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cDNA cloning and analysis of the expression of nucleoporin p451
Gene 221 (1998) 245–253
cDNA cloning and analysis of the expression of nucleoporin p451 Tianhua Hu 2, Larry Gerace * Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Received 5 November 1997; received in revised form 7 August 1998; accepted 11 August 1998; Received by U.K. Laemmli
Abstract The p62 complex is an assembly of four O-linked glycoproteins (p62, p58, p54, and p45) localized in the central region of the nuclear pore complex. It has been suggested to provide a substrate binding site near the central gated channel of the pore during nuclear protein import. The sequences of p62, p58, and p54 from rat have been reported previously. We have now carried out cDNA cloning of rat p45. The authenticity of the p45 clone was confirmed by two-dimensional gel analysis of the in vitro translated product of this clone. Sequence comparison showed that p45 is mostly identical to the amino terminal four-fifths of p58. p45 contains an N-terminal FG (Phe-Gly) repeat region, a middle coiled-coil region, and a truncated C-terminal FG repeat region (compared to p58). The sequence data and genomic Southern hybridization results strongly support the possibility that p45 and p58 are generated by mRNA alternative splicing. The sequences of three other p58-related cDNA clones indicate that the p58/p45 gene transcript gives rise to additional alternatively spliced mRNAs in mammalian cells. Interestingly, the expression level of p45 relative to p58 varies in different cultured cell lines, indicating that the p62 complex is heterogeneous with respect to these two subunits. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Nuclear pore complex; Nuclear transport; p62 complex; Alternative splicing
1. Introduction The nuclear pore complex is a large supramolecular assembly spanning the nuclear envelope that provides a channel for molecular exchanges between the nucleus and the cytoplasm (reviewed by Pante´ and Aebi, 1996; Go¨rlich and Mattaj, 1996; Doye and Hurt, 1997; Nigg, 1997). While small molecules can diffuse passively through the NPC, most proteins and RNAs are transported by energy and signal-dependent mechanisms. Nuclear protein import is specified by NLSs in the transported molecules. Many NLSs are short sequences of amino acids highly enriched in basic residues, although other classes of NLS also have been described (see Go¨rlich and Mattaj, 1996; Nigg, 1997). * Corresponding author. Tel: +1 619 784 8514; Fax: +1 619 7849132; e-mail: [email protected] 1 Sequence data reported in this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. AF000898-AF000901. 2 Present address: Department of Biochemistry and Biophysics, University of California, San Fransisco, CA 94143, USA. Abbreviations: NE, nuclear envelope; NLS, nuclear localization sequence; NRK, normal rat kidney; NPC, Nuclear pore complex; ORF, open reading frame; PCR, polymerase chain reaction.
Nuclear protein import involves interactions between soluble cytosolic factors and certain proteins of the NPC (reviewed by Sweet and Gerace, 1995; Go¨rlich and Mattaj, 1996; Nigg, 1997). So far, four important cytosolic factors involved in the import of proteins containing basic amino acid-type NLSs have been characterized in detail: the NLS receptor/importin a/karyopherin a (Adam and Gerace, 1991; Go¨rlich et al., 1994; Moroianu et al., 1995; Weis et al., 1995), p97/importin b/karyopherin b (Adam and Adam, 1994; Chi et al., 1995; Go¨rlich et al., 1995; Radu et al., 1995), the small GTPase Ran/TC4(Melchior et al., 1993; Moore and Blobel, 1993), and NTF2/p10(Moore and Blobel, 1994; Paschal and Gerace, 1995). A combination of in vitro and in vivo studies have suggested that substrates with basic amino acid-type NLSs are imported by a complex multistep process: after the import substrate initially binds to the NLS receptor in the cytosol, the substrate complex interacts with the cytoplasmic periphery of the NPC, moves to the central region of the NPC, and is translocated through a central gated channel to the nuclear interior (reviewed by Melchior and Gerace, 1995; Go¨rlich and Mattaj, 1996; Nigg, 1997). During transit through the NPC, the import substrate/receptor complex appears to bind to a group
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of NPC proteins containing FG (Phe-Gly) repeat motifs (Iovine et al., 1995; Rexach and Blobel, 1995; Hu et al., 1996). In vertebrate cells, at least most of the FG repeat proteins are modified with O-linked N-acetylglucosamine (Snow et al., 1987). Four of these FG repeatcontaining O-linked glycoproteins (p62, p58, p54 and p45) are present in a discrete complex, called the p62 complex (Finlay et al., 1991; Kita et al., 1993; Guan et al., 1995). Whereas some FG repeat proteins are localized peripherally and are restricted to one side of the NPC (Pante´ and Aebi, 1996), the p62 complex is localized centrally and on both the nucleoplasmic and cytoplasmic sides of the pore (Guan et al., 1995). Biochemical analysis indicates that the p62 complex has a mass of about 234 kDa ( Kita et al., 1993; Guan et al., 1995), and suggests that it contains one copy of each of the four subunits. The p62 complex has been suggested to be directly involved in nuclear protein import ( Finlay et al., 1991), probably serving as a substrate binding site near the central gated channel of the NPC ( Hu et al., 1996). The sequences of p62 (Starr et al., 1990; Carmo-Fonseca et al., 1991; Cordes et al., 1991) and p58 and p54 have been reported previously (Hu et al., 1996). In addition to having at least one region with multiple FG repeats, they all contain a region with a high probability of forming an a-helical coiled-coil structure(s), which might be involved in protein–protein interactions that determine the structure of the p62 complex (see Buss and Stewart, 1995). Previously, we obtained several lines of evidence that p45 and p58 are closely related proteins ( Hu et al., 1996). First, the HPLC profiles of peptides produced from trypsin-digested p45 and p58 were very similar. Second, peptide sequences obtained for p45 from two peaks of the HPLC gradient were identical to those of p58 from similar elution positions, corresponding to amino acid residues 317–333 and 427–433 of p58. Third, polyclonal antibodies raised against recombinant p58 recognized both p58 and p45 on Western blots. However, antibodies against a C-terminal peptide of p58 recognized only p58 but not p45 in Western blotting, indicating that the C-terminus of p58 is not shared by p45. Northern hybridization using full-length p58 coding sequence as a probe detected two major bands of approx. 3.8 and 2.5 kb, suggesting that p45 and p58 either are generated by mRNA alternative splicing or are encoded by two closely related genes (Hu et al., 1996). The sequence of a cDNA representing a putative partial length p45 clone that we obtained previously by PCR (Hu et al., 1996) suggested that p45 and p58 are generated by alternative splicing of mRNA (Hu et al., 1996). Now, we describe the isolation of a cDNA clone that contains the full-ORF of p45. The sequence of the p45 cDNA and the genomic Southern hybridization results strongly support the alternative splicing model. We further found that the relative expression levels of
p45 vary in different cell lines. Our results provide the basis for gaining new insight into the assembly and functional regulation of the p62 complex.
2. Materials and methods The cDNA library screening, Western blotting, and Northern hybridization were performed as described previously (Hu et al., 1996), except as noted below. 2.1. cDNA cloning of p45 To clone the p45 cDNA, a rat hepatoma cDNA library (Stratagene, La Jolla, CA) was screened with a probe generated from the p58 cDNA (see Results). Positive clones from a first round of screening were further screened by PCR to identify putative p45 clones (see Results). For this, each positive plaque from the first screening was obtained by picking an agarose plug from the plate at the position of the positive signal. Aliquots containing phage particles released from the agarose plug into solution were used directly in PCR reactions. Positive clones were then isolated by a second round of screening using the same probe as in the first round. Plasmid excision and DNA sequencing were performed as described previously (Hu et al., 1996). 2.2. Determination of the authenticity of a putative p45 clone To determine whether a putative p45 cDNA clone encodes the full-length ORF of p45, in vitro transcription and translation of this clone was performed using a TNT Coupled Reticulocyte Lysate System (Promega Corp., Madison, WI ) according to the manufacturer’s instructions. The translation products were analyzed by SDS–PAGE and by two-dimensional electrophoresis. An Immobiline DryStrip kit (Pharmacia LKB Biotechnology Inc., Piscataway, NJ ) was used to conduct two-dimensional electrophoresis according to the manufacturer’s instructions. After electrophoresis, proteins were transferred to a nitrocellulose membrane and analyzed by Western blotting and subsequently by fluorography (Sambrook et al., 1989). 2.3. Southern hybridization Southern hybridization experiments were performed essentially as described by Sambrook et al. (1989). Rat genomic DNA (Clontech, Palo Alto, CA) was digested overnight separately with the restriction enzymes BclI, BglII, DraI, and XbaI (GIBCO BRL, Gaithersburg, MD) and electrophoresed on a 0.7% agarose gel in 0.5×TBE. Ten micrograms of digested DNA was loaded on each lane. DNA fragments were then transferred to
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a nylon membrane and fixed to it by UV crosslinking with a Bio-Rad GS Gene Linker, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). The membrane was first hybridized with a probe that is shared by p58 and p45 cDNA clones, and exposed to an X-ray film. Probe molecules that associated with the membrane were then removed, and the membrane was hybridized to another probe specific for the p45 cDNA. Both probes were generated by PCR using radiolabeled nucleotide. 2.4. Analysis of the p45 expression pattern To investigate the expression of p45 in different cells, cultured cells or rat liver NEs were directly dissolved in SDS–PAGE loading buffer and boiled for 5 min. A sample containing 5×105 cells was loaded on each lane. p45 and p58 were detected by Western blotting using polyclonal antibodies that recognize both of these proteins (Hu et al., 1996).
3. Results 3.1. cDNA cloning of p45 Previously, we obtained a cDNA clone by PCR that encodes a putative partial length p45 ORF (Hu et al., 1996). To obtain a full-length cDNA clone encoding p45, we re-screened the rat macrophage cDNA library from which p58 was cloned (Hu et al., 1996) using a middle region of the p58 coding sequence as a probe (nucleotides 774–1328). This probe contains a region coding for one of the two peptides shared by p45 and p58, and therefore should be able to detect both p58 and p45 clones. Forty-eight positive clones were identified from approx. 2 million phage plaques. In addition to p58 clones, three new groups of clones were identified. However, none of them encode p45, based on the features of their ORFs (see below). We then used a rat hepatoma cDNA library to search for p45 cDNAs. As in our previous analysis (Hu et al., 1996), we first screened the rat hepatoma library by PCR under conditions that would be expected to yield a 3∞ portion of the p45 cDNA. A p58 cDNA segment located between the two peptides shared by p58 and p45 was chosen as the 5∞ primer, and a sequence from the library vector at the 3∞ end of the cDNA inserts was used as the 3∞ primer ( Fig. 1). A product of approx. 600 bp , named Ga1, was amplified from the cDNA library in two independent PCR reactions (data not shown). This is distinct from the approx. 2.5 kb cDNA fragment of p58 that was predicted to be generated by this procedure. It is also different from the approx. 400 bp product obtained previously with the rat macrophage cDNA library (Hu et al., 1996). DNA sequencing
Fig. 1. Strategy for cloning the 3∞ region of the p45 ORF by PCR. Shown is a diagram of the p58 ORF inserted into the library vector. The positions of the 5∞ and 3∞ primers relative to the p58 ORF and the vector are indicated. Regions I and II represent the two peptides shared by p45 and p58. Region C indicates the C-terminal region of p58 that is not present in p45.
of Ga1 showed that the 5∞ half of this PCR product perfectly matched the p58 cDNA sequence between nucleotides 1346–1687 of the p58 clone (Hu et al., 1996), after which the two sequences diverged at a consensus sequence for mRNA splicing (see Fig. 3). This common region corresponds to amino acid residues 417–530 of p58, and contains the expected shared peptide of p58 and p45 (Fig. 1). These results, together with the finding that the 3∞ end of the Ga1 ORF does not contain the C-terminus of p58, suggested that Ga1 could be part of the p45 cDNA. We therefore screened the rat hepatoma cDNA library by plaque hybridization in an effort to obtain a fulllength p45 cDNA. The probe used for this was the p58 cDNA corresponding to the middle of the p58 sequence (nucleotides 774–1328), which should be able to detect both p58 and p45 clones. Forty-five positive clones were identified from approx. 1.2 million phage plaques. These clones were further screened by PCR to distinguish putative p45 clones from p58 clones. The 5∞ primer was the same as used to obtain Ga1 by PCR, whereas a DNA segment downstream of the potential splice site in the Ga1 sequence was chosen as the 3∞ primer. Four positive clones were identified by this approach. Sequence analysis indicated that all are derivatives of the same cDNA. The longest clone contains an ORF coding for a polypeptide with all the known characteristics of p45, including the presence of the two peptide sequences shared by p45 and p58, very high sequence homology to p58 (see below), and a C-terminus different from p58. The 3∞ portion of this clone is identical in sequence to the putative p45 cDNA fragment Ga1 obtained by PCR (discussed above). However, it was not clear whether this putative p45 clone contains the complete ORF for the protein, because an in-frame stop codon is not present in the 5∞ untranslated region. To investigate whether this clone has the full-length coding sequence of p45, we conducted in vitro transcription and translation experiments using the putative p45
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cDNA as a template. The products were analyzed by conventional SDS–PAGE ( Fig. 2A) and two-dimensional electrophoresis ( Fig. 2B). We found that the largest in vitro translated product co-migrated with rat liver p45 in both one- and two-dimensional gel systems (Fig. 2). This product was detected only when the plasmid DNA of the clone was added to the in vitro transcription and translation reaction ( Fig. 2A). Furthermore, this product was recognized by an antip58/p45 polyclonal antibody (Hu et al., 1996) on Western blots of in vitro translated p45 (data not
Fig. 2. Determination of the identity of a putative p45 clone. A DNA sample of a putative p45 clone was added to an in vitro transcription and translation reaction. The products were analyzed by SDS–PAGE (A) and two-dimensional electrophoresis (B). (A) In vitro transcription and translation products from a reaction without the addition of DNA ( lane 1) or with added DNA from a putative p45 clone ( lane 2) were analyzed by SDS–PAGE, together with rat-liver NE proteins ( lane 3). The signals in lanes 1 and 2 were detected by fluorography, while those in lane 3 were by Western blotting with polyclonal antibodies against p58/p45. (B) The in vitro translation products of a putative p45 clone were mixed with rat liver NE proteins and analyzed by twodimensional electrophoresis followed by fluorography and Western blotting as indicated. Most of the signal on the Western blot of (B) was due to p45 from the rat liver NEs, because of the relatively small quantity of in vitro translated p45.
shown). Considered together, these results indicate that the clone contains the full-length coding sequence of p45. The sequence of the p45 cDNA clone and the predicted ORF is shown in Fig. 3. The clone is 1707 bp long, and contains an ORF encoding a protein of 513 amino acids with a predicted molecular weight of 51 827 daltons. The 5∞ and 3∞ untranslated regions are 22 bp and 153 bp long, respectively. Further 3∞ untranslated sequence was not obtained despite repeated efforts. The 5∞ untranslated region and the N-terminus of the p45 ORF are identical to these portions of p58, and this identity continues almost to the end of p45, except that p45 has a deletion of 22 amino acids corresponding to residues 224–245 of p58 (Fig. 6). The two sequences diverge at a position five amino acids before the C-terminus of p45. At this point, p45 continues with a stretch of five unique amino acids, whereas p58 continues with a stretch of 55 unique amino acids. Interestingly, the nucleotide sequences of p45 at both the deletion and divergent sites (compared to p58) closely resemble the consensus sequence for mRNA splicing sites (C/A A G G/A) (Breathnach and Chambon, 1981; Sharp, 1981; Cech, 1983), indicating that p45 and p58 are generated by mRNA alternative splicing. However, we still could not rule out the possibility that the two proteins are encoded by two closely related genes. To further distinguish these two models, we conducted Southern hybridization experiments with rat genomic DNA samples digested separately with the restriction enzymes BclI, BglII, DraI, and XbaI. The nylon membrane containing rat genomic DNA fragments was first hybridized to a DNA probe corresponding to nucleotides 1487–1543 of p45 cDNA, which is shared by p58 and p45 cDNA clones ( Fig. 4A). Membrane-bound probe was then removed, and the membrane was hybridized to a second probe corresponding to nucleotides 1546–1707 of p45 cDNA, which is specific to p45 (Fig. 4B). The hybridization results from the two different probes are identical ( Fig. 4A and B), indicating that p45 and p58 transcripts are from the same gene, not from two closely related genes. Furthermore, both probes hybridized to only one band on each lane, indicating that there is likely only one copy of the p58/p45 gene in the genome. Therefore, we conclude that p45 and p58 are generated by mRNA alternative splicing. In comparison to p58, p45 contains the complete N-terminal FG repeat region and middle coiled-coil region, as well as half of the C-terminal FG repeat region (Fig. 5). The 22 residue deletion occurs in a segment linking the N-terminal FG repeat region to the coiled-coil region. 3.2. Additional species of mRNA derived from the p58/p45 gene As discussed above, in our first attempt to clone the p45 cDNA by screening a rat macrophage cDNA
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Fig. 3. cDNA and deduced amino acid sequence from the p45 clone. Underlined amino acid sequences represent the two peptides shared by p45 and p58, as determined by peptide sequencing (Hu et al., 1996). The two putative mRNA splice sites are shown in boxes.
library, three new clones were identified. The first cDNA clone, whose 3∞ portion was obtained by PCR and originally thought to be a portion of the p45 cDNA (Hu et al., 1996), occurred four times in the 48 positive clones. The clone has an in-frame stop codon in the 5∞ untranslated region and contains a complete ORF that encodes a protein of approx. 23 kDa ( Fig. 5). This cDNA clone contains three regions: a middle region that is identical to an internal region in p58 (nucleotides 216–1492 in p58), and the 5∞ and 3∞ regions that are distinct from p58. Sequences at both junction sites strongly suggest that this clone is another alternatively spliced form of the p58/p45 gene transcript ( Fig. 5). Except for a unique stretch of three amino acids at its
C-terminus, the deduced amino acid sequence of p23 is identical to a middle region of p58 (amino acids 261–465) ( Figs. 5 and 6). This part of p58 contains the complete coiled-coil region, but does not contain any FG repeats ( Fig. 6). We have not detected the putative p23 protein in either rat liver NEs or NRK cells by Western blotting using polyclonal antibodies raised against the full-length p58 protein. This could be due to a very low abundance of the p23 mRNA in these cell types. The best candidate for a p23 mRNA is the minor 1.6 kb band seen in the Northern blot analysis of Fig. 7B (see below). The second clone, H6, occurred twice and has a size of approx. 5 kb. Partial sequence of H6 showed that the
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stop codon in the 5∞ untranslated region of both clones, both ORFs might be incomplete. 3.3. Variable expression of p45 in different cells
Fig. 4. Analysis of the p58/p45 gene by Southern hybridization. Ten micrograms of rat genomic DNA, digested with different restriction enzymes as indicated, was loaded on each lane, electrophoresed and transferred to a nylon membrane. The membrane was first hybridized to a radiolabeled DNA probe that is shared by p58 and p45 cDNA clones (A). The bound probe was then removed from the membrane, and the membrane was hybridized to another probe that is specific to p45 (B).
472 bp at the 5∞ end of this clone is identical to the region of p58 between nucleotides 1021 and 1492, whereas the 131 bp at the 3∞ end of H6 (excluding a polyA sequence) is identical to the 3∞ end of the p23 cDNA (data not shown). The rest of the sequence that we determined for H6 (approx. 2 kb), which extends inward from the 5∞ and 3∞ regions discussed above, is novel. DNA sequences at both junction sites closely match the consensus sequence for mRNA splice sites, indicating that this clone also is generated by alternative splicing from the p58/p45 gene transcript. The third clone, H7, occurred nine times in the 48 positives and has a size of approx. 1.2 kb. It is identical to a region of the p58 cDNA between nucleotides 908 and 2124 [the full-length p58 cDNA is 3749 bp long (Hu et al., 1996)] and is followed by a polyA sequence. This clone could be another spliced product of the p58/p45 gene transcript. The 5∞ region of clone H6 encodes a short (99 amino acid) ORF, 93 residues of which are identical to p58 between amino acids 373 and 465, whereas H7 encodes a 213 amino acid ORF that is identical to the C-terminal end of p58. Due to the lack of an in-frame
When we examined the relative levels of p58 and p45 in NRK cells and rat liver NEs by Western blotting with a polyclonal antibody that recognizes both p45 and p58, we found that the p45/p58 ratio varies in these two sources. In NRK cells, the p45 band is substantially weaker than the p58 band, whereas in rat liver NEs the intensities of the p45 and p58 bands are very similar ( Fig. 7A). These results are consistent with Northern hybridization experiments with mRNA preparations from NRK cells and rat liver. A probe corresponding to the p58 coding sequence strongly recognizes bands at 2.5 and 3.8 kb in these two samples, which very likely correspond to mRNAs for p45 and p58, respectively. In rat-liver mRNA, the relative intensities of the major 2.5 and 3.8 kb bands are very similar, whereas in NRK cell mRNA the 3.8 kb band is significantly stronger than the 2.5 kb band. This pattern parallels the relative intensities of p45 and p58 seen by Western blotting ( Fig. 7B). In addition to the major 2.5 and 3.8 kb bands, a number of minor hybridizing species are seen, which could correspond to other apparent alternative splice forms of the p58/p45 transcript, such as the mRNA for putative p23 (see above). To further investigate the expression of p45 in different cells, we carried out Western blotting experiments using cell extracts from several rat and mouse cell lines ( Fig. 8). We found that the relative intensities of the p45 and p58 bands varied from cell line to cell line, and that the p45 signal was usually weaker than the p58 signal. In B16A (a mouse melanoma line), P19 (a mouse teratocarcinoma line), and P19MES (a differentiated line derived from P19) cells, the p45 signal was significantly weaker than that of p58, although it was clearly detectable. However, in HTC cells (a rat hepatoma line) the p45 band was almost undetectable, whereas the p58 signal was as strong as in the other cell lines analyzed ( Fig. 8). This variation does not seem to be related to the cell-cycle status of the cells, because all cultures were maintained in exponential growth. In fact, no difference was found in the levels of p45 in dividing vs. nondividing NRK cells (data not shown). A clear difference in the expression of nuclear lamin isotypes has been observed in P19 vs. P19MES cells, analogous to the regulation of lamin expression during differentiation that is seen in various tissues in vivo (see Martin et al., 1995). While no differences in the relative expression levels of p45 and p58 were detected in this cultured cell model, it is plausible that p45 expression, or the expression of p58/p45 related proteins from other alternatively spliced mRNAs, is regulated in some cell types in vivo at certain stages of development.
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Fig. 5. cDNA and deduced amino acid sequences from a p23 clone. The two putative mRNA splice sites are shown in boxes. The region between the two boxed sequences is identical to a region of the p58 cDNA, whereas the region downstream of the second boxed sequence (CAGG) is identical to a 3∞ terminal region of clone H6.
Fig. 6. Schematic comparison of p58, p45, and p23. FG repeats and regions with a high probability of forming coiled-coil structures are indicated. Each vertical line represents one FG repeat. The unique C-termini of p45 and p23 are indicated by distinct hatched patterns.
4. Discussion Previously, several lines of evidence suggested that p45 and p58 are generated by alternative mRNA splicing (Hu et al., 1996). The cDNA sequence of p45 described herein strongly supports this model. It is further indicated by genomic Southern hybridization results. To our knowledge, this is the only example up to now of NPC proteins that are encoded by alternatively spliced
Fig. 7. Expression patterns of p45 in rat liver and NRK cells. (A) Western blotting with polyclonal antibodies that recognize both p58 and p45. A whole-cell lysate from NRK cells or rat-liver NE proteins were analyzed as indicated. (B) Northern hybridization with a probe generated from the full-length coding sequence of p58. 25 mg of NRK cell polyA+ RNA or 10 mg of rat liver polyA+ RNA were examined as indicated.
mRNAs. Although mRNAs for p58 and p45 are the predominant products resulting from this alternative splicing, our cDNA cloning and Northern hybridization
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Fig. 8. Expression pattern of p45 in several mouse and rat cell lines. Whole cell lysates from different cell lines ( lanes 1–4) or proteins from rat liver NEs ( lane 5) were analyzed by Western blotting using polyclonal antibodies against p58/p45. Lane 1, B16A cells (mouse melanoma); lane 2, P19 cells (mouse teratocarcinoma); lane 3, P19MES cells (differentiated P19); lane 4, HTC cells (rat hepatoma).
data strongly suggest that several additional alternatively spliced mRNAs are expressed from the p58/p45 gene transcripts in some cells, including a transcript that encodes putative p23. However, we have not detected any of these protein products by Western blotting in the cells that we examined. Notwithstanding the similar sequence and domain organization of p45 and p58, the fact that both proteins are present in the same cells indicates that they probably each have some functionally distinct features. The C-terminal FG repeat region of p58 interacts in vitro with a complex consisting of import substrate (containing a basic amino acid-type NLS), NLS receptor, and p97, whereas the N-terminal FG repeat region of p58 does not (Hu et al., 1996). Since p45 contains only half of the C-terminal FG repeat region of p58, it may interact differently than p58 with a substrate/NLS receptor/p97 complex or, for that matter, with other nuclear import or export complexes. Moreover, p45 contains a 22-residue deletion (relative to p58) adjacent to its coiled-coil region. Since this coiled-coil region might be involved in protein–protein interactions, this deletion could affect the association of p45 with other subunits of the p62 complex or with other NPC components. p45 contains a unique C-terminal sequence not shared by p58, LCASA, which resembles the consensus site (CAAX ) for protein isoprenylation (Clarke, 1992). This raises the intriguing possibility that p45 could be isoprenylated at its C-terminus. Such a modification could regulate the interaction of p45 with cytosolic factors or have some other function. We investigated whether p45 is isoprenylated by immunoprecipitating p45 from cultured cells that had been incubated with a radioactive isoprenylation substrate. We also attempted to label p45 in vitro by incubating recombinant p45 with a radioactive isoprenylation substrate in the presence of cytosol, and by
including a radioactive isoprenylation substrate in an in vitro transcription/translation reaction with the p45 cDNA clone. No label was obtained in p45 in any these experiments (Hu and Gerace, unpublished observations), so it remains unclear whether p45 is isoprenylated. The p62 complex of rat liver NEs is suggested to contain one copy each of p62, p58, p54 and p45, based on its mass of approx. 234 kDa and the roughly stoichiometric levels of these four subunits in purified preparations (Guan et al., 1995). However, the very low expression of p45 in HTC cells implies that the p62 complex does not always contain p45. This indicates that more than one type of p62 complex exists. The strong homology between p45 and p58 suggests that the two proteins might be able to substitute for each other in the p62 complex. If two copies of the p58/p45 pair were in each p62 complex, then different p62 complexes might contain p58:p45 ratios of 2:0, 1:1 and 0:2. Considering the potential functional differences between p58 and p45 (see above), these different types of p62 complex could have different biological properties. In this regard, the possibility should be considered that proteins produced by other alternatively spliced mRNAs of the p58/p45 gene transcript may be assembled into some versions of the p62 complex. For example, the putative p23 protein, which contains the complete coiledcoil domain of p58/p45 but lacks the FG repeat regions, might be incorporated in the p62 complex but be unable to bind the cytosolic factors targeted to FG repeats. Thus, it is possible that alternative splicing of p58/p45 gene transcript is used to produce different types of p62 complexes with different functional properties. This could provide a mechanism to regulate the nuclear transport activity of the NPC in different cell types. In conclusion, with the cloning of the p45 cDNA, the coding sequences for all subunits of the rat liver p62 complex are now available. This provides the tools to obtain different types of recombinant p62 complexes for functional studies, and to further investigate the specific role of the p62 complex in nuclear transport.
Acknowledgement We would like to thank all members of our laboratory for their help and valuable discussions. We are very grateful to Dr Dwayne Stupack for his help with twodimensional gel electrophoresis, and thank Dr Thomas Mo¨hler for providing B16A cells. This work was supported by a Rockefeller Foundation fellowship to T. Hu, a grant from the NIH to L. Gerace (GM41955), and a grant from the Lucille P. Markey Charitable Trust.
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References Adam, E.J., Adam, S.A., 1994. Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope. J. Cell Biol. 125, 547–555. Adam, S.A., Gerace, L., 1991. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837–847. Breathnach, R., Chambon, P., 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349–383. Buss, F., Stewart, M., 1995. Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62. J. Cell Biol. 128, 251–261. Carmo-Fonseca, M., Kern, H., Hurt, E.C., 1991. Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization. Eur. J. Cell Biol. 55, 17–30. Cech, T.R., 1983. RNA splicing: three themes with variations. Cell 34, 713–716. Chi, N.C., Adam, E.J., Adam, S.A., 1995. Sequence and characterization of cytoplasmic nuclear protein import factor p97. J. Cell Biol. 130, 265–274. Clarke, S., 1992. Protein isoprenylation and methylation at carboxylterminal cysteine residues. Annu. Rev. Biochem. 61, 355–386. Cordes, V., Waizenegger, I., Krohne, G., 1991. Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region. Eur. J. Cell Biol. 55, 31–47. Doye, V., Hurt, E., 1997. From nucleoporins to nuclear pore complexes. Curr. Opin. Cell Biol. 9, 401–411. Finlay, D.R., Meier, E., Bradley, P., Horecka, J., Forbes, D.J., 1991. A complex of nuclear pore proteins required for pore function. J. Cell Biol. 114, 169–183. Go¨rlich, D., Kostka, S., Kraft, R., Dingwall, C., Laskey, R.A., Hartmann, E., Prehn, S., 1995. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr. Biol. 5, 383–392. Go¨rlich, D., Mattaj, I.W., 1996. Nucleocytoplasmic transport. Science 271, 1513–1518. Go¨rlich, D., Prehn, S., Laskey, R.A., Hartmann, E., 1994. Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767–778. Guan, T., Muller, S., Klier, G., Pante´, N., Blevitt, J.M., Haner, M., Paschal, B., Aebi, U., Gerace, L., 1995. Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex. Mol. Biol. Cell 6, 1591–1603. Hu, T., Guan, T., Gerace, L., 1996. Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins. J. Cell Biol. 134, 589–601. Iovine, M.K., Watkins, J.L., Wente, S.R., 1995. The GLFG repetitive
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region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor. J. Cell Biol. 131, 1699–1713. Kita, K., Omata, S., Horigome, T., 1993. Purification and characterization of a nuclear pore glycoprotein complex containing p62. J. Biochem. 113, 377–382. Martin, L., Crimaudo, C., Gerace, L., 1995. cDNA cloning and characterization of lamina-associated polypeptide 1C (LAP1C ), an integral protein of the inner nuclear membrane. J. Biol. Chem. 270, 8822–8828. Melchior, F., Gerace, L., 1995. Mechanisms of nuclear protein import. Curr. Opin. Cell Biol. 7, 310–318. Melchior, F., Paschal, B., Evans, J., Gerace, L., 1993. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J. Cell Biol. 123, 1649–1659. Moore, M.S., Blobel, G., 1993. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663. Moore, M.S., Blobel, G., 1994. Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc. Natl. Acad. Sci. USA 91, 10212–10216. Moroianu, J., Blobel, G., Radu, A., 1995. Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. Proc. Natl. Acad. Sci. USA 92, 2008–2011. Nigg, E.A., 1997. Nucleoplasmic transport signals, mechanisms and regulation. Nature 386, 779–787. Pante´, N., Aebi, U., 1996. Toward the molecular dissection of protein import into nuclei. Curr. Opin. Cell Biol. 8, 397–406. Paschal, B.M., Gerace, L., 1995. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J. Cell Biol. 129, 925–937. Radu, A., Blobel, G., Moore, M.S., 1995. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc. Natl. Acad. Sci. USA 92, 1769–1773. Rexach, M., Blobel, G., 1995. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683–692. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sharp, P.A., 1981. Speculations on RNA splicing. Cell 23, 643–646. Snow, C.M., Senior, A., Gerace, L., 1987. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 104, 1143–1156. Starr, C.M., D’Onofrio, M., Park, M.K., Hanover, J.A., 1990. Primary sequence and heterologous expression of nuclear pore glycoprotein p62. J. Cell Biol. 110, 1861–1871. Sweet, D.J., Gerace, L., 1995. Taking from the cytoplasm and giving to the pore: soluble transport factors in nuclear protein import. Trends Cell Biol. 5, 444–447. Weis, K., Mattaj, I.W., Lamond, A.I., 1995. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science 268, 1049–1053.
cDNA cloning and analysis of the expression of nucleoporin p451 Tianhua Hu 2, Larry Gerace * Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Received 5 November 1997; received in revised form 7 August 1998; accepted 11 August 1998; Received by U.K. Laemmli
Abstract The p62 complex is an assembly of four O-linked glycoproteins (p62, p58, p54, and p45) localized in the central region of the nuclear pore complex. It has been suggested to provide a substrate binding site near the central gated channel of the pore during nuclear protein import. The sequences of p62, p58, and p54 from rat have been reported previously. We have now carried out cDNA cloning of rat p45. The authenticity of the p45 clone was confirmed by two-dimensional gel analysis of the in vitro translated product of this clone. Sequence comparison showed that p45 is mostly identical to the amino terminal four-fifths of p58. p45 contains an N-terminal FG (Phe-Gly) repeat region, a middle coiled-coil region, and a truncated C-terminal FG repeat region (compared to p58). The sequence data and genomic Southern hybridization results strongly support the possibility that p45 and p58 are generated by mRNA alternative splicing. The sequences of three other p58-related cDNA clones indicate that the p58/p45 gene transcript gives rise to additional alternatively spliced mRNAs in mammalian cells. Interestingly, the expression level of p45 relative to p58 varies in different cultured cell lines, indicating that the p62 complex is heterogeneous with respect to these two subunits. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Nuclear pore complex; Nuclear transport; p62 complex; Alternative splicing
1. Introduction The nuclear pore complex is a large supramolecular assembly spanning the nuclear envelope that provides a channel for molecular exchanges between the nucleus and the cytoplasm (reviewed by Pante´ and Aebi, 1996; Go¨rlich and Mattaj, 1996; Doye and Hurt, 1997; Nigg, 1997). While small molecules can diffuse passively through the NPC, most proteins and RNAs are transported by energy and signal-dependent mechanisms. Nuclear protein import is specified by NLSs in the transported molecules. Many NLSs are short sequences of amino acids highly enriched in basic residues, although other classes of NLS also have been described (see Go¨rlich and Mattaj, 1996; Nigg, 1997). * Corresponding author. Tel: +1 619 784 8514; Fax: +1 619 7849132; e-mail: [email protected] 1 Sequence data reported in this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. AF000898-AF000901. 2 Present address: Department of Biochemistry and Biophysics, University of California, San Fransisco, CA 94143, USA. Abbreviations: NE, nuclear envelope; NLS, nuclear localization sequence; NRK, normal rat kidney; NPC, Nuclear pore complex; ORF, open reading frame; PCR, polymerase chain reaction.
Nuclear protein import involves interactions between soluble cytosolic factors and certain proteins of the NPC (reviewed by Sweet and Gerace, 1995; Go¨rlich and Mattaj, 1996; Nigg, 1997). So far, four important cytosolic factors involved in the import of proteins containing basic amino acid-type NLSs have been characterized in detail: the NLS receptor/importin a/karyopherin a (Adam and Gerace, 1991; Go¨rlich et al., 1994; Moroianu et al., 1995; Weis et al., 1995), p97/importin b/karyopherin b (Adam and Adam, 1994; Chi et al., 1995; Go¨rlich et al., 1995; Radu et al., 1995), the small GTPase Ran/TC4(Melchior et al., 1993; Moore and Blobel, 1993), and NTF2/p10(Moore and Blobel, 1994; Paschal and Gerace, 1995). A combination of in vitro and in vivo studies have suggested that substrates with basic amino acid-type NLSs are imported by a complex multistep process: after the import substrate initially binds to the NLS receptor in the cytosol, the substrate complex interacts with the cytoplasmic periphery of the NPC, moves to the central region of the NPC, and is translocated through a central gated channel to the nuclear interior (reviewed by Melchior and Gerace, 1995; Go¨rlich and Mattaj, 1996; Nigg, 1997). During transit through the NPC, the import substrate/receptor complex appears to bind to a group
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of NPC proteins containing FG (Phe-Gly) repeat motifs (Iovine et al., 1995; Rexach and Blobel, 1995; Hu et al., 1996). In vertebrate cells, at least most of the FG repeat proteins are modified with O-linked N-acetylglucosamine (Snow et al., 1987). Four of these FG repeatcontaining O-linked glycoproteins (p62, p58, p54 and p45) are present in a discrete complex, called the p62 complex (Finlay et al., 1991; Kita et al., 1993; Guan et al., 1995). Whereas some FG repeat proteins are localized peripherally and are restricted to one side of the NPC (Pante´ and Aebi, 1996), the p62 complex is localized centrally and on both the nucleoplasmic and cytoplasmic sides of the pore (Guan et al., 1995). Biochemical analysis indicates that the p62 complex has a mass of about 234 kDa ( Kita et al., 1993; Guan et al., 1995), and suggests that it contains one copy of each of the four subunits. The p62 complex has been suggested to be directly involved in nuclear protein import ( Finlay et al., 1991), probably serving as a substrate binding site near the central gated channel of the NPC ( Hu et al., 1996). The sequences of p62 (Starr et al., 1990; Carmo-Fonseca et al., 1991; Cordes et al., 1991) and p58 and p54 have been reported previously (Hu et al., 1996). In addition to having at least one region with multiple FG repeats, they all contain a region with a high probability of forming an a-helical coiled-coil structure(s), which might be involved in protein–protein interactions that determine the structure of the p62 complex (see Buss and Stewart, 1995). Previously, we obtained several lines of evidence that p45 and p58 are closely related proteins ( Hu et al., 1996). First, the HPLC profiles of peptides produced from trypsin-digested p45 and p58 were very similar. Second, peptide sequences obtained for p45 from two peaks of the HPLC gradient were identical to those of p58 from similar elution positions, corresponding to amino acid residues 317–333 and 427–433 of p58. Third, polyclonal antibodies raised against recombinant p58 recognized both p58 and p45 on Western blots. However, antibodies against a C-terminal peptide of p58 recognized only p58 but not p45 in Western blotting, indicating that the C-terminus of p58 is not shared by p45. Northern hybridization using full-length p58 coding sequence as a probe detected two major bands of approx. 3.8 and 2.5 kb, suggesting that p45 and p58 either are generated by mRNA alternative splicing or are encoded by two closely related genes (Hu et al., 1996). The sequence of a cDNA representing a putative partial length p45 clone that we obtained previously by PCR (Hu et al., 1996) suggested that p45 and p58 are generated by alternative splicing of mRNA (Hu et al., 1996). Now, we describe the isolation of a cDNA clone that contains the full-ORF of p45. The sequence of the p45 cDNA and the genomic Southern hybridization results strongly support the alternative splicing model. We further found that the relative expression levels of
p45 vary in different cell lines. Our results provide the basis for gaining new insight into the assembly and functional regulation of the p62 complex.
2. Materials and methods The cDNA library screening, Western blotting, and Northern hybridization were performed as described previously (Hu et al., 1996), except as noted below. 2.1. cDNA cloning of p45 To clone the p45 cDNA, a rat hepatoma cDNA library (Stratagene, La Jolla, CA) was screened with a probe generated from the p58 cDNA (see Results). Positive clones from a first round of screening were further screened by PCR to identify putative p45 clones (see Results). For this, each positive plaque from the first screening was obtained by picking an agarose plug from the plate at the position of the positive signal. Aliquots containing phage particles released from the agarose plug into solution were used directly in PCR reactions. Positive clones were then isolated by a second round of screening using the same probe as in the first round. Plasmid excision and DNA sequencing were performed as described previously (Hu et al., 1996). 2.2. Determination of the authenticity of a putative p45 clone To determine whether a putative p45 cDNA clone encodes the full-length ORF of p45, in vitro transcription and translation of this clone was performed using a TNT Coupled Reticulocyte Lysate System (Promega Corp., Madison, WI ) according to the manufacturer’s instructions. The translation products were analyzed by SDS–PAGE and by two-dimensional electrophoresis. An Immobiline DryStrip kit (Pharmacia LKB Biotechnology Inc., Piscataway, NJ ) was used to conduct two-dimensional electrophoresis according to the manufacturer’s instructions. After electrophoresis, proteins were transferred to a nitrocellulose membrane and analyzed by Western blotting and subsequently by fluorography (Sambrook et al., 1989). 2.3. Southern hybridization Southern hybridization experiments were performed essentially as described by Sambrook et al. (1989). Rat genomic DNA (Clontech, Palo Alto, CA) was digested overnight separately with the restriction enzymes BclI, BglII, DraI, and XbaI (GIBCO BRL, Gaithersburg, MD) and electrophoresed on a 0.7% agarose gel in 0.5×TBE. Ten micrograms of digested DNA was loaded on each lane. DNA fragments were then transferred to
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a nylon membrane and fixed to it by UV crosslinking with a Bio-Rad GS Gene Linker, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). The membrane was first hybridized with a probe that is shared by p58 and p45 cDNA clones, and exposed to an X-ray film. Probe molecules that associated with the membrane were then removed, and the membrane was hybridized to another probe specific for the p45 cDNA. Both probes were generated by PCR using radiolabeled nucleotide. 2.4. Analysis of the p45 expression pattern To investigate the expression of p45 in different cells, cultured cells or rat liver NEs were directly dissolved in SDS–PAGE loading buffer and boiled for 5 min. A sample containing 5×105 cells was loaded on each lane. p45 and p58 were detected by Western blotting using polyclonal antibodies that recognize both of these proteins (Hu et al., 1996).
3. Results 3.1. cDNA cloning of p45 Previously, we obtained a cDNA clone by PCR that encodes a putative partial length p45 ORF (Hu et al., 1996). To obtain a full-length cDNA clone encoding p45, we re-screened the rat macrophage cDNA library from which p58 was cloned (Hu et al., 1996) using a middle region of the p58 coding sequence as a probe (nucleotides 774–1328). This probe contains a region coding for one of the two peptides shared by p45 and p58, and therefore should be able to detect both p58 and p45 clones. Forty-eight positive clones were identified from approx. 2 million phage plaques. In addition to p58 clones, three new groups of clones were identified. However, none of them encode p45, based on the features of their ORFs (see below). We then used a rat hepatoma cDNA library to search for p45 cDNAs. As in our previous analysis (Hu et al., 1996), we first screened the rat hepatoma library by PCR under conditions that would be expected to yield a 3∞ portion of the p45 cDNA. A p58 cDNA segment located between the two peptides shared by p58 and p45 was chosen as the 5∞ primer, and a sequence from the library vector at the 3∞ end of the cDNA inserts was used as the 3∞ primer ( Fig. 1). A product of approx. 600 bp , named Ga1, was amplified from the cDNA library in two independent PCR reactions (data not shown). This is distinct from the approx. 2.5 kb cDNA fragment of p58 that was predicted to be generated by this procedure. It is also different from the approx. 400 bp product obtained previously with the rat macrophage cDNA library (Hu et al., 1996). DNA sequencing
Fig. 1. Strategy for cloning the 3∞ region of the p45 ORF by PCR. Shown is a diagram of the p58 ORF inserted into the library vector. The positions of the 5∞ and 3∞ primers relative to the p58 ORF and the vector are indicated. Regions I and II represent the two peptides shared by p45 and p58. Region C indicates the C-terminal region of p58 that is not present in p45.
of Ga1 showed that the 5∞ half of this PCR product perfectly matched the p58 cDNA sequence between nucleotides 1346–1687 of the p58 clone (Hu et al., 1996), after which the two sequences diverged at a consensus sequence for mRNA splicing (see Fig. 3). This common region corresponds to amino acid residues 417–530 of p58, and contains the expected shared peptide of p58 and p45 (Fig. 1). These results, together with the finding that the 3∞ end of the Ga1 ORF does not contain the C-terminus of p58, suggested that Ga1 could be part of the p45 cDNA. We therefore screened the rat hepatoma cDNA library by plaque hybridization in an effort to obtain a fulllength p45 cDNA. The probe used for this was the p58 cDNA corresponding to the middle of the p58 sequence (nucleotides 774–1328), which should be able to detect both p58 and p45 clones. Forty-five positive clones were identified from approx. 1.2 million phage plaques. These clones were further screened by PCR to distinguish putative p45 clones from p58 clones. The 5∞ primer was the same as used to obtain Ga1 by PCR, whereas a DNA segment downstream of the potential splice site in the Ga1 sequence was chosen as the 3∞ primer. Four positive clones were identified by this approach. Sequence analysis indicated that all are derivatives of the same cDNA. The longest clone contains an ORF coding for a polypeptide with all the known characteristics of p45, including the presence of the two peptide sequences shared by p45 and p58, very high sequence homology to p58 (see below), and a C-terminus different from p58. The 3∞ portion of this clone is identical in sequence to the putative p45 cDNA fragment Ga1 obtained by PCR (discussed above). However, it was not clear whether this putative p45 clone contains the complete ORF for the protein, because an in-frame stop codon is not present in the 5∞ untranslated region. To investigate whether this clone has the full-length coding sequence of p45, we conducted in vitro transcription and translation experiments using the putative p45
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cDNA as a template. The products were analyzed by conventional SDS–PAGE ( Fig. 2A) and two-dimensional electrophoresis ( Fig. 2B). We found that the largest in vitro translated product co-migrated with rat liver p45 in both one- and two-dimensional gel systems (Fig. 2). This product was detected only when the plasmid DNA of the clone was added to the in vitro transcription and translation reaction ( Fig. 2A). Furthermore, this product was recognized by an antip58/p45 polyclonal antibody (Hu et al., 1996) on Western blots of in vitro translated p45 (data not
Fig. 2. Determination of the identity of a putative p45 clone. A DNA sample of a putative p45 clone was added to an in vitro transcription and translation reaction. The products were analyzed by SDS–PAGE (A) and two-dimensional electrophoresis (B). (A) In vitro transcription and translation products from a reaction without the addition of DNA ( lane 1) or with added DNA from a putative p45 clone ( lane 2) were analyzed by SDS–PAGE, together with rat-liver NE proteins ( lane 3). The signals in lanes 1 and 2 were detected by fluorography, while those in lane 3 were by Western blotting with polyclonal antibodies against p58/p45. (B) The in vitro translation products of a putative p45 clone were mixed with rat liver NE proteins and analyzed by twodimensional electrophoresis followed by fluorography and Western blotting as indicated. Most of the signal on the Western blot of (B) was due to p45 from the rat liver NEs, because of the relatively small quantity of in vitro translated p45.
shown). Considered together, these results indicate that the clone contains the full-length coding sequence of p45. The sequence of the p45 cDNA clone and the predicted ORF is shown in Fig. 3. The clone is 1707 bp long, and contains an ORF encoding a protein of 513 amino acids with a predicted molecular weight of 51 827 daltons. The 5∞ and 3∞ untranslated regions are 22 bp and 153 bp long, respectively. Further 3∞ untranslated sequence was not obtained despite repeated efforts. The 5∞ untranslated region and the N-terminus of the p45 ORF are identical to these portions of p58, and this identity continues almost to the end of p45, except that p45 has a deletion of 22 amino acids corresponding to residues 224–245 of p58 (Fig. 6). The two sequences diverge at a position five amino acids before the C-terminus of p45. At this point, p45 continues with a stretch of five unique amino acids, whereas p58 continues with a stretch of 55 unique amino acids. Interestingly, the nucleotide sequences of p45 at both the deletion and divergent sites (compared to p58) closely resemble the consensus sequence for mRNA splicing sites (C/A A G G/A) (Breathnach and Chambon, 1981; Sharp, 1981; Cech, 1983), indicating that p45 and p58 are generated by mRNA alternative splicing. However, we still could not rule out the possibility that the two proteins are encoded by two closely related genes. To further distinguish these two models, we conducted Southern hybridization experiments with rat genomic DNA samples digested separately with the restriction enzymes BclI, BglII, DraI, and XbaI. The nylon membrane containing rat genomic DNA fragments was first hybridized to a DNA probe corresponding to nucleotides 1487–1543 of p45 cDNA, which is shared by p58 and p45 cDNA clones ( Fig. 4A). Membrane-bound probe was then removed, and the membrane was hybridized to a second probe corresponding to nucleotides 1546–1707 of p45 cDNA, which is specific to p45 (Fig. 4B). The hybridization results from the two different probes are identical ( Fig. 4A and B), indicating that p45 and p58 transcripts are from the same gene, not from two closely related genes. Furthermore, both probes hybridized to only one band on each lane, indicating that there is likely only one copy of the p58/p45 gene in the genome. Therefore, we conclude that p45 and p58 are generated by mRNA alternative splicing. In comparison to p58, p45 contains the complete N-terminal FG repeat region and middle coiled-coil region, as well as half of the C-terminal FG repeat region (Fig. 5). The 22 residue deletion occurs in a segment linking the N-terminal FG repeat region to the coiled-coil region. 3.2. Additional species of mRNA derived from the p58/p45 gene As discussed above, in our first attempt to clone the p45 cDNA by screening a rat macrophage cDNA
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Fig. 3. cDNA and deduced amino acid sequence from the p45 clone. Underlined amino acid sequences represent the two peptides shared by p45 and p58, as determined by peptide sequencing (Hu et al., 1996). The two putative mRNA splice sites are shown in boxes.
library, three new clones were identified. The first cDNA clone, whose 3∞ portion was obtained by PCR and originally thought to be a portion of the p45 cDNA (Hu et al., 1996), occurred four times in the 48 positive clones. The clone has an in-frame stop codon in the 5∞ untranslated region and contains a complete ORF that encodes a protein of approx. 23 kDa ( Fig. 5). This cDNA clone contains three regions: a middle region that is identical to an internal region in p58 (nucleotides 216–1492 in p58), and the 5∞ and 3∞ regions that are distinct from p58. Sequences at both junction sites strongly suggest that this clone is another alternatively spliced form of the p58/p45 gene transcript ( Fig. 5). Except for a unique stretch of three amino acids at its
C-terminus, the deduced amino acid sequence of p23 is identical to a middle region of p58 (amino acids 261–465) ( Figs. 5 and 6). This part of p58 contains the complete coiled-coil region, but does not contain any FG repeats ( Fig. 6). We have not detected the putative p23 protein in either rat liver NEs or NRK cells by Western blotting using polyclonal antibodies raised against the full-length p58 protein. This could be due to a very low abundance of the p23 mRNA in these cell types. The best candidate for a p23 mRNA is the minor 1.6 kb band seen in the Northern blot analysis of Fig. 7B (see below). The second clone, H6, occurred twice and has a size of approx. 5 kb. Partial sequence of H6 showed that the
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stop codon in the 5∞ untranslated region of both clones, both ORFs might be incomplete. 3.3. Variable expression of p45 in different cells
Fig. 4. Analysis of the p58/p45 gene by Southern hybridization. Ten micrograms of rat genomic DNA, digested with different restriction enzymes as indicated, was loaded on each lane, electrophoresed and transferred to a nylon membrane. The membrane was first hybridized to a radiolabeled DNA probe that is shared by p58 and p45 cDNA clones (A). The bound probe was then removed from the membrane, and the membrane was hybridized to another probe that is specific to p45 (B).
472 bp at the 5∞ end of this clone is identical to the region of p58 between nucleotides 1021 and 1492, whereas the 131 bp at the 3∞ end of H6 (excluding a polyA sequence) is identical to the 3∞ end of the p23 cDNA (data not shown). The rest of the sequence that we determined for H6 (approx. 2 kb), which extends inward from the 5∞ and 3∞ regions discussed above, is novel. DNA sequences at both junction sites closely match the consensus sequence for mRNA splice sites, indicating that this clone also is generated by alternative splicing from the p58/p45 gene transcript. The third clone, H7, occurred nine times in the 48 positives and has a size of approx. 1.2 kb. It is identical to a region of the p58 cDNA between nucleotides 908 and 2124 [the full-length p58 cDNA is 3749 bp long (Hu et al., 1996)] and is followed by a polyA sequence. This clone could be another spliced product of the p58/p45 gene transcript. The 5∞ region of clone H6 encodes a short (99 amino acid) ORF, 93 residues of which are identical to p58 between amino acids 373 and 465, whereas H7 encodes a 213 amino acid ORF that is identical to the C-terminal end of p58. Due to the lack of an in-frame
When we examined the relative levels of p58 and p45 in NRK cells and rat liver NEs by Western blotting with a polyclonal antibody that recognizes both p45 and p58, we found that the p45/p58 ratio varies in these two sources. In NRK cells, the p45 band is substantially weaker than the p58 band, whereas in rat liver NEs the intensities of the p45 and p58 bands are very similar ( Fig. 7A). These results are consistent with Northern hybridization experiments with mRNA preparations from NRK cells and rat liver. A probe corresponding to the p58 coding sequence strongly recognizes bands at 2.5 and 3.8 kb in these two samples, which very likely correspond to mRNAs for p45 and p58, respectively. In rat-liver mRNA, the relative intensities of the major 2.5 and 3.8 kb bands are very similar, whereas in NRK cell mRNA the 3.8 kb band is significantly stronger than the 2.5 kb band. This pattern parallels the relative intensities of p45 and p58 seen by Western blotting ( Fig. 7B). In addition to the major 2.5 and 3.8 kb bands, a number of minor hybridizing species are seen, which could correspond to other apparent alternative splice forms of the p58/p45 transcript, such as the mRNA for putative p23 (see above). To further investigate the expression of p45 in different cells, we carried out Western blotting experiments using cell extracts from several rat and mouse cell lines ( Fig. 8). We found that the relative intensities of the p45 and p58 bands varied from cell line to cell line, and that the p45 signal was usually weaker than the p58 signal. In B16A (a mouse melanoma line), P19 (a mouse teratocarcinoma line), and P19MES (a differentiated line derived from P19) cells, the p45 signal was significantly weaker than that of p58, although it was clearly detectable. However, in HTC cells (a rat hepatoma line) the p45 band was almost undetectable, whereas the p58 signal was as strong as in the other cell lines analyzed ( Fig. 8). This variation does not seem to be related to the cell-cycle status of the cells, because all cultures were maintained in exponential growth. In fact, no difference was found in the levels of p45 in dividing vs. nondividing NRK cells (data not shown). A clear difference in the expression of nuclear lamin isotypes has been observed in P19 vs. P19MES cells, analogous to the regulation of lamin expression during differentiation that is seen in various tissues in vivo (see Martin et al., 1995). While no differences in the relative expression levels of p45 and p58 were detected in this cultured cell model, it is plausible that p45 expression, or the expression of p58/p45 related proteins from other alternatively spliced mRNAs, is regulated in some cell types in vivo at certain stages of development.
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Fig. 5. cDNA and deduced amino acid sequences from a p23 clone. The two putative mRNA splice sites are shown in boxes. The region between the two boxed sequences is identical to a region of the p58 cDNA, whereas the region downstream of the second boxed sequence (CAGG) is identical to a 3∞ terminal region of clone H6.
Fig. 6. Schematic comparison of p58, p45, and p23. FG repeats and regions with a high probability of forming coiled-coil structures are indicated. Each vertical line represents one FG repeat. The unique C-termini of p45 and p23 are indicated by distinct hatched patterns.
4. Discussion Previously, several lines of evidence suggested that p45 and p58 are generated by alternative mRNA splicing (Hu et al., 1996). The cDNA sequence of p45 described herein strongly supports this model. It is further indicated by genomic Southern hybridization results. To our knowledge, this is the only example up to now of NPC proteins that are encoded by alternatively spliced
Fig. 7. Expression patterns of p45 in rat liver and NRK cells. (A) Western blotting with polyclonal antibodies that recognize both p58 and p45. A whole-cell lysate from NRK cells or rat-liver NE proteins were analyzed as indicated. (B) Northern hybridization with a probe generated from the full-length coding sequence of p58. 25 mg of NRK cell polyA+ RNA or 10 mg of rat liver polyA+ RNA were examined as indicated.
mRNAs. Although mRNAs for p58 and p45 are the predominant products resulting from this alternative splicing, our cDNA cloning and Northern hybridization
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Fig. 8. Expression pattern of p45 in several mouse and rat cell lines. Whole cell lysates from different cell lines ( lanes 1–4) or proteins from rat liver NEs ( lane 5) were analyzed by Western blotting using polyclonal antibodies against p58/p45. Lane 1, B16A cells (mouse melanoma); lane 2, P19 cells (mouse teratocarcinoma); lane 3, P19MES cells (differentiated P19); lane 4, HTC cells (rat hepatoma).
data strongly suggest that several additional alternatively spliced mRNAs are expressed from the p58/p45 gene transcripts in some cells, including a transcript that encodes putative p23. However, we have not detected any of these protein products by Western blotting in the cells that we examined. Notwithstanding the similar sequence and domain organization of p45 and p58, the fact that both proteins are present in the same cells indicates that they probably each have some functionally distinct features. The C-terminal FG repeat region of p58 interacts in vitro with a complex consisting of import substrate (containing a basic amino acid-type NLS), NLS receptor, and p97, whereas the N-terminal FG repeat region of p58 does not (Hu et al., 1996). Since p45 contains only half of the C-terminal FG repeat region of p58, it may interact differently than p58 with a substrate/NLS receptor/p97 complex or, for that matter, with other nuclear import or export complexes. Moreover, p45 contains a 22-residue deletion (relative to p58) adjacent to its coiled-coil region. Since this coiled-coil region might be involved in protein–protein interactions, this deletion could affect the association of p45 with other subunits of the p62 complex or with other NPC components. p45 contains a unique C-terminal sequence not shared by p58, LCASA, which resembles the consensus site (CAAX ) for protein isoprenylation (Clarke, 1992). This raises the intriguing possibility that p45 could be isoprenylated at its C-terminus. Such a modification could regulate the interaction of p45 with cytosolic factors or have some other function. We investigated whether p45 is isoprenylated by immunoprecipitating p45 from cultured cells that had been incubated with a radioactive isoprenylation substrate. We also attempted to label p45 in vitro by incubating recombinant p45 with a radioactive isoprenylation substrate in the presence of cytosol, and by
including a radioactive isoprenylation substrate in an in vitro transcription/translation reaction with the p45 cDNA clone. No label was obtained in p45 in any these experiments (Hu and Gerace, unpublished observations), so it remains unclear whether p45 is isoprenylated. The p62 complex of rat liver NEs is suggested to contain one copy each of p62, p58, p54 and p45, based on its mass of approx. 234 kDa and the roughly stoichiometric levels of these four subunits in purified preparations (Guan et al., 1995). However, the very low expression of p45 in HTC cells implies that the p62 complex does not always contain p45. This indicates that more than one type of p62 complex exists. The strong homology between p45 and p58 suggests that the two proteins might be able to substitute for each other in the p62 complex. If two copies of the p58/p45 pair were in each p62 complex, then different p62 complexes might contain p58:p45 ratios of 2:0, 1:1 and 0:2. Considering the potential functional differences between p58 and p45 (see above), these different types of p62 complex could have different biological properties. In this regard, the possibility should be considered that proteins produced by other alternatively spliced mRNAs of the p58/p45 gene transcript may be assembled into some versions of the p62 complex. For example, the putative p23 protein, which contains the complete coiledcoil domain of p58/p45 but lacks the FG repeat regions, might be incorporated in the p62 complex but be unable to bind the cytosolic factors targeted to FG repeats. Thus, it is possible that alternative splicing of p58/p45 gene transcript is used to produce different types of p62 complexes with different functional properties. This could provide a mechanism to regulate the nuclear transport activity of the NPC in different cell types. In conclusion, with the cloning of the p45 cDNA, the coding sequences for all subunits of the rat liver p62 complex are now available. This provides the tools to obtain different types of recombinant p62 complexes for functional studies, and to further investigate the specific role of the p62 complex in nuclear transport.
Acknowledgement We would like to thank all members of our laboratory for their help and valuable discussions. We are very grateful to Dr Dwayne Stupack for his help with twodimensional gel electrophoresis, and thank Dr Thomas Mo¨hler for providing B16A cells. This work was supported by a Rockefeller Foundation fellowship to T. Hu, a grant from the NIH to L. Gerace (GM41955), and a grant from the Lucille P. Markey Charitable Trust.
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