A PY-NLS Nuclear Targeting Signal Is Required for Nuclear Localization and Function of the Saccharomyces cerevisiae mRNA-binding Protein Hrp1

Proteins destined for import into the nucleus contain nuclear localization signals (NLSs) that are recognized by import receptors termed karyopherins or importins. Until recently, the only nuclear import sequence that had been well defined and characterized was the classical NLS (cNLS), which is recognized by importin α. However, Chook and coworkers (Lee, B. J., Cansizoglu, A. E., Süel, K. E., Louis, T. H., Zhang, Z., and Chook, Y. M. (2006) Cell 126, 543–558) have provided new insight into nuclear targeting with their identification of a novel NLS, termed the PY-NLS, that is recognized by the human karyopherin β2/transportin (Kapβ2) receptor. Here, we demonstrate that the PY-NLS is conserved in Saccharomyces cerevisiae and show for the first time that the PY-NLS is a functional nuclear targeting sequence in vivo. The apparent ortholog of Kapβ2 in yeast, Kap104, has two known cargos, the mRNA-binding proteins Hrp1 and Nab2, which both contain putative PY-NLS-like sequences. We find that the PY-NLS-like sequence within Hrp1, which closely matches the PY-NLS consensus, is both necessary and sufficient for nuclear import and is also required for receptor binding and protein function. In contrast, the PY-NLS-like sequences in Nab2, which vary from the PY-NLS consensus, are not required for proper import or protein function, suggesting that Kap104 may interact with different cargos using multiple mechanisms. Dissection of the PY-NLS consensus reveals that the minimal PY-NLS in yeast consists of the C-terminal portion of the human consensus, R/H/KX2–5PY, with upstream basic or hydrophobic residues enhancing the targeting function. Finally, we apply this analysis to a bioinformatic search of the yeast proteome as a preliminary search for new potential Kap104 cargos. Proteins destined for import into the nucleus contain nuclear localization signals (NLSs) that are recognized by import receptors termed karyopherins or importins. Until recently, the only nuclear import sequence that had been well defined and characterized was the classical NLS (cNLS), which is recognized by importin α. However, Chook and coworkers (Lee, B. J., Cansizoglu, A. E., Süel, K. E., Louis, T. H., Zhang, Z., and Chook, Y. M. (2006) Cell 126, 543–558) have provided new insight into nuclear targeting with their identification of a novel NLS, termed the PY-NLS, that is recognized by the human karyopherin β2/transportin (Kapβ2) receptor. Here, we demonstrate that the PY-NLS is conserved in Saccharomyces cerevisiae and show for the first time that the PY-NLS is a functional nuclear targeting sequence in vivo. The apparent ortholog of Kapβ2 in yeast, Kap104, has two known cargos, the mRNA-binding proteins Hrp1 and Nab2, which both contain putative PY-NLS-like sequences. We find that the PY-NLS-like sequence within Hrp1, which closely matches the PY-NLS consensus, is both necessary and sufficient for nuclear import and is also required for receptor binding and protein function. In contrast, the PY-NLS-like sequences in Nab2, which vary from the PY-NLS consensus, are not required for proper import or protein function, suggesting that Kap104 may interact with different cargos using multiple mechanisms. Dissection of the PY-NLS consensus reveals that the minimal PY-NLS in yeast consists of the C-terminal portion of the human consensus, R/H/KX2–5PY, with upstream basic or hydrophobic residues enhancing the targeting function. Finally, we apply this analysis to a bioinformatic search of the yeast proteome as a preliminary search for new potential Kap104 cargos. In eukaryotic cells, the genetic material is separated from the cellular machinery in the cytoplasm by a nuclear membrane that is studded with regulated channels termed nuclear pore complexes (1Fahrenkrog B. Aebi U. Nat. Rev. Mol. Cell Biol. 2003; 4: 757-766Crossref PubMed Scopus (336) Google Scholar, 2Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (297) Google Scholar, 3Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar). Nuclear pores prevent passive diffusion of macromolecules larger than ∼40 kDa (4Paine P.L. Moore L.C. Horowitz S.B. Nature. 1975; 254: 109-114Crossref PubMed Scopus (578) Google Scholar, 5Bonner W.M. Protein Migration and Accumulation in Nuclei. Academic Press, New York1978Google Scholar), so this division can facilitate the precise management of gene expression by controlling access to the genome. However, the presence of the nuclear membrane also necessitates the involvement of nuclear import machinery that can specifically recognize cargo in the cytoplasm, mediate transport through nuclear pores, and effect delivery in the nucleus. For many import cargos, these import factors are members of the importin/karyopherin β transport receptor superfamily, which has 14 members in Saccharomyces cerevisiae and over 20 members in humans (6Mosammaparast N. Pemberton L.F. Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). These import receptors recognize protein cargo bearing targeting sequences called nuclear localization signals (NLSs) 2The abbreviations used are: NLS, nuclear localization signal; cNLS, classical NLS; GFP, green fluorescent protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; 5-FOA, 5-fluoroorotic acid. and mediate interactions with the nuclear pore, allowing translocation of the cargo into the nucleus (7Cook A. Bono F. Jinek M. Conti E. Annu. Rev. Biochem. 2007; 76: 647-671Crossref PubMed Scopus (413) Google Scholar). Once inside the nucleus, the import complex encounters RanGTP, a small Ras family GTPase that regulates the directionality of nuclear transport (8Quimby B.B. Dasso M. Curr Opin. Cell Biol. 2003; 15: 338-344Crossref PubMed Scopus (161) Google Scholar). RanGTP binds to the karyopherin β, causing a conformational change and triggering cargo release (9Lee S.J. Matsuura Y. Liu S.M. Stewart M. 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Stewart M. Devine S.E. Corbett A.H. J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). A large number of classical substrates have been experimentally verified, and examination of their nuclear targeting signals has led to the development of a useful consensus sequence (12Lange A. Mills R.E. Lange C.J. Stewart M. Devine S.E. Corbett A.H. J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). In contrast to the classical nuclear import pathway, for most karyopherin β receptors only a handful of substrates have been identified making generalization of binding schemes difficult. Compounding the problem, these cargos often lack sequence or structural homology and, in some cases, even interact with a particular karyopherin β receptor using distinct binding sites and diverse interaction modes (for example, Refs. 13Lee S. Sekimoto T. Yamashita E. Nagoshi E. Nakagawa A. Imamoto N. Yoshimura M. Sakai H. Chong K. Tsukihara T. Yoneda Y. Science. 2003; 302: 1571-1575Crossref PubMed Scopus (173) Google Scholar, 14Cingolani G. Petosa C. Weis K. Müller C.W. Nature. 1999; 399: 221-229Crossref PubMed Scopus (452) Google Scholar, 15Cingolani G. Bednenko J. Gillespie M.T. Gerace L. Mol. Cell. 2002; 10: 1345-1353Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, a mechanism underlying recognition of a specific class of cargos by a transport receptor that does not mediate cNLS protein import was recently determined. Chook and coworkers (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google the of a human karyopherin β called karyopherin (Kapβ2) or to the NLS of its characterized the mRNA-binding protein nuclear the structural with known Kapβ2 cargos, then a of that the for a putative Kapβ2 the NLS is the NLS has a basic and the NLS has a hydrophobic or basic upstream of a C-terminal The and residues the of the consensus led to these Kapβ2 recognition sequences these then the human identified putative Kapβ2 cargos, and that a of these PY-NLS proteins to Kapβ2 in a in These binding that the proteins are substrates of in functional and localization are necessary to demonstrate that these proteins are by Kapβ2 in a The apparent ortholog of Kapβ2 in S. cerevisiae is Kap104 which has two known cargos, the mRNA-binding proteins protein and Hrp1 nuclear Blobel G. Science. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). has defined the of these cargo proteins that are required for nuclear import J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R.E. H. Mol. Cell. Biol. PubMed Scopus Google Scholar). In Nab2, a an binding of a of is necessary and sufficient for binding to Kap104 and for nuclear import in J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R.E. H. Mol. Cell. Biol. PubMed Scopus Google Scholar). of the of also binding to Kap104 in J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In Hrp1, the of the protein is required for binding to Kap104 and is sufficient to mediate import in J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the protein in Hrp1 and that mediate Kap104 binding have been specific nuclear import sequences have been many Kapβ2 cargos (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), Hrp1 and are in with Hrp1 to and of E. J. S. Moore 11: PubMed Scopus Google and in and R.E. S. S.M. L. EMBO J. 2002; PubMed Scopus Google Scholar, Zhang Corbett A.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the human protein most closely to Hrp1 is nuclear The of this was to the of the novel PY-NLS sequence in vivo. of the sequences of Hrp1 and putative PY-NLS-like sequences in both proteins. a of function, and binding we that the PY-NLS-like sequence within Hrp1 is necessary and sufficient for nuclear import of the protein and is required for protein function. However, the conserved PY-NLS-like sequences within are not required for nuclear of These demonstrate that the PY-NLS consensus sequence is conserved in S. cerevisiae also that Kap104 of cargo Finally, identification of the functional PY-NLS in yeast to the yeast proteome to new putative cargos of we with a preliminary of the of the PY-NLS in S. and from or and to A. T. in Press, Scholar, J. D. A Press, Scholar). yeast and used in this are in and used in this F. C. 11: PubMed Scopus Google A. PubMed Google A. Zhang Corbett A.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Zhang Corbett A.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google A.E. M.T. Corbett A.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google E. J. S. Moore 11: PubMed Scopus Google in a new fluorescent protein proteins in using The was using a an with a For most from their in in and for to localization the of the in with as a in with as a and for to localization the of the in in and for to localization the required an of to the In binding Kap104 was in as a protein Zhang Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar), and then with yeast from or the E. in with each of and and by by and with of and with yeast or in with as a and in with as a to expression of the proteins. and in and in was in with and by and protein was by One of yeast and of with The was and the was with and with and a The proteins to a membrane and with either and to the proteins or and to the In in of each of the Hrp1 and was using a J. G. PubMed Scopus Google Scholar). or Hrp1 or proteins into either or a or to in in and or 5-fluoroorotic or for For from the Hrp1 that either or Hrp1 as the only of Hrp1 to in a and for over time using an with and the was was with different PY-NLS the consensus sequence for a hydrophobic PY-NLS (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), the sequence for a basic PY-NLS (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), and the sequence for the C-terminal of the PY-NLS, was then used with A. E. A. 2002; Google to two the yeast proteome by the proteins in the S. cerevisiae J. 2007; PubMed Scopus Google and the proteins to either the nucleus or the to a localization using the yeast L.C. Nature. 2003; PubMed Scopus Google Scholar). The are in a with in a A of is the Corbett Hrp1 and PY-NLS-like the protein sequences of the known Kap104 cargos Hrp1 and Blobel G. Science. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that Hrp1 one sequence and two sequences that are to the Kapβ2 binding sequence termed the PY-NLS (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google The PY-NLS-like sequence of Hrp1 is the of the protein within the to required for import J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The PY-NLS-like sequences of are both in the C-terminal PY-NLS-like sequence the and and sequence is the and The sequence in Hrp1 closely the PY-NLS, the C-terminal portion of the consensus sequence by a basic of amino In contrast, the putative sequences in both contain upstream basic or hydrophobic vary from the PY-NLS consensus in the C-terminal as both sequences contain a within two to residues by a and a than the change in the the Hrp1 and are both cargos of Kap104 Blobel G. Science. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), may interact with the receptor using different of interactions or even in different Hrp1 and by that nuclear localization of Hrp1 and Kap104 Blobel G. Science. 1996; PubMed Scopus Google Scholar), the localization of Hrp1 or was in or Hrp1 was with the of the protein to the C-terminal of the PY-NLS-like was the with this protein has been to in and to to the nucleus Zhang Corbett A.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In cells, both and are to the in cells, both and are the that proper nuclear localization of Hrp1 and is also that of the proteins are A Blobel G. Science. 1996; PubMed Scopus Google Scholar, Zhang Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar), is within the nucleus in both and cells, that not nuclear import is in The PY-NLS-like within Hrp1 and for Nuclear and for Kap104 the putative PY-NLS-like sequence within Hrp1 is necessary for the import of Hrp1 into the nucleus, specific amino in the C-terminal of the PY-NLS-like sequence of and the in was to the either the or both the upstream and the residues to was to the cytoplasm, that the PY-NLS-like sequence of Hrp1 is necessary for nuclear of the and the in These not to with particular nuclear or and their is that of the proteins not the PY-NLS-like sequence within Hrp1 is required for the interaction Hrp1 and Kap104, we interact with either or in yeast For this with yeast from either or to that the PY-NLS-like sequence within Hrp1 is for receptor the PY-NLS-like sequence within Hrp1 is sufficient to mediate import of a protein, either the C-terminal or the PY-NLS-like sequence of Hrp1 was to used to a protein that was large to through the nuclear pore into the nucleus. to in the nucleus these proteins an system of was to the The C-terminal to nuclear with some The PY-NLS-like sequence to nuclear targeting with the protein to the nucleus. These that the PY-NLS-like sequence of Hrp1 is sufficient to mediate import into the nucleus and that even the small C-terminal the upstream basic or hydrophobic residues can mediate nuclear targeting to some Hrp1 to the cytoplasm in Blobel G. Science. 1996; PubMed Scopus Google not that import of these proteins is The PY-NLS-like within for into the to that for Hrp1 was to the PY-NLS-like sequences within Nab2, which contain residues than are required for nuclear either or both of these PY-NLS-like sequences within are required for nuclear and in in is to the nucleus. with amino in either or both of the two putative PY-NLS-like sequences is also to the nucleus, that PY-NLS-like sequence is required for import. either of the putative PY-NLS-like sequences within is sufficient to mediate nuclear either PY-NLS-like sequence or PY-NLS-like sequence was to and these proteins in the that PY-NLS-like sequence is sufficient to mediate import into the nucleus. The PY-NLS-like for Hrp1 for the PY-NLS-like of Hrp1 is both necessary and sufficient to mediate Hrp1 nuclear this sequence required for the of the nuclear Hrp1 protein E. J. S. Moore 11: PubMed Scopus Google in vivo. In contrast, of the PY-NLS-like sequences within is required for we that of the sequences required for function. to the PY-NLS-like sequences within Hrp1 or are for protein in or a with Hrp1 or Nab2, PY-NLS Hrp1 or Nab2, or and or is a of that J. G. PubMed Scopus Google Scholar). and are only a functional of or are to in Hrp1 or Hrp1 are not that the PY-NLS-like sequence within Hrp1 is for protein function, import of Hrp1 is required for proper E. J. S. Moore 11: PubMed Scopus Google Scholar). Nab2, Nab2, or as the only of as well as Nab2, that PY-NLS-like within is required for Dissection of the PY-NLS the of the and the residues to the of the PY-NLS we within the PY-NLS of we the to In the of Kapβ2 to nuclear the of the PY-NLS hydrophobic and with the receptor (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). change to a these amino change also the PY-NLS-like sequences of Nab2, allowing a of can for in S. cerevisiae PY-NLS sequences. we the to The of the nuclear PY-NLS hydrophobic with Kapβ2 (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google and may to the of the NLS to is a this amino change interactions and structural are required for proper PY-NLS function. Hrp1 and Hrp1 the with and in the Hrp1 PY-NLS was to the cytoplasm, that proper of the PY-NLS-like sequence was for in the Hrp1 PY-NLS-like was to the cytoplasm also nuclear that the may a in the Hrp1 that the not The of the and Hrp1 was using the Hrp1 as the only of Hrp1 are that the in the C-terminal of the Hrp1 PY-NLS-like sequence is required for Hrp1 function. Hrp1 are have a Hrp1 as the only of Hrp1 are for a was of the analysis demonstrate that Hrp1 than an The of the PY-NLS in S. has the PY-NLS-like sequences of the Kap104 cargos and The PY-NLS-like sequences of are not functional NLSs in is to that not have been to contain a putative of the PY-NLS-like sequences within vary from the consensus in the a that we have to for proper PY-NLS in A and In contrast, we find that Hrp1 does contain a functional PY-NLS, that the PY-NLS is conserved in The of the PY-NLS of yeast NLS consensus sequences and, with a to a preliminary search for new putative Kap104 cargos in S. in we not only that the PY-NLS-like of Hrp1 mediate nuclear import also that a minimal C-terminal effect nuclear that both of these sequences can mediate nuclear targeting in to the of putative proteins in yeast we two using the hydrophobic and basic PY-NLS consensus sequences as well as a sequence of the we the yeast proteome as by the proteins in the S. cerevisiae J. 2007; PubMed Scopus Google to the of potential Kap104 cargos. we the proteins to either the nucleus or the as by a localization the yeast L.C. Nature. 2003; PubMed Scopus Google Scholar). was nuclear proteins contain nuclear targeting a of which contain one the C-terminal sufficient to mediate proteins or of the yeast proteome contain one PY-NLS nuclear of the nuclear contain putative A of proteins that are for Kap104 import are in which a of yeast proteins that are nuclear or contain a putative hydrophobic or basic PY-NLS, and lack a putative classical NLS (12Lange A. Mills R.E. Lange C.J. Stewart M. Devine S.E. Corbett A.H. J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). A of the is or yeast proteins that contain putative hydrophobic or basic lack putative classical from the Saccharomyces of the of function, required for localization of and for of and with and small required for and in the of in the of in diverse and that the complex to into of the required for of in of regulates with and protein of the small required for and 20 protein in and of a complex required for the of complexes protein that in the C-terminal of which is the of of the with to the the of protein A in required for and in required for complex of and in required for of two that the in of in in and the of the and complex required for of in a new In this we demonstrate that the PY-NLS, a human Kapβ2 binding nuclear targeting sequence (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), is conserved in S. cerevisiae and show for the first time in that the PY-NLS is a functional nuclear import in vivo. function, and binding we show that the PY-NLS-like sequence within Hrp1, a cargo of the yeast ortholog of is necessary and sufficient for nuclear import and is required for receptor binding and for protein function. These that the PY-NLS-like sequence within Hrp1 is a NLS and, in binding to demonstrate that this PY-NLS can mediate import of a protein cargo in vivo. In contrast, we that the putative PY-NLS-like sequences within are not functional NLS suggesting that have for into the nucleus. These may different import and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R.E. H. Mol. Cell. Biol. PubMed Scopus Google show that proper localization Kap104, these that Kap104 can interact with different of cargo proteins using different mechanisms. In the first of Kap104 recognizes cargo proteins Hrp1 PY-NLS and in the of Kap104 recognizes cargo proteins interactions that within the of J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R.E. H. Mol. Cell. Biol. PubMed Scopus Google Scholar). also the of the residues of the PY-NLS, the and to the of the Hrp1 We that the to an of Hrp1 by suggesting not the is required for proper localization and of the Hrp1 effect Hrp1 localization and may to the of the PY-NLS only two residues from the of residues are to to the of NLSs to their The within the PY-NLS of Hrp1 may not required for PY-NLS the of the protein may lack However, a to PY-NLS sequences within cargos the is in a of the the in the Hrp1 PY-NLS to a PY-NLS function, suggesting that the change in the of the PY-NLS-like sequences within may for the lack of of these putative in vivo. the PY-NLS sequence of Hrp1 does not the consensus by (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google for human Kapβ2 the C-terminal the of upstream basic residues from the with the of the C-terminal of Hrp1 can mediate this that the for recognition of yeast by Kap104 may than the for recognition of a PY-NLS by human is to that the Hrp1 does not mediate proper Hrp1 is in to the nuclear For proteins that nuclear the C-terminal may not sufficient for proper even in for many a nuclear presence to may sufficient for the protein to its in the we that the minimal PY-NLS in yeast consists of the C-terminal portion of the human PY-NLS, R/H/KX2–5PY, with upstream basic or hydrophobic residues enhancing the of the NLS to mediate nuclear import. the in the of the consensus sequence for the PY-NLS, used in PY-NLS However, a preliminary of the of the PY-NLS in yeast using the consensus sequence for the minimal yeast PY-NLS as well as the consensus sequences for basic and hydrophobic that of the S. cerevisiae proteome and of the proteins that to the nucleus or contain a putative this analysis that Hrp1 contain a putative PY-NLS, and, show that this PY-NLS is functional in vivo. In contrast, the PY-NLS-like sequences in are not identified in this we only the hydrophobic or basic PY-NLS consensus of the yeast proteome and of the nuclear proteins contain one For of the yeast proteome and of nuclear proteins contain a putative classical NLS (12Lange A. Mills R.E. Lange C.J. Stewart M. Devine S.E. Corbett A.H. J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). the of putative proteins is than the of putative the of putative yeast PY-NLS cargos is of the yeast proteins that are contain a putative hydrophobic or basic PY-NLS, and lack a cNLS that are in or Kapβ2 cargos are also in in (16Lee B.J. Cansizoglu A.E. Süel K.E. Louis T.H. Zhang Z. Chook Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the that nuclear import a PY-NLS may a to in However, functional of PY-NLS cargos are required we can to the of this import to the nuclear proteome in vivo. We for of the and for of the for the of the for the of the and for the of the