Many of the selected sequences exhibited a significant level of nuclear import activity and could be classified into five classes of monopartite NLS when aligned ( Fig. ), we selected 218 and 253 sequences from 12X and XBBX IVV libraries, which could encode monopartite NLSs, after five and six rounds of screening, respectively. The optimal round was selected to satisfy both high sequence diversity and high rate of functional NLS in the pool from the respective rounds.
At least 10 clones from each screening round (except for the first round) were sequenced and assayed for nuclear import activity. Finally, the amplified DNAs were digested with BamHI and XbaI and cloned into the pTUE-GFP3 vector for sequencing and transfection assay. As the screening cycle was increased, the amount of library and the amplification cycles of PCR were decreased to 10–20 pmol and 23–25 cycles, respectively. The amplified DNA fragments were purified with a QIAquick PCR purification kit (Qiagen) and used as a library for the subsequent round of screening. The selected DNAs in the eluate were PCR-amplified with SP6-SD2 and Flag2 primers ( supplemental Table S1) and HotStarTaq DNA polymerase (Qiagen). The volume of the washing buffer was increased to 600–800 μl with each cycle of the screening. These columns were sequentially washed with 200 μl of binding buffer, 200 μl of washing buffer (20 m m Hepes-NaOH (pH 7.8), 0.1 m NaCl, 1 m m EDTA, 0.2% Nonidet P-40), and 200 μl of washing buffer lacking NaCl and eluted with 30 μl of 20 m m glutathione and 20 m m maltose. For the rice maltose-binding protein-importin α (10 μg) the incubation with the library was conducted in solution for 30 min at room temperature, and the solution was repeatedly passed through a 200-μl pipette tip column containing 25 μl of amylose resin. Library Screening-The random peptide IVV library (40 pmol) of cDNA/mRNA-peptide fusion was diluted in 60 μl of binding buffer (20 m m Hepes-NaOH (pH 7.8), 0.15 m NaCl, 1 m m EDTA, 0.2% Nonidet P-40, 0.5% skim milk) and incubated for 30 min with the rice, human, and yeast importin α (20 μg) fused with GST, which had been bound to 20–25 μl of glutathione beads packed in a minicolumn made of a 200-μl pipette tip. The defined consensus patterns and properties of importin α-dependent NLSs provide useful information for identifying NLSs. These results explain the causes of the NLS diversity. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin α, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin α and identified six classes of NLSs, including three novel classes. We report here six different NLS classes that specifically bind to distinct binding pockets of importin α. Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. The importin α/β pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Glycobiology and Extracellular Matrices.
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