Agrobacterium VirE2 gets the VIP1 treatment in plant nuclear import.

Agrobacterium tumefaciens transforms plant cells by targeting a large single-stranded DNA molecule (T-strand) to the plant nucleus. The host cell contribution to nuclear import and transformation is the focus of several current articles. Recently, plant proteins have been identified that promote nuclear import of the T-strand. In particular, VIP1 might couple transformation to the importin-dependent nuclear import pathway and deliver the T-strand to chromatin, thereby promoting integration into the host genome.     

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.     

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.     

VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.     

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.     

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.     

Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import

Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.     

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.     

A GTPase distinct from Ran is involved in nuclear protein import

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.     

VirE2, a type IV secretion substrate,interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens

Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.     

Isolation, purification, and identification of virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.     

New centromeric component CENP-W is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein

CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.     

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice

Background

Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated.

Methodology/Principal Findings

The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum.

Conclusions/Significance

SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.     

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.