Because many cell proteins are too large to diffuse passively through the NPCs, the nuclear compartment and the cytosol can maintain different protein compositions. Mature cytosolic ribosomes, for example, are about 30 nm in diameter and thus cannot diffuse through the NPC, confining protein synthesis to the cytosol. But how does the nucleus export newly made ribosomal subunits or import large molecules, such as DNA polymerases and RNA polymerases, which have subunit molecular masses of 100,000–200,000 daltons? As we discuss next, these and most other transported protein and RNA molecules bind to specific receptor proteins that actively ferry large molecules through NPCs. Even small proteins like histones frequently use receptor-mediated mechanisms to cross the NPC, thereby increasing transport efficiency.
nuclear localization signals Direct nuclear proteins to the nucleus
When proteins are experimentally extracted from the nucleus and reintroduced into the cytosol, even the very large ones reaccumulate efficiently in the nucleus. Sorting signals called nuclear localization signals (NLSs) are responsible for the selectivity of this active nuclear import process. The signals have been precisely defined by using recombinant DNA technology for numerous nuclear proteins, as well as for proteins that enter the nucleus only transiently (Figure 12–9). In many nuclear proteins, the signals consist of one or two short sequences that are rich in the positively charged amino acids lysine and arginine (see Table 12–3, p. 648), with the precise sequence varying for different proteins. Other nuclear proteins contain different signals, some of which are not yet characterized.
Nuclear localization signals can be located almost anywhere in the amino acid sequence and are thought to form loops or patches on the protein surface. Many function even when linked as short peptides to lysine side chains on the surface of a cytosolic protein, suggesting that the precise location of the signal within the amino acid sequence of a nuclear protein is not important. Moreover, as long as one of the protein subunits of a multicomponent complex displays a nuclear localization signal, the entire complex will be imported into the nucleus.
Figure 12–7 The nuclear envelope. The double-membrane envelope is penetrated by pores in which nuclear pore complexes (not shown) are positioned. The outer nuclear membrane is continuous with the endoplasmic reticulum (er). The ribosomes that are normally bound to the cytosolic surface of the er membrane and outer nuclear membrane are not shown. The nuclear lamina is a fibrous protein meshwork underlying the inner membrane.
One can visualize the transport of nuclear proteins through NPCs by coating gold particles with a nuclear localization signal, injecting the particles into the cytosol, and then following their fate by electron microscopy (Figure 12–10). The particles bind to the tentaclelike fibrils that extend from the scaffold nucleoporins at the rim of the NPC into the cytosol, and then proceed through the center of the NPC. Presumably, the unstructured regions of the nucleoporins that form a diffusion barrier for large molecules (mentioned earlier) are pushed away to allow the coated gold particles to squeeze through.
Macromolecular transport across NPCs differs fundamentally from the transport of proteins across the membranes of other organelles, in that it occurs (B) 0.1 µm inner nuclear membrane nuclear envelope outer nuclear membrane 50 nm scaffold nucleoporins membrane ring proteins channel nucleoporinsnuclear basket disordered region of channel nucleoporins cytosolic fbrils 0.1 µm 0.1 µm