Molecular Cloning and Targeting of a Fibrillarin Homolog from Arabidopsis

Abstract

Fibrillarin is a nucleolar protein known to be involved in the processing of ribosomal RNA precursors. We isolated AtFbr1, a cDNA encoding a homolog of fibrillarin in Arabidopsis. The cDNA is 1.2 kb in size and encodes a polypeptide of 310 amino acid residues with a molecular mass of 33 kD. AtFbr1 is expressed at high levels in the flower and root tissue and at a slightly lower level in leaf tissue, whereas it was nearly undetectable in siliques. Expression of AtFbr1 was compared with that of the FLP (fibrillarin-like protein) gene identified by the Arabidopsis genome project. Abscisic acid treatment resulted in the down-regulation of the expression of both AtFbr1 and FLP genes in seedlings, although the degree of suppression was higher for FLP than for AtFbr1. In addition, the expression level of FLP decreased with the age of the seedlings, whereas AtFbr1 did not exhibit any detectable change. The subcellular localization of AtFbrl was studied with an in vivo targeting approach using a fusion protein, and was found to be correctly targeted to the nucleolus in protoplasts when expressed as a green fluorescent fusion protein (GFP). Deletion experiments showed that the N-terminal glycine-and arginine-rich region is necessary and sufficient to target AtFbr1 to the nucleolus.

Characterization of Karyopherin Cargoes Reveals Unique Mechanisms of Kap121p-Mediated Nuclear Import

“…However, Kap121p mediates its import through a direct interaction with the N-terminal rg-NLS. This establishes Nop1p as the first member of a novel class of Kap121p cargoes and extends previous studies implicating the Nop1p GAR domain in nuclear localization (40,49). Interestingly, this novel Kap121p-recognized rg-NLS sequence is similar to the rg-NLS sequences of the Kap104p import cargoes Nab2p and Nab4p.…”

“…Moreover, localization studies of Nop1p homologues identified the evolutionarily conserved N-terminal domain as possessing nuclear localization activity. In the case of human Nop1p (fibrillarin), the domain was required, but not sufficient, for nuclear import (49), whereas in Arabidopsis the GAR domain of AtFbr1 was both necessary and sufficient to target this protein to nucleoli (40). These data suggested that if Kap121p indeed mediates the import of Nop1p, it may do so through a kind of NLS that is different from those previously characterized.…”

The Pentratricopeptide Repeat Protein DELAYED GREENING1 Is Involved in the Regulation of Early Chloroplast Development and Chloroplast Gene Expression in Arabidopsis

“…5E; Lee et al, 2002). In contrast, GFP signals accumulated specifically in the nucleus when GFP was fused to the nuclear localization signal of the fibrillarin protein from Arabidopsis (Pih et al, 2000). In addition, GFP signals were found to accumulate in both the cytoplasm and the nucleus when Arabidopsis was transformed with the control vector without a specific targeting sequence, and no GFP signal was detected in wild-type, untransformed plants.…”

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…DNA encoding the full-length LTD protein was amplified by PCR using prime LTDGFPF and prime LTDGFPR, and the PCR products were ligated into the GFP fusion vector pUC18-35s-sGFP with GFP as a reporter. The constructs for nuclear, chloroplast and mitochondria localization were constructed according to the previous methods 31,32,33 . The resulting fusion constructs and the control vector were introduced into wild-type Arabidopsis.…”

“…31). GFP fusion proteins with the targeting signals of fibrillarin 32 and FRO1 (ref. 33) of Arabidopsis were located specifically in the nucleus and mitochondria, respectively (Fig.…”