Macromolecular Transport Across the Nuclear Envelope

Once the processing of an mRNA is completed in the nucleus, it remains associated with specific hnRNP proteins in a messenger ribonuclear protein complex, or mRNP. Before it can be translated into the encoded protein, it must be exported out of the nucleus into the cytoplasm. The nucleus is separated from the cytoplasm by two membranes, which form the nuclear envelope (see Figure 5-19). Like the plasma membrane surrounding cells, each nuclear membrane consists of a water-impermeable phospholipid bilayer and vari ous associated proteins. Transport of macromolecules including mRNPs, tRNAs, and ribosomal subunits out of the nucleus and transport of all nuclear proteins translated in the cytoplasm into the nucleus occur through nuclear pores in a process that differs fundamentally from the transport of small molecules and ions across other cellular membranes (Chapter 7). Insight into the mechanisms of transport through nuclear pores first came from studies of the nuclear pores and of import and export of individual proteins, which we consider first, before returning to the export of mRNPs.

Large and Small Molecules Enter and Leave the Nucleus via Nuclear Pore Complexes

Numerous pores perforate the nuclear envelope in all eu-karyotic cells. Each nuclear pore is formed from an elaborate structure termed the nuclear pore complex (NPC), which is immense by molecular standards, «125 million daltons in vertebrates, or about 30 times larger than a ribosome. An NPC is made up of multiple copies of some 50 (in yeast) to 100 (in vertebrates) different proteins called nucleoporins. Electron micrographs of nuclear pore complexes reveal a roughly octagonal, membrane-embedded structure from which eight «100-nm-long filaments extend into the nucleoplasm (Figure 12-18). The distal ends of these filaments are joined by the terminal ring, forming a structure called the

Spoke

Outer nuclear membrane

Nuclear envelope

Inner nuclear membrane

Cytoplasmic filaments

Proximal filaments

Central transporter Cytoplasmic ring

Spoke

Cytoplasmic filaments

Nuclear envelope

Inner nuclear membrane

Central transporter Cytoplasmic ring

Outer spoke ring

Inner spoke ring Nucleoplasmic ring

Nuclear basket

Outer spoke ring

Inner spoke ring Nucleoplasmic ring

Nuclear basket

▲ FIGURE 12-18 Nuclear pore complex. (a) Nuclear envelopes microdissected from the large nuclei of Xenopus oocytes visualized by field emission in-lens scanning electron microscopy. Top: View of the cytoplasmic face reveals octagonal shape of membrane-embedded portion of nuclear pore complexes. Bottom: View of the nucleoplasmic face shows the nuclear basket that extends from the membrane portion. (b) Cut-away model of the pore complex. [Part (a) from V Doye and E. Hurt, 1997, Curr. Opin. Cell Biol. 9:401; courtesy of M. W. Goldberg and T D. Allen. Part (b) adapted from M. P Rout and J. D. Atchison, 2001, J. Biol. Chem. 276:16593.]

nuclear basket. The membrane-embedded portion also is attached directly to the nuclear lamina, a network of lamin intermediate filaments that extends over the inner surface of the nuclear envelope (see Figure 21-16). Cytoplasmic filaments extend from the cytoplasmic side of the NPC into the cytosol.

Ions, small metabolites, and globular proteins up to =60 kDa can diffuse through a water-filled channel in the nuclear pore complex; these channels behave as if they are =0.9 nm in diameter. However, large proteins and ribonucleoprotein complexes cannot diffuse in and out of the nucleus. Rather, these macromolecules are selectively transported in and out of the nucleus with the assistance of soluble transporter proteins that bind macromolecules and also interact with certain nucleoporins. The principles underlying macromolecular transport through nuclear pore complexes were first determined for the import of individual proteins into the nucleus, which we discuss first before turning to the question of how fully processed mRNAs are transported into the cytoplasm.

Importins Transport Proteins Containing Nuclear-Localization Signals into the Nucleus

All proteins found in the nucleus are synthesized in the cytoplasm and imported into the nucleus through nuclear pore complexes. Such proteins contain a nuclear-localization signal (NLS) that directs their selective transport into the nucleus. NLSs were first discovered through the analysis of mutants of simian virus 40 (SV40) that produced an abnormal form of the viral protein called large T-antigen. The wild-type form of this protein is localized to the nucleus in virus-infected cells, whereas some mutated forms of large Tantigen accumulate in the cytoplasm. The mutations responsible for this altered cellular localization all occur within a specific seven-residue sequence rich in basic amino acids near the C-terminus of the protein: Pro-Lys-Lys-Lys-Arg-Lys-Val. Experiments with engineered hybrid proteins in which this sequence was fused to a cytosolic protein demonstrated that it directs transport into the nucleus, and consequently functions as an NLS (Figure 12-19). NLS sequences subsequently were identified in numerous other proteins imported into the nucleus. Many of these are similar to the basic NLS in SV40 large T-antigen, whereas other NLSs are chemically quite different. For instance, an NLS in the hnRNP A1 protein is hydrophobic.

Early work on the mechanism of nuclear import focused on proteins containing a basic NLS, similar to the one in SV40 large T-antigen. A digitonin-permeabilized cell system provided an in vitro assay for analyzing soluble cytosolic components required for nuclear import (Figure 12-20). Using this assay system, four required proteins were purified: Ran, nuclear transport factor 2 (NTF2), importin a, and importin p. Ran is a monomeric G protein that exists in two conformations, one when complexed with GTP and an alternative one when the GTP is hy-drolyzed to GDP (see Figure 3-29). The two importins

▲ EXPERIMENTAL FIGURE 12-19 Fusion of a nuclear-localization signal (NLS) to a cytoplasmic protein causes the protein to enter the cell nucleus. (a) Normal pyruvate kinase, visualized by immunofluorescence after treating cultured cells with a specific antibody (yellow), is localized to the cytoplasm. This very large cytosolic protein functions in carbohydrate metabolism. (b) When a chimeric pyruvate kinase protein containing the SV40 NLS at its N-terminus was expressed in cells, it was localized to the nucleus. The chimeric protein was expressed from a transfected engineered gene produced by fusing a viral gene fragment encoding the SV40 NLS to the pyruvate kinase gene. [From D. Kalderon et al., 1984, Cell 39:499; courtesy of Dr. Alan Smith.]

form a heterodimeric nuclear-import receptor: the a subunit binds to a basic NLS in a "cargo" protein to be transported into the nucleus, and the p subunit interacts with a class of nucleoporins called FG-nucleoporins. These nu-cleoporins, which line the channel of the nuclear pore complex and also are found in the nuclear basket and the cytoplasmic filaments, contain multiple repeats of short hy-drophobic sequences rich in phenylalanine (F) and glycine (G) residues (FG-repeats). More recently, several importin p homologs have been found that function in the nuclear import of proteins with other classes of NLSs. These importins interact directly with their cognate NLSs without the need for an adapter protein like importin a.

A current model for the import of cytoplasmic cargo proteins mediated by a monomeric importin is shown in Figure 12-21. Free importin in the cytoplasm binds to its cognate NLS in a cargo protein, forming a bimolecular cargo complex. The cargo complex then translocates through the NPC channel as the importin binds transiently to successive individual FG-repeats in the FG-nucleoporins that line the channel. The FG-repeats are thought to act like "stepping stones" as the cargo complex diffuses from one FG-nucleoporin to another on its way through the channel by a process that does not require a direct input of energy from ATP hydrolysis or other mechanisms. The

▲ EXPERIMENTAL FIGURE 12-20 The failure of nuclear transport to occur in permeabilized cultured cells in the absence of lysate demonstrates the involvement of soluble cytosolic components in the process. (a) Phase-contrast micrographs of untreated and digitonin-permeabilized HeLa cells. Treatment of a monolayer of cultured cells with the mild, nonionic detergent digitonin permeabilizes the plasma membrane so that cytosolic constituents leak out, but leaves the nuclear envelope and NPCs intact. (b) Fluorescence micrographs of digitonin-permeabilized HeLa cells incubated with a fluorescent protein chemically coupled to a synthetic SV40 T-antigen NLS peptide in the presence and absence of cytosol (lysate). Accumulation of this transport substrate in the nucleus occurred only when cytosol was included in the incubation (lower right). [From S. Adam et al., 1990, J. Cell. Biol. 111:807; courtesy of Dr. Larry Gerace.]

hydrophobic FG-repeats are thought to occur in regions of extended, otherwise hydrophilic polypeptide chains that fill the central transporter channel. The extended chains are proposed to form a meshwork of hydrophilic strands associated through hydrophobic interactions between the FG-repeats. This molecular meshwork is thought to restrict the diffusion of proteins larger than =60 kD. However, importins carrying cargo proteins are thought to penetrate the molecular meshwork by making successive interactions with the FG-repeats.

When the cargo complex reaches the nucleoplasm, the importin interacts with Ran-GTP, causing a conforma-tional change in the importin that decreases its affinity for the NLS, releasing the cargo protein into the nucleoplasm.

The importin-Ran-GTP complex then diffuses back through the NPC, again, through transient interactions of the importin with FG repeats. Once the importin-Ran-GTP complex reaches the cytoplasmic side of the NPC, Ran interacts with a specific GTPase-accelerating protein (RanGAP) that is a component of the NPC cytoplasmic filaments. This stimulates Ran to hydrolyze its bound GTP to GDP, causing it to convert to a conformation that has low affinity for the importin, so that the free importin is released into the cytoplasm, where it can participate in another cycle of import.

Ran is returned to the nucleus by NTF2. The NTF2 dimer binds specifically to Ran-GDP and also interacts with the FG repeats of FG-nucleoporins. Consequently, the NTF2-Ran-GDP complex can diffuse through the pore via transient

Ran . GDP Ran . GTP Importin Cargo

P P NLS

Nucleoplasm

Cytoplasm

Nucleoplasm

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