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 alpha. 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 beta2/transportin (Kapbeta2) 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 Kapbeta2 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.

FIGURE 1.

Hrp1 and Nab2 contain putative PY-NLS-like sequences. A, domain structures of Hrp1 and Nab2. The positions of the PY-NLS-like sequence(s) are indicated by asterisks, and sequences are listed below each protein. B, nuclear localization of Hrp1 and Nab2 is dependent on Kap104. Wild-type and ΔKAP104 cells expressing GFP-Hrp1, Nab2-GFP, or the control protein Nop1-GFP were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown.

FIGURE 2.

The PY-NLS-like sequence within Hrp1 is necessary and sufficient for Hrp1 import and is necessary for Kap104 binding. A, wild-type cells expressing GFP alone, wild-type GFP-Hrp1, P531A/Y532A GFP-Hrp1, or R525A/P531A/Y532A GFP-Hrp1 were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown. B, in vitro binding between GST-Kap104 and either wild-type GFP-Hrp1 or R525A/P531A/Y532A GFP-Hrp1 was examined using glutathione beads as described under “Experimental Procedures.” The unbound (U) and bound (B) fractions were probed with an anti-GFP antibody to detect GFP-Hrp1 fusion proteins or with an anti-GST antibody to detect GST-Kap104. C, wild-type cells expressing a GFP-GFP control, GFP-GFP-Hrp1-(522–534) (containing the Hrp1 PY-core), or GFP-GFP-Hrp1-(503–534) (containing the entire Hrp1 PY-NLS) were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown.

FIGURE 3.

The PY-NLS-like sequences within Nab2 are neither necessary nor sufficient for Nab2 nuclear localization. A, wild-type cells expressing GFP alone, wild-type Nab2-GFP, P332A Nab2-GFP, P407A Nab2-GFP, or P332A/P407A Nab2-GFP were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown. B, wild-type cells expressing a GFP-GFP control, GFP-GFP-Nab2-(320–333), or GFP-GFP-Nab2-(389–408) were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown.

FIGURE 4.

The PY-NLS-like sequence within Hrp1 is required for protein function. Protein function in vivo was assessed by a plasmid shuffle assay as described under “Experimental Procedures.” A, ΔHRP1 cells (ACY1571) maintained by a plasmid encoding wild-type Hrp1 and expressing either wild-type or mutant Hrp1 proteins were serially diluted, spotted onto control or 5-FOA plates, and grown at 30 °C for 3 days. B, ΔNAB2 cells (ACY427) maintained by a plasmid encoding wild-type Nab2 and expressing either wild-type or mutant Nab2 proteins were serially diluted, spotted onto control or 5-FOA plates, and grown at 30 °C for 3 days.

FIGURE 5.

Functional dissection of the PY-NLS motif. A, wild-type cells expressing GFP alone, wild-type GFP-Hrp1, GFP-Hrp1 P531A/Y532A, GFP-Hrp1 Y532V, or GFP-Hrp1 P531A were examined by direct fluorescence microscopy (GFP). Corresponding differential interference contrast (DIC) images are shown. B, ΔHRP1 cells (ACY1571) maintained by a plasmid encoding wild-type Hrp1 and expressing either wild-type or mutant Hrp1 protein were spotted onto control or 5-FOA plates and grown at 30 °C for 3 days. C, wild-type Hrp1 or P531A Hrp1 cells were monitored for growth over time at 37 °C as described under “Experimental Procedures.”

FIGURE 6.

The prevalence of predicted PY-NLS proteins in S. cerevisiae. Algorithms for hydrophobic and basic PY-NLSs or for the C-terminal PY-NLS core motif (see “Experimental Procedures”) were used to search the 5,850 proteins in the yeast proteome (Proteome) and the 1,515 proteins that are nuclear or nucleolar at steady state (Nuclear). The results of this preliminary analysis are plotted in a Venn diagram and summarized in the chart below. Hydrophobic and basic PY-NLSs, by definition, also contain the C-terminal PY core motif, so proteins denoted as containing “Hydrophobic”or“Basic” PY-NLSs also contain the PY-NLS core.