A classic experiment that addressed this problem used several forms of radioactive nucleoplasmin, which is a large pentameric protein involved in chromatin assembly. In this experiment, either the intact protein or the nucleoplasmin heads, tails, or heads with a single tail were injected into the cytoplasm of a frog oocyte or into the nucleus (Figure Q12–1). All forms of nucleoplasmin, except heads, accumulated in the nucleus when injected into the cytoplasm, and all forms were retained in the nucleus when injected there.
A. What portion of the nucleoplasmin molecule is responsible for localization in the nucleus?
nucleoplasmin nuclear cytoplasmic Figure Q12–1 Cellular preparation injection injection location of injected nucleoplasmin components (problem 12–7). schematic diagrams of autoradiographs show the cytoplasm and nucleus with the location of nucleoplasmin indicated by the red areas.
B. How do these experiments distinguish between active transport, in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion, in which a binding site for a nuclear component allows accumulation in the nucleus?
12–8 Assuming that 32 million histone octamers are required to package the human genome, how many his-tone molecules must be transported per second per nuclear pore complex in cells whose nuclei contain 3000 nuclear pores and are dividing once per day?
12–9 The nuclear pore complex (NPC) creates a barrier to the free exchange of molecules between the nucleus and cytosol, but in a way that remains mysterious. In yeast, for example, the central pore of the NPC has a diameter of 35 nm and is 30 nm long, which is somewhat smaller than its vertebrate counterpart. Even so, it is large enough to accommodate virtually all components of the cytosol. Yet the pore allows passive diffusion of molecules only up to about 40 kd; entry of anything larger requires help from a nuclear import receptor. Selective permeability is controlled by protein components of the NPC that have unstructured, polar tails extending into the central pore. These tails are characterized by periodic repeats of the hydrophobic amino acids phenylalanine (F) and glycine (G).
At high enough concentration (~50 mM), the FG-repeat domains of these proteins can form a gel, with a meshwork of interactions between the hydrophobic FG repeats (Figure Q12–2A). These gels allow passive diffusion of small molecules, but they prevent entry of larger proteins such as the fluorescent protein mCherry fused to maltose binding protein (MBP) (Figure Q12–2B). (The fusion to MBP makes mCherry too large to enter the nucleus by passive diffusion.) However, if the nuclear import receptor, importin, is fused to a similar protein, MBP-GFP, the importin-MBP-GFP fusion readily enters the gel (Figure Q12–2B).
Figure Q12–2 fG-repeat gel and influx of proteins into the nucleus (problem 12–9). (a) Cartoon of the meshwork (gel) formed by pairwise interactions between hydrophobic fG repeats. for fG-repeats separated by 17 amino acids, as is typical, the mesh formed by extended amino acid side chains would correspond to about 4 nm on a side, which would be large enough to account for the characteristic passive diffusion of proteins through nuclear pores. (B) Diffusion of mBp-mCherry and importin-mBp-Gfp into a gel of fG-repeats. in each group, the solution is shown at left and the gel at right. The bright areas indicate regions that contain the fluorescent proteins.