Gap

interactions between NTF2 and the FG repeats. When this complex reaches the nucleoplasm, it encounters a specific guanine nucleotide-exchange factor (Ran-GEF) that causes Ran to release its bound GDP and rebind GTP that is present at much higher concentration. The resulting conformational change in Ran decreases its affinity for NTF2 so that free Ran-GTP is released into the nucleoplasm and NTF2 is free to diffuse back through the pore.

The import complex travels through the pore by diffusion, a random process. Yet transport is unidirectional. The direction of transport is a consequence of the rapid dissociation of the import complex when it reaches the nu-cleoplasm. As a result, there is a concentration gradient of the importin-cargo complex across the NPC: high in the cytoplasm where the complex assembles and low in the nucleoplasm where it dissociates. This concentration gradient is responsible for the unidirectional nature of nuclear import. A similar concentration gradient is responsible for driving the importin in the nucleus back into the cytoplasm. The concentration of the importin-Ran-GTP complex is higher in the nucleoplasm, where it assembles, than on the cytoplasmic side of the NPC, where it dissociates. Ultimately, the direction of the transport processes is dependent on the asymmetric distribution of the Ran-GEF and the Ran-GAP. Ran-GEF in the nucleoplasm maintains Ran in the Ran-GTP state, where it promotes dissociation of the cargo complex. Ran-GAP on the cytoplasmic side of the NPC converts Ran-GTP to Ran-GDP, dissociating the importin-Ran-GTP complex and releasing free importin into the cytosol.

▲ EXPERIMENTAL FIGURE 12-22 The movement of human hnRNP A1 protein between nuclei in a heterokaryon shows that it can cycle in and out of the cytoplasm, but human hnRNP C protein, which showed no such movement, cannot. Cultured HeLa cells and Xenopus cells were fused by treatment with polyethylene glycol, producing heterokaryons containing nuclei from each cell type. The hybrid cells were treated with cycloheximide immediately after fusion to prevent protein synthesis. After 2 hours, the cells were fixed and stained with fluorescent-labeled antibodies specific for human hnRNP C and A1 proteins. These antibodies do not bind to the homologous Xenopus proteins. (a) A fixed preparation viewed by phase-contrast microscopy includes unfused HeLa cells (arrowhead) and Xenopus cells (dotted arrow), as well as fused heterokaryons

Exportins Transport Proteins Containing Nuclear-Export Signals out of the Nucleus

A very similar mechanism is used to export proteins, tRNAs, and ribosomal subunits from the nucleus to the cytoplasm. This mechanism initially was elucidated from studies of certain hnRNP proteins that "shuttle" between the nucleus and cytoplasm. For instance, the cell-fusion experiments described in Figure 12-22 first showed that some hnRNP proteins cycle in and out of the cytoplasm, whereas others remain localized in the nucleus. Such "shuttling" proteins contain a nuclear-export signal (NES) that stimulates their export from the nucleus to the cytoplasm through nuclear pores, in addition to an NLS that results in their reuptake into the nucleus. Experiments with engineered hybrid genes encoding a nucleus-restricted protein fused to various segments of a protein that shuttles in and out of the nucleus have identified at least three different classes of NESs: a leucine-rich sequence found in PKI (an inhibitor of protein kinase A) and in the Rev protein of human immunodeficiency virus (HIV), a 38-residue sequence in hnRNP A1, and a sequence in hnRNP K. To date, no functionally significant structural features (e.g., repeated amino acids) have been identified in the latter two classes.

The mechanism whereby shuttling proteins are exported from the nucleus is best understood for those containing a leucine-rich NES. According to the current model shown in Figure 12-23, a specific nuclear-export receptor in the nucleus, exportin 1, first forms a complex with Ran-GTP and then binds the NES in a cargo protein. Binding of exportin 1

(solid arrow). In the heterokaryon in this micrograph, the round HeLa-cell nucleus is to the right of the oval-shaped Xenopus nucleus. (b, c) When the same preparation was viewed by fluorescence microscopy, the stained hnRNP C protein appeared green and the stained hnRNP A1 protein appeared red. Note that the unfused Xenopus cell on the left is unstained, confirming that the antibodies are specific for the human proteins. In the heterokaryon, hnRNP C protein appears only in HeLa-cell nuclei (b), whereas the A1 protein appears in both nuclei (c). Since protein synthesis was blocked after cell fusion, some of the human hnRNP A1 protein must have left the HeLa-cell nucleus, moved through the cytoplasm, and entered the Xenopus nucleus in the heterokaryon. [See S. Pinol-Roma and G. Dreyfuss, 1992, Nature 355:730; courtesy of G. Dreyfuss.]

Ran . GDP Ran . GTP Exportin 1 Cargo

P P NES

Cargo complex u

Nucleoplasm

Cytoplasm

Nucleoplasm

Cytoplasm

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