Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

Abstract

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-alpha-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-alpha/beta pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.

Fig. 1.

Activity-based profiles of classical and noncanonical classes of NLSs optimized for yeast. (A) A profile of a classical class 2 monopartite NLS. A single amino acid residue within the template sequence indicated at the top in the matrix was replaced with various other residues indicated in the left column, and the nuclear import activity was assayed in yeast. Activity scores were determined as in Fig. S1 -GUS-GFP reporters fused to NLSs with scores (1 and 2), (–5), (6 and 7), and (–10) exhibit nuclear, partially nuclear, nuclear plus cytoplasmic, and cytoplasmic phenotypes, respectively. This template NLS has an activity score of 4, a middle level of NLS activity. Scores with higher, slightly higher, and lower activities than an average value for each position are shown in red, orange, and blue, respectively. At several mutational positions, modified templates with a different level of basal activity were used to obtain more dispersed scores. Blanks represent undetermined scores. A value that may overlap the scores of class 4 NLSs is given in parentheses. (B) A profile of noncanonical class 3 monopartite NLS. Values that may overlap the scores of class 2 NLS are given in parentheses. (C) A profile of a noncanonical class 4 monopartite NLS.

Fig. 2.

Previously unidentified cell cycle-regulated nucleocytoplasmic shuttling proteins. (A) Different patterns of importin-α-dependent NLSs overlapping the consensus CDK site. The core basic residues of the monopartite and bipartite NLSs and potential CDK phosphorylation sites are marked in red and blue, respectively. “B” represents Lys or Arg. (B) List of previously unidentified cell cycle-regulated nucleocytoplasmic shuttling proteins. (C) Cell cycle-regulated nuclear transport depending on predicted CDK1 sites. The subcellular localization of the indicated GFP-fused proteins was observed in exponentially growing (Asyn, asynchronous) and synchronized yeast cells (α-factor, arrested in G₁ with α-factor; Release, cultured for 1 h after release of G₁ arrest; Left). The previously reported Acm1 and Whi5 were analyzed to confirm our predicted NLS sites. The potential CDK phosphorylation sites within and around a predicted NLS were converted to alanine, and these mutants were similarly analyzed (Right). GFP-Pds1 was expressed from a galactose-inducible pYES-GFP vector to alleviate its overexpression-related toxic effects, and the induced yeast cells were arrested by 3-h incubation with nocodazole instead of the release of G₁ arrest. The other GFP fusion proteins were expressed from a constitutive expression vector, pGAD-GFP or pAUA-GFP. (D) Cell cycle-regulated nuclear transport depends on CDK1 activity. cdc28-as1 cells expressing the GFP fusions of the indicated proteins were synchronized in S or G₂ by arrest and release with the α-factor in the presence or absence of 1 μM 1NM-PP1, a specific inhibitor of Cdc28p-as1.

Fig. 3.

CDK1-regulated nucleocytoplasmic shuttling involves nuclear export activities mediated by different export receptors. Subcellular localization of GFP-fused proteins with the indicated gene products was observed in exponentially growing cells of either the wild type, an msn5Δ mutant, or a Crm1-T539C strain treated with leptomycin B. The results for which there was no observed difference between the wild-type and mutant strains are not shown.