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

Abstract

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 ␣. 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 ␤2/transportin (Kap␤2) 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 Kap␤2 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/KX 2-5 PY, 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 (1-3). Nuclear pores prevent passive diffusion of macromolecules larger than ϳ40 kDa (4, 5), 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 ␤ transport receptor superfamily, which has 14 members in Saccharomyces cerevisiae and over 20 members in humans (6). These import receptors recognize protein cargo bearing targeting sequences called nuclear localization signals (NLSs) 2 and mediate interactions with the nuclear pore, allowing translocation of the cargo into the nucleus (7). Once inside the nucleus, the import complex encounters RanGTP, a small Ras family GTPase that regulates the directionality of nuclear transport (8). RanGTP binds to the karyopherin ␤, causing a conformational ch...

Nuclear localization signals and human disease

“…Using these guidelines, the human proteome was searched and 81 potential cargoes for Kapb2 were identified, (49,50) some of which they demonstrated interact directly with Kapb2 in vitro (35). A follow-up study provided evidence that the PY-NLS motif is conserved in budding yeast in the RNA binding protein, Hrp1 (Table 1), and also that the PY-NLS functions as a nuclear targeting signal in vivo (36).…”

“…Although cNLS motifs likely mediate the majority of nuclear protein import (36), it is critical to note that there are many additional transport routes facilitated by nonclassical transport pathways. Hence, there are likely many unidentified and undefined NLS motifs that interact with various transport receptors.…”

Nucleocytoplasmic Distribution Is Required for Activation of Resistance by the Potato NB-LRR Receptor Rx1 and Is Balanced by Its Functional Domains

“…It should be noted that in yeast, only 57% of the nuclear proteins contains a classical NLS (Lange et al, 2007). The remaining 43% of nuclear proteins is thought to pass the nuclear pore complex via alternative mechanisms, like direct interactions with importin b or nucleoporins, nonclassical nuclear targeting signals, or cotransport via interactions with proteins that do contain NLSs, the socalled piggyback mechanism (Ursula Stochaj, 1999;Christophe et al, 2000;Dostie et al, 2000;Cingolani et al, 2002;Xu and Massagué , 2004;Lee et al, 2006;Chuderland et al, 2008;Lange et al, 2008).…”

Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export

“…Consistent with this hypothesis, we showed that recombinant Nab2-N-F73D does not bind to Mlp1, whereas Nab2-N-F72D is still competent to bind Mlp1, in an in vitro bead binding assay. To assess the impact of amino acid substitutions at Nab2 Phe 72 or Phe 73 on the relative binding affinity of Nab2 for Mlp1, we measured the binding of S-Tag-Mlp1-NBD to GST-Nab2-N mutants F72D, F73D, or F73W at increasing concentrations of S-TagMlp1-NBD in the solid-phase binding assay. We find that the interaction between Nab2-N-F73D and Mlp1-NBD is undetectable in this assay, whereas Mlp1-NBD binds to Nab2-N-F72D with roughly 2-fold lower relative affinity (K d ϳ 2.3 Ϯ 1.1 M) compared with wild-type (Fig.…”