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First published online 19 August 2003
doi: 10.1242/jcs.00690

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The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal

Eva Kiesler¹, Francesc Miralles^1,*, Ann-Kristin Östlund Farrants² and Neus Visa^1,

¹ Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden
² Department of Zoological Cell Biology, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden

View larger version (28K): [in a new window]   Fig. 1. Hrp65-encoding clones isolated in yeast two-hybrid screening. (A) Graphical depictions of the three Hrp65 isoforms. The sequence between amino acids 1-499 is common to all isoforms, but the C-terminal sequences are variable. (B) Identification of the Hrp65 isoforms isolated in the yeast 2-hybrid screening. Plasmids isolated from each Hrp65-encoding clone were used as templates for multiplex PCR reactions with one sense oligonucleotide that is common to all known Hrp65 isoforms and two antisense oligonucleotides that discriminate between the three Hrp65 isoforms. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. The figures above the lanes indicate the first amino acid encoded in each cDNA. (C) Schematic representation of individual Hrp65 cDNAs isolated in the two-hybrid screening. For each clone, the first encoded amino acid downstream of the GAL4 AD is indicated to the left. The differences in length at the C-terminus indicate the isoform as determined in (B).

View larger version (49K): [in a new window]   Fig. 2. In vitro binding of the three Hrp65 isoforms. Recombinant His-tagged Hrp65-1, -2 and -3 were purified on Ni-NTA-agarose and incubated in the presence of 35S-methionine labeled Hrp65-1, -2 or -3 obtained by in vitro translation in rabbit reticulocyte lysate (lanes 7-15). As a negative control, Ni-NTA agarose beads without His-tagged proteins were incubated in the presence of each 35S-labeled Hrp65 isoform (lanes 4-6). The bound proteins were eluted, resolved in a 10% SDS-PAGE gel and autoradiographed. 35S-labeled Hrp65-1, -2 and -3 proteins were loaded as inputs (lanes 1-3). The multiple Hrp65 bands observed in each input lane (bracket) are due to partial degradation and to alternative translation initiation sites. The mobility of molecular mass standards is indicated on the right (kDa).

View larger version (48K): [in a new window]   Fig. 3. Mapping of the self-interaction domain. Truncations and deletions of the Hrp65 protein (or PSF in part B) were expressed as GAL4-AD or -BD fusions and assayed for their ability to interact with full-length Hrp65 in the yeast two-hybrid system. Graphical representations of the assayed constructs are shown on the left. (A) Mapping of the Hrp65 self-interaction domain. Yeast cells were transformed with a full-length Hrp65 construct plus a truncation/deletion construct. The protein-protein interactions were detected by plating serial dilutions of the co-transformants onto double selective -His -Ade medium. (B) ClustalW alignment of the protein binding domains (PBDs) of C. tentans Hrp65 (residues 259-415), Drosophila melanogaster NonA (residues 448-603) and Homo sapiens PSF (residues 443-601). Residues identical in all three sequences are shown in black boxes, identical residues in two out of three compared sequences are shown in dark grey and similar residues in two out of three compared sequences are shown in light grey. (C) Interaction of the PBDs of Hrp65 and PSF with the full-length Hrp65. Protein-protein interactions were detected as in (A), except that the transformants were selected on -His plates.

View larger version (35K): [in a new window]   Fig. 4. Chromatographic analysis of Hrp65 oligomers. Recombinant Hrp65-1 purified on Ni-NTA agarose was fractionated on a Superose HR6TM gel filtration column and the protein in each fraction was analyzed by 12% SDS-PAGE and Coomassie staining. Hrp65 was detected from fraction 27 to fraction 37. A major part of the protein fractionated as a broad peak between fractions 29-33. The fractionation of molecular mass standards in the gel filtration column is indicated at the top. The mobilities of molecular mass standards in the SDS-PAGE are shown on the left, in kDa.

View larger version (23K): [in a new window]   Fig. 5. Subcellular localization of the Hrp65-2 isoform in C. tentans cells. (A) Immunoreactivity of the anti-Hrp65-2 antibody analyzed by western blot analysis of nuclear and cytoplasmic protein extracts prepared from C. tentans tissue culture cells. The proteins in each extract were separated by SDS-PAGE and blotted to transfer membranes. The membranes were cut into strips and incubated with the anti-Hrp65-2 antibody. The mobilities of molecular mass standards, in kDa, are shown on the left. (B) Semi-thin cryosections of C. tentans salivary gland cells were stained with the anti-Hrp65-2 specific antibody followed by a FITC-conjugated secondary antibody. An illustration of the subcellular structures that are visible after immunofluorescent labeling is shown in the middle. Cp, cytoplasm; chrom, polytene chromosomes; n, nucleolus; np, nucleoplasm. The broken line represents the nuclear envelope and the solid line represents the cell surface. The pre-immune serum was used in parallel as a negative control, and the picture was overlaid with a broken line to demarcate the border between nucleus and cytoplasm.

View larger version (50K): [in a new window]   Fig. 6. Nuclear import of Hrp65 isoforms analyzed by transient transfection assays. GFP fused to either Hrp65-1 or to Hrp65 amino acids 1-499, and Hrp65-2 fused to a Flag epitope were expressed in human HeLa cells. The Flag-Hrp65-2 signal was detected by immunostaining using monoclonal anti-Flag antibody followed by a Texas-red conjugated secondary antibody. (A) Single transfection of HeLa cells with constructs encoding GFP-Hrp65-1, Flag-Hrp65-2 and GFP-Hrp65(1-499). (B) Flag-Hrp65-2 was expressed together with either GFP-Hrp65-1 or GFP-Hrp65(1-499) by cotransfection of two constructs into HeLa cells. Transfected cells are shown in phase contrast on the right.