Importin alpha from Arabidopsis thaliana is a nuclear import receptor that recognizes three classes of import signals.

Protein import into the nucleus is a two-step process. In vitro import systems from vertebrate cell extracts have shown several soluble factors are required. One of these factors is the receptor importin alpha, which binds to nuclear localization signals (NLS) in vitro. We previously cloned an importin alpha homolog from Arabidopsis thaliana (At-IMP alpha) and demonstrated that this protein was not depleted from tobacco (Nicotiana tabacum) protoplasts after permeabilization of the plasma membrane, (Hicks et al., 1996). To determine if At-IMP alpha is functional, we used an in vitro NLS-binding assay. We found that At-IMP alpha is specific, and the receptor is able to recognize three classes of NLS identified in plants. Purified antibodies to At-IMP alpha were used to determine the in vivo location of importin alpha in tobacco protoplasts. Importin alpha is found in the cytoplasm and nucleus, and it is most highly concentrated at the nuclear envelope. The biochemical properties of nuclear importin alpha and localization studies using purified nuclei demonstrate that importin alpha is tightly associated with the plant nucleus. Moreover, these results suggest that a fraction of nuclear importin alpha interacts with the nuclear pore complex.

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.

Three classes of nuclear import signals bind to plant nuclei.

Three nuclear localization signals (NLS), including an unusual Mat alpha 2-like NLS from maize (Zea mays) R, were found to compete for binding to plant nuclei. In addition, the authentic yeast Mat alpha 2 NLS, which does not function in mammals, was shown to function in plants in vivo. Our results indicate that plants possess a site at the nuclear pore complex that recognizes the three known classes of NLSs.

Nuclear localization signal binding proteins in higher plant nuclei.

The import of proteins into the nucleus is a vital process that is mediated by proteins which specifically recognize nuclear localization signals (NLSs). These factors have not been identified in plants. Previously, we demonstrated that higher plants possess a low-affinity binding site at the nuclear pore that specifically binds to several classes of functional NLSs. By the use of crosslinking reagents and a radiolabeled peptide to the bipartite NLS from the endogenous plant transcription factor Opaque2, two NLS binding proteins (NBPs) of 50-60 kDa and at least two NBPs of 30-40 kDa were identified. Competition studies indicated that labeling was specific for the functional NLS but not a mutant NLS impaired in vivo or a peptide unrelated to NLSs. Also, the apparent dissociation constant (100-300 microM) for labeling was similar to that of the binding site. Proteins of similar mass were labeled with two different crosslinking reagents, and concentration and time studies indicated that these NBPs were distinct proteins and not aggregates. Treatment with salt, detergent, or urea before or during NLS binding demonstrated that the properties of the binding site and the NBPs were identical. This tight correlation strongly indicates that some or all of the NBPs constitute the nuclear pore binding site. Overall, our results indicate that some components of NLS recognition are located at the nuclear pores in higher plants.

Protein import into the nucleus: an integrated view.

The directed movement of macromolecules into and out of the nucleus is a fundamental process in eukaryotes and occurs through the nuclear pore complex (NPC). A diverse array of molecules are transported across the nuclear envelope including proteins, mRNAs, tRNAs, snRNP complexes, ribosomal subunits, and in specialized cases, DNA. The structural and functional differences between these molecules point to the mechanistic complexity of NPCs and other components of the nuclear transport apparatus. This machinery must not only recognize within transported molecules specific targeting signals that differ between proteins, RNA, and RNA/protein complexes, it must translocate these molecules across the nuclear envelope. Additional levels of complexity are necessary because molecules such as proteins may continually undergo bidirectional transport across the envelope. Beyond these basic functions, the nuclear transport apparatus is regulated at the level of individual substrates and at more global levels such as coupling to cell cycle regression.

Specific binding of nuclear localization sequences to plant nuclei.

We have begun to dissect the import apparatus of higher plants by examining the specific association of nuclear localization sequences (NLSs) with purified plant nuclei. Peptides to the simian virus 40 (SV40) large T antigen NLS and a bipartite NLS of maize were allowed to associate with tobacco and maize nuclei. Wild-type NLSs were found to compete for a single class of low-affinity binding sites having a dissociation constant (Kd) of approximately 200 microM. Peptides to mutant NLSs, which are inefficient in stimulating import, were poor competitors, as were reverse wild-type and non-NLS peptides. The NLS binding site was proteinaceous and resistant to extraction under conditions where pores were still associated. In addition, immunofluorescence and immunoelectron microscopy indicated that binding was at the nuclear envelope. Overall, plant nuclei may be an excellent system to identify components of the import apparatus.

Characterization of auxin-binding proteins from zucchini plasma membrane.

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin.

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

The diageotropica mutant of tomato lacks high specific activity auxin binding sites.

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.