Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2.

Abstract

The maize regulatory protein Opaque-2 (02) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy-and amino-terminal fusions of 0 2 to reporter protein p-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of 0 2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the 0 2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basiclleucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino-versus carboxy-terminal GUS fusions is discussed.

The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization

“…Maize transcription factors Opaque-2 and R, as well as yeast MATch2 have been shown to contain at least two distinct regions that are sufficient to direct nuclear localization [ 12,36,44,49]. It has been proposed that multiple NLSs may function to increase the efficiency of translocation or may regulate the nuclear import of the factor in different signalling pathways [31,36].…”

“…This was done in order to insert an optimal plant translation initiation site. [49] with the following modifications: tungsten particles were coated with the appropriate plasmid as described [9], 76 MPa delivery disks were used during the bombardment. Five independent bombardments were performed per construct.…”

“…Bombarded onion slices were assayed for GUS activity as described by Varagona et al [49]. Epidermal peels were then placed on a slide containing 50 mM sodium phosphate buffer, pH 7.2 and 4,6-diamidin-2-menylindol-dihyrochloride (DAPI).…”

“…Research on basic-leucine-zipper (bZIP) transcription factors, such as T G A l a from tobacco and Opaque-2 from maize, has demonstrated that the basic DNA-binding domain of these proteins is sufficient for nuclear localization [47,49]. Similarly, the yeast MAT~2 homeodomain protein contains two NLSs.…”

Isolation of an Arabidopsis homologue of the maize homeobox Knotted-1 gene

“…If so, MERIHB1 may form dimers or heterodimers that perform various and/or specific functions in development. A putative nuclear localization signal found in KN1, KNAT1, and KNAT2 [12,19] is located at the N-terminus of the ELK region in the MERIHB 1 peptide.…”