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

The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 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 O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.     

Arabidopsis thaliana Atrab28: a nuclear targeted protein related to germination and toxic cation tolerance

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues.Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.     

The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals.

The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport.     

Tus,an E. coli Protein,Contains Mammalian Nuclear Targeting and Exporting Signals

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

Plastid targeting of E. coli β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells

Summary Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target -glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.Abbreviations BMS maize Black Mexican Sweet suspension culture cells - CTP chloroplast targeting peptide - glgC16 an allosteric variant of E. coli ADP-glucose pyrophosphorylase - GUS -glucuronidase - LUX luciferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSU small subunit of ribulose bisphosphate carboxylase     

Exclusive cytosolic localization and broad tRNA<Superscript>Ser</Superscript> specificity of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seryl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of Arabidopsis thaliana seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAs^Ser, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNA^Ser specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

Agrobacterium VirE2 Proteins Can Form a Complex with T Strands in the Plant Cytoplasm

Wild-type VirE2 and VirD2 proteins from Agrobacterium tumefaciens contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An A. tumefaciens virE2 virD2ΔNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.     

Structural requirements of oleosin domains for subcellular targeting to the oil body.

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability.     

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes

 The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L. Received: 19 July 1999 / Accepted: 19 February 2000     

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.