植物体内核定位信号介导的蛋白质核转运

介绍了植物体内核定位信号的类型和结构特点及由核定位信号介导的蛋白质核转运机理 .

Localization of Two GFP_tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli

Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.

Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B （LMB）, a specific inhibitor of Crml, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals （NESs） in pericentrin with diverse export activities. All of the five NESs could bind to Crml in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal （NLS） consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Expression and localization of VCX/Y proteins and their possible involvement in regulation of ribosome assembly during spermatogenesis

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromosomes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST) fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated and the localization of VCY protein in human testis was determined by immunohistochemistry. In the testis seminiferous epithelium. VCY proteins were highly expressed in nuclei of germ cells. Using propidium iodide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainly localized in the uucleoli of COST cells. In addition, the colocalization for VCY and VCX-8r in COS7 cells was also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtained using yeast two-hybrid system. All the information above indicates that VCX/Y protein family might be involved in the regulation of ribosome assembly (hiring spermatogenesis.

Identification of novel nuclear localization signal within the ErbB-2 protein

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis ofthe extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal(NLS)-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR (aa655-668) contains three clusters of basicamino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of thisNLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for thenuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nucleartranslocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanismunderlying ErbB2 nuclear trafficking and localization.

A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present,among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot,immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of Western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase.Our data indicated that the PSTAIRE CDK- like protein has a direct bearing on the mitosis.

Immunolcocalization of actin in intact and DNA-and histone-depleted nuclei and chromosomes of allium cepa

The presence of actin in eukaryotic nuclei and chromosomes, and especially in higher plant nuclei and chromosomes, has not been well established. We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes, indicating that actin is present in the nuclei and chromosomes of the higher plant. We labeled sections of the meristematic cells of A. cepa with immunogold technique, gold particles were found over the whole nuclei and a number of gold particles were concentrated in condensed chromatin and nucleoli, confirming the results of the immunofluoresence observations. We treated the nuclei and chromosomes of A.cepa with DNase I and 2M NaCl and obtained DNA- and histone-depleted nuclei and chromosomes. Indirect immunofluorescence tests showed that the DNA- and histonedepleted nuclei and chromosomes reacted positively with the anti-actin antibodies. These results demonstrate that actin exists not only in intact nuclei and chromosomes,but also in DNA- and histone-depleted nuclei and chrmosomes of the plant. In addition, our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A. cepa.

Nuclear localization of Sindbis virus nonstructural protein nsP2

In early infection, approximately 10% of nonstruc-tural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear nsP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile, a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were discussed in relation to the inhibition of host macro-molecular synthesis.