation is the clear subnuclear localisation of the fusion protein. Counter-staining with DAPI shows strong staining of the areas occupied by the PrnA protein. Identical results were obtained whether the cultures were grown in the presence or the absence of proline (only mycelia

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The Tripartite NLS of the Proline Pathway Activator PrnA

Figure 5. Localisation of the GFP fusion proteins carrying the following sequences of PrnA: (a) 1-74 (comprising the 1, 2, 3 and 4 basic amino acid residue clusters); (b) 1-41 (clusters 1, 2 and 3); (c) 1-74 (clusters 1, 2, 3 and 4) carrying the prnA1 mutation of the Zn cluster; (d) 1-41 (clusters 1, 2 and 3) carrying two mutations in cluster 3: (Arg34Gly), (Arg35Gly). (e)-(h) Effect of the prnA121 mutation on a strong arti®cial NLS. (e) and (f) Sequences 1-27 (comprising clusters 1-2) three times in tandem, at 25  C and 37  C, respectively. (g) and (h) The same as (e) and (f) but carrying the prnA121 mutation. Colour photography results in different intensities of GFP ¯uorescence to appear as different tones of yellow-green, with more intense ¯uorescence resulting in a more yellowish appearance of the nuclei (observe differences between (a), (b) and (c) on the one hand and (e), (f) and (h) on the other). This corresponds to the difference in intensities shown in Figure 4 between (d) and (f) on the one hand and (g) on the other.

grown in the absence of proline are shown in Figures 6 and 7). Thus, neither nuclear nor subnuclear localisation depend on a conformational change of the PrnA protein subsequent to induction with proline.

GFP nuclear localisation is not lost during mitosis It has been reported that a construction containing the putative NLS of StuA fused to the carboxy terminus of the GFP is well localised in the nuclei, but that the localisation is lost during mitosis (Suelmann et al., 1997). We have failed to observe a similar phenomenon with any of our constructions. We observed both synchronised and non-synchronised mitotic ®gures in the transition from two to four and four to eight nuclei in germinating spores. Examples of this can be seen in Figure 8. We show retention in the nuclei in late anaphase-telophase (Figure 8(a)) and in telophase (Figure 8(b) and (c)). This is true for all constructions tested, and we have not seen the disappearance of nuclear stain-

ing in any of the constructions showing nuclear localisation.

Discussion This work is the ®rst analysis of the NLS corresponding to a Zn-binuclear cluster transcriptional activator. We have discovered a new class of NLSs and obtained data that suggest that nuclear entry is distinct from nuclear retention. We have shown that different bipartite combinations of clusters of basic sequences found in the amino terminus of the PrnA transcriptional activator of A. nidulans fail to drive entry of the cognate GFP fusion proteins into the nucleus. The minimal requirement for transport into the nucleus is that of a sequence of three basic clusters (1, 2 and 3). Of particular signi®cance is the fact, that, at least in the context of the PrnA amino-terminal sequence, the paradigmatic nucleoplasmin NLS does not result in nuclear localisation. This is, as far as we are aware, the ®rst demonstration of a tripartite nuclear localisation sequence. The agreement between the cryosensitivity of PrnA function

The Tripartite NLS of the Proline Pathway Activator PrnA

Figure 6. (a) Localisation of the GFP protein without any PrnA sequences. (b) Nuclear localisation of the GFP fusion with PrnA sequence 1-130 (see Figure 3) comprising the 1 and 2 basic amino acid residue clusters, the whole Zn-binuclear cluster domain (and thus basic sequences 3 and 4) and all sequences up to and including the putative dimerisation domain. (c) and (d) Subnuclear localisation of the whole PrnA protein fused to GFP and driven by the gpdA promoter; (c) the GFP ¯uorescence and (d) the DAPI staining of the same ®lament.

(assessed by the utilisation of proline as a nitrogen source) and the cryosensitivity of nuclear entry is particularly satisfying, as it bridges the macroscopical and phenotypic level with the sub-cellular level of observation. A GFP construction including the 1, 2, 3 and 4 motifs and carrying the prnA121 mutation has exactly the same temperature dependence vis aÁ vis nuclear entry as that shown by growth test of strains carrying the prnA29 and prnA121 mutations. The identical temperature dependence of both the growth phenotype and the nuclear entry process demonstrates that the second basic sequence is necessary for nuclear localisation of the intact prnA protein and thus that the DNAbinding domain, including its putative dimerisa-

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Figure 7. Subnuclear localisation of the whole PrnA protein fused to GFP. (a) and (b) The localisation of this fusion protein driven by the gpdA promoter; (c) and (d) by the weak physiological prnA promoter. For each construct, the ¯uorescence of GFP ((a) and (c)) and DAPI ((b) and (d)) is shown. Not all the nuclei are in the same focal plane.

tion element, is not suf®cient to drive nuclear localisation. The restoration of the basicity of cluster 2 in all revertants further supports this conclusion (see below). The very strong arti®cial NLSs obtained when sequences 1 and 2 are repeated two or three times in tandem cannot be explained as a simple quantitative effect, as the nuclear entry observed for the construction containing non-repeated sequences 1 and 2 is nil (identical to the control carrying the GFP without additional sequences). Two other interpretations are possible. One, is that a new, arti®cial, tripartite sequence, composed by sequences 1-2-1 and/or 2-1-2 is recognised by, presumably, karyopherin a. The second interpretation is that sequences 2-1 constitute an acceptable bipartite NLS while sequences 1-2 do not. Sequences 2 and 1 in the tandem constructions are separated by 17 residues. The distance between the two basic sequences of the nucleoplasmin NLS has been increased up to 22 residues without loss of nuclear localisation (Robbins et al., 1991) while the distance found between the two basic sequences of the BP1 polymerase from in¯uenza virus is of 16 residues.

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The Tripartite NLS of the Proline Pathway Activator PrnA

Figure 8. Mitotic nuclei retain GFP-fusion protein ¯uorescence. (a) A synchronised anaphase-telophase event visualised with the GFPPrnA (1-74) fusion protein. (b) and (c) A non-synchronised telophase event: (b) GFP ¯uorescence, (c) DAPI staining of the same ®lament. The construction used for this observation contains the PrnA sequence 1-130 comprising the 1 and 2 basic amino acid residue clusters, the whole Zn-binuclear cluster domain (and thus basic sequences 3 and 4) and all sequences up to and including the putative dimerisation domain.

The fact that the nucleoplasmin NLS fails to drive the GFP construction into the nucleus, supports the ®rst interpretation. Whatever the basis of the ability of these, arti®cially strong NLSs, to drive entry into the nucleus, proteins carrying these are translocated by a mechanism including at least one step (presumably the NLS recognition step) identical to that operating with the natural tripartite NLS. This is demonstrated by the cryosensitivity of nuclear localisation observed in tandem constructions carrying the prnA121 Arg(23)Pro mutation. An important observation is that nuclear localisation and nuclear retention seem to be functionally different. We have improved nuclear localisation by duplicating or triplicating in tandem a bipartite sequence, which is per se unable to drive localisation into the nucleus, and separately nuclear retention by adding a sequence, which presumably permits the protein to maintain a stable quaternary structure. This sequence may act in two different, albeit non-exclusive ways. The addition of this sequence may result in the attachment of the protein to a sub-nuclear structure (Schmidt-Zachmann et al., 1993) or simply displace an equilibrium towards the nuclear-internalised form. The latter may be the somewhat trivial result of protein dimerisation and the resulting increase in protein size. Proteins of the binuclear Zn cluster class that have been characterised to date bind DNA, with one possible exception, as dimers (Marmorstein et al., 1992; Marmorstein & Harrisson, 1994; Swaminathan et al., 1997; Lenouvel et al., 1997; Strauss et al., 1998). A dimerisation element, separated by a short number of residues from the DNA binding domain sensu strictu, is almost universally present in these proteins (Reece & Ptashne, 1993; Schjerling & Holmberg, 1996). This dimerisation element could thus be considered functionally as part of the DNA-binding domain. The putative dimerisation element of PrnA is between amino acid residues 109 and 121. Its location was determined employing the PepCoil algorithm (Cazelle et al., 1998). This algorithm predicts very accurately

the dimerisation elements of Gal4p, Ppr1p and Put3p, elements that are known by crystallography (Marmorstein et al., 1992; Marmorstein & Harrison, 1994; Lewis et al., 1996; Swaminathan et al., 1997). The construction carrying the motifs 1, 2, 3 and 4 (1-74), while carrying a complete Zn binuclear cluster, does not carry the putative dimerisation element. The most straightforward interpretation of the different behaviour of the strong arti®cial NLSs (1-27 repeated in tandem) and the 1-130 construction is that basic sequences in tandem only increase the ef®ciency of nuclear localisation, while an intact DNA binding domain improves nuclear retention. It should be stressed that between residues 74 and 130 there are no basic clusters that could act as additional NLSs. The conclusion that sequences carboxy terminal to the binuclear Zn cluster improve nuclear retention holds irrespective of whether residues 109-121 turn out to be a genuine dimerisation sequence. It is, however, relevant that a peptide carrying residues 1-150 has been shown to recognise correctly in vitro PrnA binding sites which are inverted repeats (D. GoÂmez, B. Cubero, & C. S., unpublished results). It is unlikely that retention is due to DNA binding per se, as the whole prn cluster (comprising all the genes known to require PrnA for transcription, for a review, see Scazzocchio, 1994) contains only ®ve PrnA binding sites (D. GoÂmez, B. Cubero, & C. S., unpublished results). A similar retention pattern is observed in A. nidulans when the Gal4p 1-120 sequence is fused to the GFP (FernaÂndez-Abalos et al., 1998). On the other hand, the results shown in Figure 1 by Suelmann et al. (1997), where a bipartite nuclear localisation sequence from StuA is fused to the carboxy terminus of the GFP, suggests that this sequence is not suf®cient to ensure fully nuclear retention. Protein shuttling between nucleus and cytoplasm has been explained as the interplay between NLSs and NESs (nuclear export sequences; Wen et al., 1995; Gerace, 1995) or by the absence of retention in the nucleus. The latter has been shown to be the case for a number of proteins

The Tripartite NLS of the Proline Pathway Activator PrnA

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in Xenopus oocytes (Schmidt-Zachmann et al., 1993). No results bearing on this process have been previously presented for ascomycetes. The behaviour of constructions involving nested sequences of the PrnA protein supports, at least for this protein, a model in which nuclear entry and nuclear retention are separate and separable functions. No putative NES sequences are present in PrnA. It is particularly interesting that a complete and functional PrnA protein driven by its own weak, or by a strong constitutive promoter, is retained in the nucleus in the absence of inducer. Zn-binuclear cluster proteins could be thought of as analogous to hormone receptors. They are speci®c for a given group of organisms (hormone receptors for metazoans and Zn-binuclear cluster proteins for fungi) and respond to signals external to the cell. Hormone receptors are shuttled between the cytoplasm and the nucleus (Madan & DeFranco, 1993). This is surely not the case for the Zn-binuclear cluster proteins Gal4p, and Put3p, as these proteins are bound to their cognate URFs also under noninducing conditions (Selleck & Majors, 1987; Siddiqui & Brandriss, 1989). We show here, by a different methodology, that PrnA is not shuttled between cytoplasm and nucleus in response to induction. The subnuclear localisation of PrnA is intriguing. Sublocalisation of the human Zn ®nger protein ZNF74 in the nuclear matrix is re¯ected in immuno¯uorescent patches rather than discrete dots, as is seen for PrnA (Grondin et al., 1997). Further work will be necessary to determine if the sub-localisation of PrnA has functional signi®cance. The fact that the regions occupied by PrnA stain strongly with DAPI may suggest that this localisation does not occur in the nucleolus. The integrity of the fungal nuclear membrane is maintained throughout mitosis, and thus the observations of Suelmann et al. (1997) are particularly interesting. A video by these authors is available that shows clearly the simultaneous disappearance of staining from the nuclei during synchronised mitosis in germinating spores of A. nidulans (Suelmann et al., 1997). We failed to observe a similar phenomenon with any of our constructions. Apparently, loss of nuclear localisation during mitosis was not observed in A. nidulans with a construction containing the NLS of Gal4p (FernaÂndezAbalos et al., 1998). The latter, and all our constructions carry the NLS in the amino terminus of the GFP, while that tested by Suelmann et al. (1997), carries it in the carboxy terminus. We do not know whether this is the relevant factor involved in loss during mitosis. It is possible that tripartite NLSs are recognised by a protein involved in a karyopherin a-independent nuclear entry pathway. This is considered unlikely, as the best characterised of these pathways, the one involving karyopherin b2, recognises a different type of NLS not involving stretches of basic sequences (for reviews, see Pollard et al., 1996; Pemberton et al., 1998).

The pattern of the monopartite SV40 NLS recognition by karyopherin a has been elucidated by crystallographic studies (Conti et al., 1998). These show the existence of two NLS binding sites located in the ``arm'' motifs of the karyopherin a, involving a set of tryptophan-asparagine residue pairs (WXXXN). The larger binding site is located between the second and the fourth arm motif and the smaller between the seventh and eighth arm repeat. The crystallographic structure ®ts rather well with the reversion data. The larger binding site can accommodate ®ve consecutive basic sidechains; our cluster 2 contains three consecutive basic residues, thus the new basic residue present in revertants prnA121/14 and prnA29/24 can be perfectly accommodated into the published structure (see Figure 1). Revertants prnA29/62 and prnA121/6 cannot in principle be accommodated into the published structure, the fact that they result in only partial reversion may result from these structural constraints. It is satisfying that the phenotypes of the revertants correlate, at least approximately with the published structure. The crystallographic data are not compatible with recognition of bipartite NLS sequences, as the ``arm''-repeat domain of karyopherin a forms a dimer in the crystal and the dimer interface occurs in the space between the two NLS binding sites. However, the dimerisation of karyopherin a may be an artefact of crystallisation. Recent data show that in solution karyopherin a binds to both monopartite and bipartite NLSs as a monomer. Physico-chemical studies with a protein containing amino acid residues 398 to 422 show the presence of two binding sites of different af®nity in this short protein (G. Percipalle, M. M. Altamirano, J. P. Butler, A. Fersht & D. Rhodes, personal communication). Thus, it is possible that a monomer of the whole protein, which contains four additional WXXXN motifs could recognise a tripartite sequence. It must be observed that each of the distances between the 1 and 2 and between the 2 and 3 basic sequences described here are shorter that the canonical distance found between the two basic sequences of a bipartite sequence (Robbins et al., 1991). The distance between the arm motifs of karyopherin a is variable, and this may be the basis for recognition of NLSs comprising different numbers of basic repeats and there may exist a relationship between the number of basic motifs comprising an NLS and the permitted distance between them.

Materials and Methods Strains, growth conditions and A. nidulans transformation techniques The following A. nidulans strains were used for random mutagenesis: prnA29 pabaA1 wA3 niaD10; prnA121 pabaA1 fwA1 sf211. The prnA29 and prnA121 alleles are cold-sensitive mutants of the prnA gene and do not grow on proline as the sole nitrogen source at 25  C (Arst et al., 1981; Cazelle et al., 1998). Strain argB2 pantoB2 yA2 was used as a recipient for all constructions containing

594

The Tripartite NLS of the Proline Pathway Activator PrnA

sequences coding for N-terminal fragments of PrnA, and strain argB2 prnA404 pyroB4 biA1wA3 for transformation with the plasmids containing the whole prnA gene fused to GFP. prnA404 is a total loss of function mutation in the prnA gene. It is a large deletion starting in the ®rst exon and ending in the ®fth intron of the prnA gene (Cazelle et al., 1998). It results in absence of prnA mRNA (A. P., S. Demais & C. S., unpublished results). The marker symbols are de®ned by Clutterbuck (1994). Standard media and growth conditions for A. nidulans were used (Cove, 1966). Supplements were added when appropriate. Transformation experiments were carried out according to Tilburn et al. (1983). GFP fusions were introduced into A. nidulans strains by co-transformation with the pFB39 plasmid containing the argB gene (Buxton et al., 1989). The number of copies integrated in the genome was determined by Southern blots of DNA restricted with appropriate enzymes.

these constructions were driven by the strong constitutive gpd promoter and contain the trpC transcriptional terminator. The construction carrying the entire prnA gene was obtained by cloning the whole prnA open reading frame to the NcoI site of the pAN52-1-GFP plasmid or to a plasmid containing the prnA promoter and the GFP protein. In order to make these constructions, a NcoI site was introduced by PCR at the stop codon of prnA.

Random mutagenesis The two prnA mutants were mutagenised using UV light or 4-nitroquinoline-1-oxide. In both cases 4  107 spores/ml were suspended in 10 ml of 0.04 % (v/v) Tween. A 254 nm UV lamp was used. Spores were exposed to UV for 5, 15, 30 and 45 seconds and then poured onto selective medium containing proline as sole nitrogen source. 4-Nitroquinoline-1-oxide mutagenesis was carried out as described (Bal et al., 1977). Genetic techniques

Fluorescence microscopy Samples of A. nidulans mycelia used for ¯uorescence microscopy were prepared as follows. Suspensions of conidia from transformants expressing different GFP fusions were prepared in minimal medium with 1 % (w/v) glucose as the sole carbon source, 20 mM ammonium tartrate or 20 mM proline as the sole nitrogen source, and adequate supplements. Drops of these suspensions were placed on coverslips and left to grow for 16 hours at 25  C or seven hours at 37  C. Fluorescence was detected with a Zeiss ®lter (BP450-490 excitation ®lter, 510 nm dichroic and LP 520 emission ®lter). For DAPI (4,6-diamino-2-phenylindole) staining, a 0.1 mg/ml dilution in 50 % (v/v) glycerol was used. Volumes of 5 ml of this solution were added to the samples prior to visualisation under the microscope. To allow cross-comparison between different constructions, microscopic observation of strains carrying some of them are repeated in different Figures. In these cases the strains were grown, observed and photographed each time independently.

Standard genetic techniques were used (Pontecorvo et al., 1953) with modi®cations (Clutterbuck, 1994). Sequencing

Acknowledgements

Sequencing was carried out by the dideoxynucleotide termination method (Sanger et al., 1977). Revertants were sequenced directly from PCR products obtained by ampli®cation of a DNA fragment comprised between nucleotide ÿ209 and 259 of the prnA gene or by ampli®cation on a suspension of spores of the adequate strain. For PCR on spores 5 ml of the GeneReleaser were used (BioVentures, Inc.). Sequencing was performed directly on the PCR ampli®ed fragments using the Reagent Kit for Sequencing with Sequenase T7 DNA Polymerase and 7-deaza-dGTP (USB Amersham, Slough, England).

We thank Denise Zickler for her most generous help with ¯uorescence microscopy and many illuminating discussions and Simone Seror for granting us access to her Zeiss microscope. Myriam Altamirano, Piergiorgio Percipalle and Daniela Rhodes are thanked for helpful discussions. We thank them and their co-authors for communicating data before publication. We thank an anonymous referee for two interesting suggestions. This work was supported by grant number BIO4-CT96-0535 (Eurofung) of the European Union, the CNRS and the Universite Paris-Sud. A.P. was supported successively by pre-doctoral fellowships of the MinisteÁre de l'Education Nationale, de l'Enseignement SupeÂrieur et de la Recherche and of Association pour la Recherche sur le Cancer.

Plasmid construction We used the SGFP-TYG version of the green ¯uorescent protein (Chiu et al., 1996). The GFP has been cloned into the pAN52-1 vector (Punt et al., 1987). This construct was kindly provided by Corinne ClaveÂ, Bordeaux. Constructions containing different N-terminal fragments of PrnA were obtained by cloning of adequate PCR fragments to the NcoI site of the pAN52-1-GFP plasmid. A NcoI restriction site was introduced each time into the appropriate location of the prnA gene by PCR. A plasmid carrying the nucleoplasmin NLS was constructed by PCR mutagenesis of the plasmid carrying residues 1-41 of the PrnA sequence fused with GFP protein. In the resulting construction, residues 14 to 32 of PrnA were substituted by the sequence AVKRPAATKKAGQAKKK. Except when speci®cally indicated, all

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Edited by M. Yaniv (Received 18 February 2000; accepted 1 March 2000)