Nuclear Targeting in Plants

The nucleus is the site of highly active two-way macromolecular traffic between the nucleoplasm and the cytoplasm. Nuclear import and export must be highly specific processes because the content of the nucleus is distinguished compared to the cytoplasm. This is rather amazing, considering that the nuclear envelope dissolves during mitosis of animal and plant cells, necessitating reassembly of all nuclear components and reentry of nuclear proteins into the nucleus. In addition, many basic cellular processes, such as gene transcription and cell division, clearly require proper nuclear localization of regulatory and housekeeping proteins. Furthermore, experimental evidence indicates the existence of receptors and other cellular factors that mediate nuclear transport. Nuclear transport, although extensively studied in animal and lower eukaryotic systems (3, 20), has only recently been addressed in higher plants (1, 2, 5, 7, 8, 15, 24-26). This attention to protein and nucleic acid transport into the nucleus in plants was stimulated not only by a need to understand basic cell biology, but also by the desire to modulate or regulate the expression of genes via genetic engineering. The nucleus (Fig. 1) is surrounded by the nuclear envelope, which includes the inner and outer nuclear membranes and, between them, the perinuclear space. The outer nuclear membrane is contiguous with the rough ER, and its outer surface is generally studded with ribosomes. Underlying the inner nuclear membrane is the nuclear lamina, which is composed of a network of filamentous proteins. Unlike other organelles, the nuclear envelope contains NPC2, the major sites of bidirectional protein and nucleic acid traffic. Though understood on a structural level, knowledge of the biochemistry behind the NPC is limited. Only a few proteins associated with the NPC in animal systems have been identified (3); however, their actual involvement in nuclear import has not been demonstrated. Structurally, the NPC traverses the double membrane, bringing the lipid bilayers of the inner and outer membranes together around the margins of each pore. The NPC consists of eight large protein granules arranged in a circle with a large central granule or transporter that is thought to control active nucleocytoplasmic transport of large molecules (6). In addition, eight peripheral channels of a smaller diameter would allow a passive exchange of small molecules. Therefore, transport into the nucleus is fundamentally different from that into other organelles, where transported proteins pass directly through the membrane. Although most nuclear proteins carry information that simply directs them through the nuclear pore, others must continue their journey to a final subnuclear compartment (12). With regard to what is specifically responsible for proper localization of a protein inside the nucleus, very little is known. Results obtained from yeast and animal systems have led investigators to suggest some basic rules for nuclear protein targeting (3). Proteins smaller than 40 to 60 kD are thought to diffuse through the nuclear pore, though no physiologically relevant macromolecule has been shown to traverse the nuclear pore by diffusion. However, it has been shown that many proteins require at least one NLS. Two criteria are used to define these NLSs: (a) an NLS is sufficient to redirect a cytoplasmic protein to the nucleus, and (b) an NLS is necessary for directing a nuclear protein to the nucleus. However, in many cases the NLSs are not adequately characterized by both criteria. Known NLSs can be grouped into several categories (3). First, SV40-like NLSs contain a short stretch of basic amino acids (PKKKRKV). Second, MAT a2-like NLSs consist of short hydrophobic regions that contain one or more basic amino acids (KIPIK). Third, bipartite NLSs are usually a combination of two regions of basic amino acids separated by a spacer of more than four residues (SPPKAVKRPAATKKAGQAKKKKLDKEDES) (17). The first region has two basic amino acids and the second has at least three out of five basic residues. According to current speculation, the two basic regions cooperate in binding, whereas the spacer may facilitate their cooperative interaction. In addition, there are some NLSs that do not fit any of the above-mentioned groups and that are characteristic of some viral nuclear proteins (for example, the NLS of influenza ribonucleoprotein is AAFEDLRVRS). Although NLSs have been grouped into these categories, a consensus as to sequence, either between or within groups, has not been reached. This article will focus on recent advances in our understanding of NLSs recognized in plants. For earlier reviews on nuclear targeting in animal and yeast cells, see Garcia-Bustos et al. (3) and Silver (20).