Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations

Abstract

Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified two-hybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.

Figure 1.

NTT Selection against Plant Proteins. Yeast strains containing rice cDNAs in the NTT vector, pNS1, were incubated on a Leu-/His- selection medium plate for 3 d at 30°C. α1a, pNS1 carrying the rice importin-α1a (Shoji et al., 1998); NLS, pNS-NLS carrying the SV40 large T-antigen NLS as a positive control (Ueki et al., 1998); pNS1, NTT vector with no insert as a negative control; FP40, pNS-FP40 encoding a p68-like DEAD-box RNA helicase with truncation of the N terminus NLS (DDBJ/GenBank/EMBL accession number AB110202); C4A21, pNS-C4A21 encoding an SC35-like splicing factor with N-terminal truncation (accession number AB110200); YP39, pNS-YP39 encoding a neoxanthin cleavage enzyme-like protein with C-terminal truncation (accession number AB110203); C4A31, pNS-C4A31 encoding a probable acidic endochitinase with N-terminal truncation (accession number AB1102001).

Figure 2.

Nuclear Localization of GFP-Fused Proteins in the Allium Epidermal Cells. The NTT clones introduced into onion cells are indicated vertically at the left. The image of the GFP fusion protein is shown at the left, and the nucleus stained with the DNA binding dye DAPI is in the middle, and the merged image of GFP with DAPI signals is at the right. The GFP figures represent localization of the following proteins fused to GFP. (A) An uncharacterized zinc finger protein (protein ID BAA33204.1). (C) A putative ADP-ribosylation factor GAP-like zinc finger–containing protein, ZiGA4 (protein ID BAB90399.1). (E) A proteasome α subunit protein (protein ID AAB51521.1). (G) A putative member of the 14-3-3 family proteins (DDBJ/EMBL/GenBank accession number AU032196, absent in the protein databases). (I) A chlorophyll a/b binding protein used as a negative control (N.C.; protein ID AAC15992.1). (B), (D), (F), (H), and (J) Nonnuclear group proteins whose fusion proteins were detected in the nucleus. Details of these clones are shown in Table 3. Bar = 25 μm. Note that the A. cepa epidermal cell has a variety of shapes, including various grooves and invaginations (Collings et al., 2000).

Figure 3.

Localization of the GFP-Fused Proteins of the Unknown Protein Group in Allium Epidermal Cells. The images of the GFP fusion proteins are shown at the left, the nuclei stained with DAPI are in the middle, and the merged images with these two are at the right. The GFP figures represent localization of several unknown proteins fused to GFP. For detailed characteristics of these proteins, see Table 3. Bar = 25 μm. (A), (C), (E), (G), (I), and (K) Proteins localized in an inner nuclear matrix region. (B), (D), (F), (H), (J), and (L) Proteins colocalized with DNA/chromatin. (M) to (P) Figures reduced to show whole cytoplasmic area of the cells in (F), (G), (I), and (K), respectively.

Figure 4.

Primary Structures of Four Candidate Rice Nuclear Proteins. Full-length cDNAs were isolated from the original cDNA libraries and used for GFP-fusion experiments. The numbers to the right of each protein indicate the number of amino acid residues of the predicted proteins. The regions cloned by the NTT method are shown on the bottom of each protein structure by arrowed bars. The motifs and characteristic regions are shown for each protein and described on the bottom of the figure. The DDBJ/GenBank/EMBL accession numbers are as follows: NALP1 (AB110173), OsAHP1 (AB110206), OsNMCP1a (AB110204), and OsSAD1 (AB110207).

Figure 5.

Subnuclear Localization of the GFP- or DsRed2-Fused Nucleus Associating Proteins in Allium Epidermal Cells. Green and red show the GFP and the DsRed2 fusion proteins, respectively. (B), (F), (J), and (N) are counterstained DAPI images of (A), (E), (I), and (M) nuclei, respectively. (C), (G), (K), and (O) are expanded images of (A), (E), (I), and (M), respectively. (D), (H), (L), and (P) are merged and enlarged images of each corresponding DsRed2/GFP with DAPI signals. Bars in (N) and (P) = 20 and 5 μm and represent the length of the leftmost two and rightmost two figures, respectively.

Figure 6.

Tissue Specificity and Subnuclear Localization of OsAHP1 Protein in Rice. (A) Immunoblotting of the protein extracts from several rice tissues reacted with anti-OsAHP1 (top) and actin (bottom) antibodies. Fifty micrograms of total crude proteins extracted from each of the indicated tissues were loaded. Values at the bottom of each lane represent the relative expression of OsAHP1 normalized by the level of actin expression. DAP, days after pollination. (B) Immunostaining of the secondary parietal cells from the anther wall with an antibody for OsAHP1 followed by a fluorescent-labeled secondary antibody. The left panel (green) represents stained rice nuclei with the antibody, the middle panel (red) shows PI staining for chromatin, and the right panel (yellow) shows a merged figure of the antibody-stained OsAHP1 and the PI stain. Bar = 5 μm.