Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intracellular macromolecular sorting and maintenance of cellular compartments
Protein and RNA transport into and out of the nucleus

The Contents of the Cell Nucleus (nucleoplasm) are separated from the Cytoplasm by the nuclear envelope, which is formed by a double membrane. The spherical inner nuclear membrane contains specific Proteins that serve as binding sites for the nuclear lamina, a structural network that Supports the membrane and interacts with Chromosomes and nuclear RNAs. This membrane is surrounded by the outer nuclear membrane, which is very similar to—and continuous with—the membrane of The Endoplasmic reticulum (ER) (Fig. 8-19). Consequently, the outer nuclear membrane can be viewed as a specialized region of the ER membrane. Much like the membranes of the rough ER (see Section 8.6.1), the outer nuclear membrane is studded with Ribosomes engaged in Protein Synthesis. Proteins synthesized on these ribosomes are translocated into the space between the outer and inner nuclear membranes (the perinuclear space), which is directly continuous with the ER lumen (see Fig. 8-19).

The Nucleus contains numerous proteins essential for its unique Functions. These proteins—including Histones, DNA and RNA polymerases, various Gene regulatory proteins, and RNA-Processing factors—are synthesized in the Cytosol and subsequently imported into the nucleus. To reach the nuclear interior (the nuclear lumen), they must cross both the outer and inner nuclear membranes. This transport is highly selective: many proteins produced in the cytosol never gain access to the nucleus.

8.3.1. The Double Nuclear Membrane Is Pierced by Nuclear Pores [16]

In all eukaryotes, from Yeast to humans, the nuclear envelope is perforated by nuclear pores. These pores are surrounded by large, ring-like structures known as pore complexes (with an inner diameter of approximately 80 nm and a mass of 50–100 MDa). Each complex consists of a set of large protein granules arranged in an octagonal Symmetry (Figs. 8-20A and 8-21). The pore complex spans the double membrane, fusing the lipid bilayers of the inner and outer membranes together around the perimeter of the pore (Fig. 8-20B). Despite this continuity—which might theoretically allow for the free diffusion of components between the outer and inner membranes—the two membranes remain chemically distinct.

Class="center">

Fig. 8-20. Diagram showing the arrangement of nuclear pore complexes within the nuclear envelope. Top view and cross-section through the center of the pore (left). The color-highlighted "central granule" is present in some pores and absent in others; these granules may be an integral part of the pore, but they could also simply represent large particles caught in transit through the pore channel. Diagram of a small patch of the nuclear envelope (right).

Fig. 8-21. Electron micrograph of a preparation of nuclear pore complexes (negative staining). Each pore is surrounded by a ring of eight granules, each approximately the size of a ribosome. (Courtesy of A. Faberge.)

The "hole" in the center of each complex (the nuclear pore) forms an aqueous channel through which Water-soluble molecules shuttle between the nucleus and the cytoplasm. The opening often appears to be "plugged" by a large granule, which is thought to consist of newly synthesized ribosomes or other particles caught in the act of being transported to the cytoplasm (see Fig. 8-20B). The effective resting size of the pore has been determined experimentally by introducing various labeled non-nuclear molecules into the cytosol and measuring their rates of diffusion into the nucleus. Small molecules (5 kDa and under) enter the nucleus so rapidly that the nuclear envelope is essentially freely permeable to them. The concentration of a 17 kDa protein equalizes between the cytoplasm and the nucleus in about 2 minutes; for a 44 kDa protein, this takes roughly 30 minutes, whereas Globular proteins exceeding 60 kDa scarcely penetrate the nucleus at all. Quantitative analysis of such data confirms that the nuclear pore complex contains a water-filled cylindrical channel approximately 9 nm in diameter and 15 nm in length (Fig. 8-22). These dimensions are comparable to the randomly arranged channels visible in certain electron micrographs.

Apparently, the nuclear envelope is adapted to shield the contents of the nuclear compartment (the nucleoplasm) from the myriad particles, filaments, and large molecules operating in the cytoplasm. Mature cytoplasmic ribosomes, for instance, are far too large to pass through these 9-nm channels, which is why all Protein synthesis is restricted to the cytoplasm. It remains a puzzle, however, how critically important large molecules—such as DNA and RNA polymerases, whose subunits have molecular masses ranging from 100 to 200 kDa—gain entry into the nucleus. Recent evidence indicates that these and many other Nuclear Proteins interact with receptor proteins located at the periphery of the nuclear pores, which actively transport large proteins into the nucleus by temporarily diluting or expanding the pore channel.

Fig. 8-22. Cross-section of a nuclear pore (simplified diagram). The imaginary cylinder depicted in the center of the pore represents the effective size of the open channel, calculated from transport measurements. In some electron micrographs, fine filaments can be seen filling nearly the entire interior space of the pore. The presence of these filaments likely restricts the effective pore lumen to about 9 nm.

Fig. 8-23. Experiments demonstrating The entry of selected proteins into the nucleus via nuclear pores. Nucleoplasmin is a large pentameric protein with distinct "HEAD" and "tail" domains. The "heads" can be severed from the "tails" by Limited proteolysis. When intact nucleoplasmin is microinjected into the Xenopus oocyte cytoplasm, it rapidly accumulates in the nucleus, even though it is far too large to diffuse passively through the tiny channel at the center of the pore complex. The signal directing the nuclear import of this protein evidently resides in the "tail" domain, because isolated "tails" microinjected into the oocyte cytoplasm are transported into the nucleus, whereas isolated "heads" are not. The Role of nuclear pores in this signal-mediated transport is illustrated by Electron Microscopy. Nucleoplasmin "tails" were conjugated to colloidal gold particles, which are clearly visible in the Electron microscope due to their high electron density. The attached nucleoplasmin "tails" drive the passage of the colloidal gold particles through the nuclear pores.

8.3.2. Proteins Actively Enter the Nucleus Through Nuclear Pores [17]

If proteins are extracted from the nucleus and subsequently microinjected into the cytoplasm, even very large proteins will re-accumulate in the nucleus. One of the best-studied Examples is nucleoplasmin, a major nuclear protein that can be proteolytically cleaved into "head" and "tail" fragments. In microinjection experiments, the "tail" portion enters the nucleus, whereas the "head" portion does not (Fig. 8-23). If the "tails" are coupled to colloidal gold particles 20 nm in diameter (which is much larger than the inner diameter of a resting nuclear pore), the gold particles still accumulate in the nucleus and can be observed within the nuclear pores during transit (Figs. 8-23 and 8-24). Consequently, the nuclear pore is capable of "opening up" to let through an object as large and foreign as a gold particle. The pore appears to function like a valve that opens in response to a signal from a sufficiently large protein. Exactly how this occurs at THE MOLECULAR LEVEL remains a mystery.

Proteins like nucleoplasmin are actively transported through the pores, possibly even remaining in a folded state during the process. In vitro reconstitution experiments of active nuclear transport confirm that The Cell derives the energy required for this process from ATP Hydrolysis.

Fig. 8-24. Electron micrograph showing the passage of nucleoplasmin-coated colloidal gold particles (see Fig. 8-23) into the nucleus through nuclear pores, the locations of which are indicated by colored brackets. A similar result is obtained when gold particles are conjugated solely to the tail domains of nucleoplasmin molecules. These particles are larger in diameter than a resting pore; therefore, the pore must "open" to allow their passage into the nucleus. (After S. Feldherr, E. Kallenbach, N. Schultz, J. Cell Biol. 99: 2216-2222, 1984.)

8-11

8-12

8-13

8.3.3. Only Proteins Containing Special Signals Are Actively Transported into the Nucleus [18]

The selectivity of nuclear transport is conferred by nuclear import signals, which are present exclusively on nuclear proteins. As we noted earlier, in nucleoplasmin, a signal of this type resides within the tail region of the molecule. For several other nuclear proteins, nuclear import signals have been mapped with greater precision using Introduction/32.html">Genetic Engineering techniques. These signals can be located in virtually any part of a protein molecule and typically consist of a short peptide (usually four to eight amino acid residues) enriched in the positively charged Amino Acids Lysine and Arginine, and frequently containing Proline. A signal of this type was first identified in the SV40 virus T-antigen, a large (90 kDa) protein essential for Viral METABOLISM/36.html">DNA Replication in the nucleus. Normally, the T-antigen accumulates in the nucleus shortly after its synthesis in the cytosol. However, a single amino acid substitution prevents Protein transport and causes the protein to accumulate in the cytoplasm instead (Fig. 8-25). This mutation was deduced to affect the nuclear import signal sequence. In subsequent experiments, the DNA segment encoding this region of the normal T-antigen was spliced to a gene encoding a mutant cytoplasmic protein. This allowed researchers to determine the minimum sequence required to impart nuclear-entry capability to a "hybrid" protein. It was demonstrated that the nuclear import signal for the T-antigen consists of a stretch of eight consecutive amino acids located within an internal region of its polypeptide chain (see Table 8-3). Further experiments confirmed that this signal sequence functions successfully even when synthesized as a short peptide and chemically coupled to an arbitrary lysine side chain on a "cytoplasmic" mutant protein. Thus, the exact position of the nuclear import signal within a protein does not appear to be critical.

The Mechanism of Protein transport into the nucleus differs fundamentally from the Mechanisms of Protein transport into other Organelles (which will be described later). The key difference is that nuclear protein transport occurs through gated aqueous pores rather than across one or more membranes. Moreover, when the nucleus "breaks down" during mitosis, its contents mix with the cytosol, and nuclear proteins are released into the cytoplasm. When the nucleus reassembles, groups of chromosomes are initially packaged within individual double membranes that fit so tightly around them that soluble proteins—including many former nuclear components—are initially "excluded." These membrane-enclosed chromosomes then fuse to form a single nucleus, into which the required proteins must subsequently be imported from the cytosol. Perhaps because nuclear protein molecules must repeatedly undergo this post-mitotic re-import, the nuclear import signal peptide is not cleaved off after entry into the nucleus. By contrast, when a protein molecule is imported into any other membrane-bounded organelle, it is inherited generation after generation within that same compartment and is never transported into it anew. Consequently, the signal Peptides of such molecules are clipped off as soon as the protein is translocated into the compartment.

Fig. 8-25. Subcellular localization of SV40 T-antigen with and without a nuclear localization signal peptide. Wild-type T-protein contains the lysine-rich sequence shown here and is imported into the nucleus to its final destination, as demonstrated by immunofluorescence staining using Antibodies against the T-antigen (A). T-Antigens with a mutated signal peptide (e.g., with Threonine replacing lysine) remain in the cytoplasm (B). (From D. Calderon, B. Roberts, W. Richardson, A. Smith, Cell 39: 499-509, 1984.)

8.3.4. Some RNAs leave the nucleus through nuclear pores [19]

The nuclear envelope of a typical mammalian cell contains 3,000 to 4,000 pores (approximately 11 pores per 1 µm² of membrane surface area). When a cell is actively synthesizing DNA, about 10⁶ histone molecules must be transferred from the cytoplasm to the nucleus every 3 minutes to package the newly synthesized DNA into Chromatin. This means that each pore must transport approximately 100 histone molecules per minute. In a rapidly growing cell, each pore must also export about three newly assembled ribosomal subunits from the nucleus to the cytoplasm, since ribosomes are assembled in the nucleolus and function in the cytosol (see Section 9.4.17). And this represents only a fraction of the total traffic passing through the nuclear pores.

The mechanism of export of new ribosomal subunits is of particular interest. These particles are too large (about 15 nm in diameter) to pass through the 9-nm aqueous channels. They most likely traverse the nuclear pores via an Active Transport system. It is also believed that Messenger RNA molecules complexed with ribonucleoprotein particles (associated with specialized proteins) are actively transported from the nucleus to the cytoplasm. If 20-nm colloidal gold particles—similar to those used in the nucleoplasmin experiments (see Fig. 8-24)—are coupled to small RNA molecules (tRNA or 5S-RNA) and microinjected into the nucleus of a frog oocyte, they are rapidly transported through the nuclear pores into the cytoplasm. Conversely, when injected into the oocyte cytoplasm, they remain there. Apparently, In addition to receptors that recognize nuclear import signals, the pores contain one or more receptors that recognize RNA molecules (or their associated proteins) destined for the cytosol; once these receptors are engaged, the pore catalyzes active outward transport rather than inward transport. Note that although several nuclear pore proteins (including a major 190-kDa membrane protein) have recently been isolated, the precise working mechanism of the nuclear pore remains unclear.

Conclusion

The nucleus is enclosed by a double-membrane envelope consisting of two concentric membranes. The outer nuclear membrane is continuous with the ER membrane, and the space between the outer and inner nuclear membranes is continuous with the ER lumen. RNA molecules and ribosomes are synthesized in the nucleus and exported to the cytosol, whereas all proteins that function in the nucleus are synthesized in the cytosol and imported into the nucleus. Exchange of Materials between the nucleus and cytoplasm occurs through nuclear pores, which provide a direct passageway through both the inner and outer nuclear membranes.

Fig. 8-26. Major subcompartments of Mitochondria and Chloroplasts. The topology of a chloroplast can be derived from that of a mitochondrion by a simple conceptual step: if the invaginations of The inner mitochondrial membrane were to pinch off completely, they would form a compartment topologically equivalent to the thylakoids of chloroplasts.

Proteins containing nuclear import signals are actively transported into the nucleus through the pores and are recognized by short, positively charged signal peptides. Because the signal peptide is not cleaved off following transport, nuclear Proteins can be re-imported into the nucleus whenever required, such as during nuclear reassembly after mitosis. RNA molecules and, presumably, ribosomal subunits are actively transported through the pores from the nucleus to the cytoplasm.