Nuclear Targeting in Plants

Abstract

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)(25)(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

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

“…2 A-C; presumably, the sequences for the 24-kDa polypeptide and the 25S RNA were ligated together during the cloning process). The polypeptide had a basic region that could potentially serve as a nuclear localization signal (28) and an acidic C-terminal half (pI of 3.6) that might act as an acidic transcription activator domain (29). Searches of the GenBank nonredundant nucleotide, peptide, and EST databases (Aug. 25, 1995, andMar.…”

Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression

“…Relative transformation frequencies of 40430% were reproducibly achieved (Figure 2b and c) which are comparable with frequencies scored by the same method for protoplasts from cell suspension cultures of tobacco (50%; Howard et a/., 1992), Arabidopsis (44%; Axelos et a/., 1992) or soybean (25%; Rasmussen and Rasmussen, 1993), but are several magnitudes higher as compared with the transfection of rapeseed protoplasts (2 x Chapel and Glimelius, 1990) or protoplasts from pea epicotyls (2 x Abel and Theologis, unpublished). Protein-fusion reporter systems utilizing P-glucuronidase or P-galactosidase have been employed as convenient monitors to study nuclear transport and nuclear localization signals of a number of plant proteins in transient assays and in transgenic plants (for review see Raikhel, 1992). These studies also have shown the equality of both transformation systems for which correlating subcellular distributions of protein fusions have been oljserved, thereby favoring transient assays as the method of choice (Carrington et al, 1991;Restrepo etal., 1990;Shieh etal., 1993;Varagona etal., 1992).…”

“…Transient gene expression utilizing plant protoplasts has emerged as a refined method to analyze expression of chimeric gene constructs within hours after transfection. Transient assay systems have been successfully applied to define the regulatory cisacting elements in a number of plant genes and, more recently, to study nuclear transport and nuclear localization signals (NLS) of plant and plant viral proteins (for review see Raikhel, 1992).…”

Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

“…In plants severa1 nuclear-targeted proteins have been identified (Restrepo et al, 1990;Varagona et al, 1991), and it appears that, as in animals, yeast, and amphibians, nuclear targeting of proteins larger than 40 kD requires a nuclear targeting or localization sequence (Restrepo et al, 1990;van der Krol and Chua, 1991;Raikhel, 1992). Provided that similar size restrictions apply to plant nuclei, at 35 kD the monomeric molecular mass of M6PR (as determined by SDS-PAGE and matrixassisted laser desorption MS [data not shown]) is sufficiently small to allow free diffusion into the nucleus.…”