Figure 12–10 Visualizing active import through NPCs. This series of electron micrographs shows colloidal gold spheres (arrowheads) coated with peptides containing nuclear localization signals entering the nucleus through npCs. The gold particles were injected into the cytosol of living cells, which then were fixed and prepared for electron microscopy at various times after injection. (a) Gold particles are first seen in proximity to the cytosolic fibrils of the npCs. (B, C) They are then seen at the center of the npCs, exclusively on the cytosolic face. (D) They then appear on the nuclear face. These gold particles have much larger diameters than the diffusion channels in the npC and are imported by active transport. (from n. panté and U. aebi, Science 273:1729–1732, 1996. With permission from aaas.) system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. These receptors bind to both the export signal and NPC proteins to guide their cargo through the NPC to the cytosol.
Many nuclear export receptors are structurally related to nuclear import receptors, and they are encoded by the same gene family of nuclear transport receptors, or karyopherins. In yeast, there are 14 genes encoding karyopherins; in animal cells, the number is significantly larger. It is often not possible to tell from their amino acid sequence alone whether a particular family member works as a nuclear import or nuclear export receptor. As might be expected, therefore, the import and export transport systems work in similar ways but in opposite directions: the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse, while the export receptors function in the opposite fashion.
The ran GTpase imposes Directionality on Transport Through npCs
The import of nuclear proteins through NPCs concentrates specific proteins in the nucleus and thereby increases order in the cell. The cell fuels this ordering process by harnessing energy stored in concentration gradients of the GTP-bound form of the monomeric GTPase Ran, which is required for both nuclear import and export.
Like other GTPases, Ran is a molecular switch that can exist in two conformational states, depending on whether GDP or GTP is bound (discussed in Chapter 3). Two Ran-specific regulatory proteins trigger the conversion between the two states: a cytosolic GTPase-activating protein (GAP) triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) promotes the exchange of GDP for GTP and thus converts Ran-GDP to Ran-GTP. Because Ran-GAP is located in the cytosol and Ran-GEF is located in the nucleus where it is anchored to chromatin, the cytosol contains mainly Ran-GDP, and the nucleus contains mainly Ran-GTP (Figure 12–12).
Figure 12–11 Nuclear import receptors (importins). (a) Different nuclear import receptors bind different nuclear localization signals and thereby different cargo proteins. (B) Cargo protein 4 requires an adaptor protein to bind to its nuclear import receptor. The adaptors are structurally related to nuclear import receptors and recognize nuclear localization signals on cargo proteins. They also contain a nuclear localization signal that binds them to an import receptor, but this signal only becomes exposed when they are loaded with a cargo protein.
Figure 12–12 The compartmentalization of Ran-GDP and Ran-GTP. localization of ran-GDp in the cytosol and ran-GTp in the nucleus results from the localization of two ran regulatory proteins: ran GTpaseactivating protein (ran-Gap) is located in the cytosol, and ran guanine nucleotide exchange factor (ran-Gef) binds to chromatin and is therefore located in the nucleus.