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Nuclear import and the evolution of a multifunctional RNA-binding protein.

Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G - J. Cell Biol. (1998)

Bottom Line: Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA.

ABSTRACT
La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

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Human La interacts with yeast Kap60p/ Kap95p. (a) Proteins associated with Kap95–PrA were  isolated from cytosol of  strains without (left) and with  (right) a plasmid expressing  human La as a GFP fusion  (hsLa–GFP). Extracts were  passed over an IgG-Sepharose column, which was then  washed. Bound proteins were  eluted with acid and subjected to SDS-PAGE on a  10–20% gel. Proteins were  stained with Coomassie blue  R. (b) Proteins associated  with Kap95–PrA, (left) and  Kap108–PrA, (right), were  isolated from cytosol. In both  cases, the strains harbored a plasmid expressing hsLa–GFP. In this case, bound proteins were eluted with a MgCl2 gradient, separated  by SDS-PAGE, and then transferred to nitrocellulose. The GFP moiety was detected by immunoblotting with anti-GFP antibodies.  Only the relevant part of the blot is shown.
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Figure 4: Human La interacts with yeast Kap60p/ Kap95p. (a) Proteins associated with Kap95–PrA were isolated from cytosol of strains without (left) and with (right) a plasmid expressing human La as a GFP fusion (hsLa–GFP). Extracts were passed over an IgG-Sepharose column, which was then washed. Bound proteins were eluted with acid and subjected to SDS-PAGE on a 10–20% gel. Proteins were stained with Coomassie blue R. (b) Proteins associated with Kap95–PrA, (left) and Kap108–PrA, (right), were isolated from cytosol. In both cases, the strains harbored a plasmid expressing hsLa–GFP. In this case, bound proteins were eluted with a MgCl2 gradient, separated by SDS-PAGE, and then transferred to nitrocellulose. The GFP moiety was detected by immunoblotting with anti-GFP antibodies. Only the relevant part of the blot is shown.

Mentions: Two sequences similar to the consensus bipartite NLS recognized by Kapα/Kapβ1 have been identified in hsLa and termed box A and box B (Simons et al., 1996). Box A perfectly corresponds to the consensus, with 2 NH2-terminal basic residues, a 10-aa spacer, and a COOH-terminal cluster of three basic residues, whereas box B does not, having a 12-aa spacer and only two basic residues on the COOH-terminal side. Interestingly, box A was shown to be unable to confer nuclear localization on hsLa whereas box B was shown to be the hsLa NLS (Simons et al., 1996). Although we were initially drawn to the differences between box B and the consensus, the ability of human La to localize to the nucleus in the absence of Kap108p led us to consider that human La is brought to the nucleus in an α-Kap– dependent manner. We tested for this biochemically in yeast by introducing our hsLa–GFP plasmid into a strain that carried a genomic protein A fusion with the S. cerevisiae Kapβ1, Kap95p (Kap95–PrA). We then purified cytosolic Kap95p–PrA from this strain by immunoaffinity chromatography. As shown in Fig. 4 a, Kap60p, the S. cerevisiae Kapα, copurifies with Kap95–PrA from an untransformed strain. As Kap95p/Kap60p appears to be the major route to the nucleus, transporting perhaps hundreds of proteins, individual import substrates cannot generally be visualized by Coomassie blue staining (Aitchison et al., 1996; Makkerh et al., 1996). This inability to clearly see substrates most likely results from the stoichiometry of the interaction—each substrate is probably two to three orders of magnitude less abundant than the isolated Kap60p/ Kap95p dimer. Surprisingly, when Kap95–PrA is isolated from cytosol of a strain carrying the human La–GFP fusion, a clear band corresponding to the fusion can be seen by Coomassie blue staining. The ability to visualize hsLa– GFP by Coomassie staining most likely results from the high expression of this construct, making the fusion protein one of the main substrates of this pathway. That the fusion product is observable is even more remarkable when the trimeric nature of the isolated complex is taken into account; purified Kap95–PrA is bound to Kap60p, which in turn is bound to human La–GFP. The identity of the hsLa–GFP band was confirmed by Western blot analysis with anti-GFP antibodies (Fig. 4 b). Significantly, in a separate experiment performed in parallel, no hsLa–GFP copurified with Kap108–PrA (Fig. 4 b).


Nuclear import and the evolution of a multifunctional RNA-binding protein.

Rosenblum JS, Pemberton LF, Bonifaci N, Blobel G - J. Cell Biol. (1998)

Human La interacts with yeast Kap60p/ Kap95p. (a) Proteins associated with Kap95–PrA were  isolated from cytosol of  strains without (left) and with  (right) a plasmid expressing  human La as a GFP fusion  (hsLa–GFP). Extracts were  passed over an IgG-Sepharose column, which was then  washed. Bound proteins were  eluted with acid and subjected to SDS-PAGE on a  10–20% gel. Proteins were  stained with Coomassie blue  R. (b) Proteins associated  with Kap95–PrA, (left) and  Kap108–PrA, (right), were  isolated from cytosol. In both  cases, the strains harbored a plasmid expressing hsLa–GFP. In this case, bound proteins were eluted with a MgCl2 gradient, separated  by SDS-PAGE, and then transferred to nitrocellulose. The GFP moiety was detected by immunoblotting with anti-GFP antibodies.  Only the relevant part of the blot is shown.
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Related In: Results  -  Collection

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Figure 4: Human La interacts with yeast Kap60p/ Kap95p. (a) Proteins associated with Kap95–PrA were isolated from cytosol of strains without (left) and with (right) a plasmid expressing human La as a GFP fusion (hsLa–GFP). Extracts were passed over an IgG-Sepharose column, which was then washed. Bound proteins were eluted with acid and subjected to SDS-PAGE on a 10–20% gel. Proteins were stained with Coomassie blue R. (b) Proteins associated with Kap95–PrA, (left) and Kap108–PrA, (right), were isolated from cytosol. In both cases, the strains harbored a plasmid expressing hsLa–GFP. In this case, bound proteins were eluted with a MgCl2 gradient, separated by SDS-PAGE, and then transferred to nitrocellulose. The GFP moiety was detected by immunoblotting with anti-GFP antibodies. Only the relevant part of the blot is shown.
Mentions: Two sequences similar to the consensus bipartite NLS recognized by Kapα/Kapβ1 have been identified in hsLa and termed box A and box B (Simons et al., 1996). Box A perfectly corresponds to the consensus, with 2 NH2-terminal basic residues, a 10-aa spacer, and a COOH-terminal cluster of three basic residues, whereas box B does not, having a 12-aa spacer and only two basic residues on the COOH-terminal side. Interestingly, box A was shown to be unable to confer nuclear localization on hsLa whereas box B was shown to be the hsLa NLS (Simons et al., 1996). Although we were initially drawn to the differences between box B and the consensus, the ability of human La to localize to the nucleus in the absence of Kap108p led us to consider that human La is brought to the nucleus in an α-Kap– dependent manner. We tested for this biochemically in yeast by introducing our hsLa–GFP plasmid into a strain that carried a genomic protein A fusion with the S. cerevisiae Kapβ1, Kap95p (Kap95–PrA). We then purified cytosolic Kap95p–PrA from this strain by immunoaffinity chromatography. As shown in Fig. 4 a, Kap60p, the S. cerevisiae Kapα, copurifies with Kap95–PrA from an untransformed strain. As Kap95p/Kap60p appears to be the major route to the nucleus, transporting perhaps hundreds of proteins, individual import substrates cannot generally be visualized by Coomassie blue staining (Aitchison et al., 1996; Makkerh et al., 1996). This inability to clearly see substrates most likely results from the stoichiometry of the interaction—each substrate is probably two to three orders of magnitude less abundant than the isolated Kap60p/ Kap95p dimer. Surprisingly, when Kap95–PrA is isolated from cytosol of a strain carrying the human La–GFP fusion, a clear band corresponding to the fusion can be seen by Coomassie blue staining. The ability to visualize hsLa– GFP by Coomassie staining most likely results from the high expression of this construct, making the fusion protein one of the main substrates of this pathway. That the fusion product is observable is even more remarkable when the trimeric nature of the isolated complex is taken into account; purified Kap95–PrA is bound to Kap60p, which in turn is bound to human La–GFP. The identity of the hsLa–GFP band was confirmed by Western blot analysis with anti-GFP antibodies (Fig. 4 b). Significantly, in a separate experiment performed in parallel, no hsLa–GFP copurified with Kap108–PrA (Fig. 4 b).

Bottom Line: Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA.

ABSTRACT
La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

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