Limits...
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.

Show MeSH

Related in: MedlinePlus

Kap108p binds directly to both Lhp1p and  rpL11p. The binding sites  of Lhp1p and rpL11p on  Kap108p overlap. (a) Genomically expressed Kap108– PrA was isolated from cytosol of a wild-type strain (left)  and an lhp1-deletion strain  (right). After incubation with  IgG-Sepharose, unbound proteins were removed and  bound proteins were eluted  with acid (left) and a MgCl2  gradient (right). After separation on 4–20% SDS-PAGE  gels, proteins were visualized with Coomassie blue R.  (b) Genomically expressed  Lhp1–PrA was isolated from  cytosol of a wild-type strain  (left) and a KAP108-deletion strain (right). Purified  cytosol from each strain was  incubated with IgG-Sepharose, washed and eluted  with a MgCl2 step gradient.  After separation on 4–20%  SDS-PAGE gels, proteins  were visualized with Coomassie blue R. Asterisk,  Lhp1–PrA-associated proteins. (c) Cytosolic Kap108– PrA was isolated from cytosol of a strain without (left  lane), and with (right lane), a  plasmid expressing an Lhp1– GFP fusion protein. After  isolation of Kap108–PrA,  bound proteins were eluted  with acid. After separation  on a 10–20% SDS-PAGE  gel, proteins were visualized  with Coomassie blue R.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132966&req=5

Figure 1: Kap108p binds directly to both Lhp1p and rpL11p. The binding sites of Lhp1p and rpL11p on Kap108p overlap. (a) Genomically expressed Kap108– PrA was isolated from cytosol of a wild-type strain (left) and an lhp1-deletion strain (right). After incubation with IgG-Sepharose, unbound proteins were removed and bound proteins were eluted with acid (left) and a MgCl2 gradient (right). After separation on 4–20% SDS-PAGE gels, proteins were visualized with Coomassie blue R. (b) Genomically expressed Lhp1–PrA was isolated from cytosol of a wild-type strain (left) and a KAP108-deletion strain (right). Purified cytosol from each strain was incubated with IgG-Sepharose, washed and eluted with a MgCl2 step gradient. After separation on 4–20% SDS-PAGE gels, proteins were visualized with Coomassie blue R. Asterisk, Lhp1–PrA-associated proteins. (c) Cytosolic Kap108– PrA was isolated from cytosol of a strain without (left lane), and with (right lane), a plasmid expressing an Lhp1– GFP fusion protein. After isolation of Kap108–PrA, bound proteins were eluted with acid. After separation on a 10–20% SDS-PAGE gel, proteins were visualized with Coomassie blue R.

Mentions: Previously, we demonstrated that Kap108p could be isolated from yeast cytosol in a complex whose main components were Lhp1p and rpL11p (Rosenblum et al., 1997) (rpL11p was previously known as Rpl16p, for new S. cerevisiae ribosomal protein nomenclature see Mager et al., 1997). At the time we could not differentiate between two coexisting, dimeric complexes of Kap108p and a single trimeric complex of all three members. To address this issue, we generated a yeast strain that contained a genomically expressed fusion between protein A and Kap108p (Kap108– PrA) but was deleted for LHP1. We then isolated Kap108–PrA by virtue of the affinity for IgG-Sepharose of protein A. The results are shown in Fig. 1 a. In the first lane, for comparison, cytosolic Kap108–PrA was purified from cells wild type for LHP1 and the bound proteins were eluted with acid. The second panel of Fig. 1 a shows the result of a similar experiment done in parallel with cytosol from the LHP1-deletion strain. rpL11p eluted from Kap108–PrA in the LHP1 deletion strain at the same concentrations of MgCl2 that dissociate this complex isolated from a strain containing Lhp1p. As such, it is evident that Lhp1p does not mediate the rpL11p/Kap108p interaction. We have also detected other, smaller ribosomal proteins in complex with Kap108p (Rosenblum et al., 1997). In Fig. 1 a, right, these smaller ribosomal proteins, between ∼11 and 17 kD, appear to elute from the column earlier than rpL11p, peaking in the 100 mM MgCl2 fraction. Therefore, we cannot exclude the possibility that these ribosomal proteins bind to Kap108p via rpL11p.


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

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

Kap108p binds directly to both Lhp1p and  rpL11p. The binding sites  of Lhp1p and rpL11p on  Kap108p overlap. (a) Genomically expressed Kap108– PrA was isolated from cytosol of a wild-type strain (left)  and an lhp1-deletion strain  (right). After incubation with  IgG-Sepharose, unbound proteins were removed and  bound proteins were eluted  with acid (left) and a MgCl2  gradient (right). After separation on 4–20% SDS-PAGE  gels, proteins were visualized with Coomassie blue R.  (b) Genomically expressed  Lhp1–PrA was isolated from  cytosol of a wild-type strain  (left) and a KAP108-deletion strain (right). Purified  cytosol from each strain was  incubated with IgG-Sepharose, washed and eluted  with a MgCl2 step gradient.  After separation on 4–20%  SDS-PAGE gels, proteins  were visualized with Coomassie blue R. Asterisk,  Lhp1–PrA-associated proteins. (c) Cytosolic Kap108– PrA was isolated from cytosol of a strain without (left  lane), and with (right lane), a  plasmid expressing an Lhp1– GFP fusion protein. After  isolation of Kap108–PrA,  bound proteins were eluted  with acid. After separation  on a 10–20% SDS-PAGE  gel, proteins were visualized  with Coomassie blue R.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132966&req=5

Figure 1: Kap108p binds directly to both Lhp1p and rpL11p. The binding sites of Lhp1p and rpL11p on Kap108p overlap. (a) Genomically expressed Kap108– PrA was isolated from cytosol of a wild-type strain (left) and an lhp1-deletion strain (right). After incubation with IgG-Sepharose, unbound proteins were removed and bound proteins were eluted with acid (left) and a MgCl2 gradient (right). After separation on 4–20% SDS-PAGE gels, proteins were visualized with Coomassie blue R. (b) Genomically expressed Lhp1–PrA was isolated from cytosol of a wild-type strain (left) and a KAP108-deletion strain (right). Purified cytosol from each strain was incubated with IgG-Sepharose, washed and eluted with a MgCl2 step gradient. After separation on 4–20% SDS-PAGE gels, proteins were visualized with Coomassie blue R. Asterisk, Lhp1–PrA-associated proteins. (c) Cytosolic Kap108– PrA was isolated from cytosol of a strain without (left lane), and with (right lane), a plasmid expressing an Lhp1– GFP fusion protein. After isolation of Kap108–PrA, bound proteins were eluted with acid. After separation on a 10–20% SDS-PAGE gel, proteins were visualized with Coomassie blue R.
Mentions: Previously, we demonstrated that Kap108p could be isolated from yeast cytosol in a complex whose main components were Lhp1p and rpL11p (Rosenblum et al., 1997) (rpL11p was previously known as Rpl16p, for new S. cerevisiae ribosomal protein nomenclature see Mager et al., 1997). At the time we could not differentiate between two coexisting, dimeric complexes of Kap108p and a single trimeric complex of all three members. To address this issue, we generated a yeast strain that contained a genomically expressed fusion between protein A and Kap108p (Kap108– PrA) but was deleted for LHP1. We then isolated Kap108–PrA by virtue of the affinity for IgG-Sepharose of protein A. The results are shown in Fig. 1 a. In the first lane, for comparison, cytosolic Kap108–PrA was purified from cells wild type for LHP1 and the bound proteins were eluted with acid. The second panel of Fig. 1 a shows the result of a similar experiment done in parallel with cytosol from the LHP1-deletion strain. rpL11p eluted from Kap108–PrA in the LHP1 deletion strain at the same concentrations of MgCl2 that dissociate this complex isolated from a strain containing Lhp1p. As such, it is evident that Lhp1p does not mediate the rpL11p/Kap108p interaction. We have also detected other, smaller ribosomal proteins in complex with Kap108p (Rosenblum et al., 1997). In Fig. 1 a, right, these smaller ribosomal proteins, between ∼11 and 17 kD, appear to elute from the column earlier than rpL11p, peaking in the 100 mM MgCl2 fraction. Therefore, we cannot exclude the possibility that these ribosomal proteins bind to Kap108p via rpL11p.

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.

Show MeSH
Related in: MedlinePlus