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-␣-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-␣/␤ 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.CDK ͉ computational method ͉ nuclear localization signal ͉ nuclear import ͉ phosphorylation R egulated nuclear import controls the nuclear function of many proteins that shuttle between the nucleus and cytoplasm or other cellular compartments in response to certain physiological conditions or extracellular stimuli (1). Nucleocytoplasmic shuttling comprises both nuclear import and nuclear export activities. Nuclear import is mediated by the interaction of nuclear localization signals (NLSs) with transport receptor importins, among which the importin-␣ family recognizes most major classes of NLSs with 1 or 2 basic stretches, called the classical NLSs (2-4). Six different classes of monopartite and bipartite NLSs bind to distinct binding grooves of importin-␣ (5-7). Nuclear export is mediated by nuclear export signals (NESs), which are recognized by Crm1/exportin or Msn5 in yeast (1, 8). The regulation of these 2 antagonistic activities directs a protein to either the nucleus or the cytoplasm. In general, these activities are regulated by the modification of the NLSs/NESs, by intermolecular or intramolecular interactions that lead to the structural masking of NLSs/NESs, or by the supply of an NLS/NES by another protein (9).Phosphorylation within or around an NLS is the usual strategy for the regulated nuclear transport of a protein (4, 10-13). It has been shown that acidic amino acids adjacent to the basic core ...

Landscape of nuclear transport receptor cargo specificity

“…We therefore wanted to exploit our large‐scale data set in this regard. We used cNLS Mapper (Kosugi et al , 2009) to predict potential signals in the cargos selected for validation and detected these in INTS11, EXOSC10, and RPC3 (Table EV6). We also used the DILIMOT algorithm (Neduva et al , 2005) and simple regular expression pattern matching to extract short linear motifs in an unbiased way.…”

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

“…Next, we introduced point mutations into a predicted importin α-dependent nuclear-localization signal (NLS) in murine ULK1 (aa261-289) that was identified using the cNLS Mapper (Supplementary Figure S7a). 40 Individual arginine (R) and lysine (K) residues within the 29-residue NLS were replaced with alanines (A) and the resulting mutants were screened for their ability to disrupt ULK1's nuclear localization. Immunofluorescence microscopy of transiently transfected 293T cells revealed that unlike WT ULK1, the R266A-ULK1 mutant was excluded from the nuclei, even after H 2 O 2 treatment, and did not alter nuclear localization of PARP1-GFP (Figure 6c).…”

The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus

“…Next, we screened for an NLS in DPP9-l using the cNLS mapper program, which predicts importin α-dependent nuclear localization signals [40]. Using this program we identified a putative classical monopartite NLS: 2-RKVK-KLRL-9 (consensus sequence = K(K/R)X(K/R), which is located in the amino terminal extension of DPP9-l (Fig.…”

“…Silencing experiments show that DPP9 and not DPP8 is rate-limiting for the hydrolysis of prolinecontaining peptides in the cytosol suggesting that DPP9 may have a house-keeping role for amino acid homeostasis [28]. the first identified endogenous substrate of DPP9 was the tumor-related epitope RU1 [34][35][36][37][38][39][40][41][42] . Its hydrolysis by DPP9 results in its limited presentation on MhC class I molecules to the immune system linking DPP9 to the MhC class I antigen presentation pathway [28][29][30].…”