| Abstrakt: |
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. |
