Nuclear localization signal binding proteins in higher plant nuclei.
The import of proteins into the nucleus is a vital process that is mediated by proteins which specifically recognize nuclear localization signals (NLSs). These factors have not been identified in plants. Previously, we demonstrated that higher plants possess a low-affinity binding site at the nuclear pore that specifically binds to several classes of functional NLSs. By the use of crosslinking reagents and a radiolabeled peptide to the bipartite NLS from the endogenous plant transcription factor Opaque2, two NLS binding proteins (NBPs) of 50-60 kDa and at least two NBPs of 30-40 kDa were identified. Competition studies indicated that labeling was specific for the functional NLS but not a mutant NLS impaired in vivo or a peptide unrelated to NLSs. Also, the apparent dissociation constant (100-300 microM) for labeling was similar to that of the binding site. Proteins of similar mass were labeled with two different crosslinking reagents, and concentration and time studies indicated that these NBPs were distinct proteins and not aggregates. Treatment with salt, detergent, or urea before or during NLS binding demonstrated that the properties of the binding site and the NBPs were identical. This tight correlation strongly indicates that some or all of the NBPs constitute the nuclear pore binding site. Overall, our results indicate that some components of NLS recognition are located at the nuclear pores in higher plants.
Hicks
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Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
Raikhel
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D000595:Amino Acid Sequence; D000957:Antigens, Viral, Tumor / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry; D010947:Plants, Toxic; D013388:Succinimides; D014026:Nicotiana; D014157:Transcription Factors / Q000737:chemistry; D003313:Zea mays
D000957:Antigens, Viral, Tumor; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C075564:NOLC1 protein, human; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D010940:Plant Proteins; D013388:Succinimides; D014157:Transcription Factors; C067402:opaque-2 protein, Zea mays; C019358:disuccinimidyl suberate