ABSTRACT
Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import.
Subject(s)
Cell Nucleus/metabolism , Microtubules/ultrastructure , Nuclear Localization Signals/chemistry , Protein Sorting Signals , Actins/chemistry , Active Transport, Cell Nucleus , Animals , Axons/metabolism , Biological Transport , Blotting, Western , Cell-Free System , Centrosome/ultrastructure , Cytoplasm/metabolism , Cytoskeleton/chemistry , Cytoskeleton/metabolism , DNA, Single-Stranded/chemistry , Diffusion , Dyneins/chemistry , Female , Immunoblotting , Karyopherins/chemistry , Kinesins/chemistry , Microscopy, Confocal , Microtubules/metabolism , Mutation , Time Factors , Xenopus laevis , beta Karyopherins/chemistryABSTRACT
Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.
