DNA manipulations and Yeast 2-hybrid screen
The KIFC1 bait plasmid was constructed by annealing two complementary oligonucleotides (KIFC1sense and KIFC1antisense) that encode the 19 amino acid targeting sequence followed by ligation into pGBKT7 at the EcoRI and BamHI sites. The sequence of KIFC1sense is GAATTCGGAAAGGCTGCTTCAGGAGCTTCAGGGAGAGCGGCTGCA ATTGCAGGAAGAGCGGACGGATCC and KIFC1antisense is GGATCCGTCCGCTCTT CCTGCAATTGCAGCCGCTCTCCCTGAAGCTCCTGAAGCAGCCTTTCCGAATTC. The sequence of the pKIFC1bait was confirmed by sequencing. The pKIFC1bait plasmid was transformed into yeast strain AH109 and mated to a pretransformed testis cDNA library (Clontech; Palo Alto, CA) in yeast strain Y187. Positively interacting clones were selected for growth on media lacking histidine, adenine, leucine, and tryptophan and containing X-α-Gal. Diploids were restreaked on selective media and the phenotype retested. The interacting prey plasmids were rescued from each clone according to the manufacture's instructions and used to transform competent cells (Invitrogen; Carlsbad, CA). Plasmid DNA was then isolated using the Wizard Plus SV miniprep kit (Promega; Madison, WI) sequenced by the DNA sequencing facility at ECU and the sequences compared against available databases. The TLRR deletion mutants were constructed by using appropriate primers to amplify the desired fragments by PCR before transfer into pADT7 using the restriction sites EcoRI and BamHI engineered into the PCR primers. TLRRFL (full-length) was amplified with GAATTCGTTCGACTGACGGTGGATTT AATTGCC as the 5' primer and GGATCCCTCCTTTTTCTCAGACAAATTTT CC as the 3' primer. The TLRRA fragment was amplified with the TLRRFL5' primer and GGAATCCCAGA AGCTTTTCGCCTAGAGGGAGCC as the 3' primer. TLRRB was amplified with the TLRRFL3' primer and GAATTCTTTGATCCAAGAACTCTTCGTTCTCTAGCG as the 5' primer. TLRRC was amplified with TLRRFL3' primer and GAATTCTGCAGAAACCTG AGTGTTTTATATTTA as the 5' primer. TLRRD was amplified with the TLRRFL5' primer and GGATCCAGGATTT CCATTTAGATCCATTTTCC as the 3' primer. The TLRR5' deletion mutant was constructed by partial deletion of the IC#2 interacting clone with BglII followed by religation and therefore contains sequences 5' to the ATG of the putative TLRR and extends through amino acid number 166 eliminating the last leucine of the fourth TLRR motif. For characterization of the TLRR antibody, the TLRR ORF was cloned into the bacterial expression vector QE31 in frame with the His6 tag.
β-gal activity assay
β-Galactosidase activity was measured in liquid cultures of yeast using ONPG (O-nitrophenyl B-D-galactopyranoside) as substrate. Cultures of each yeast co-transformant were grown overnight at 30°C in 5 ml of SD selective medium (SD-LEU-TRP, Clontech; Palo Alto, CA), diluted with YPD medium (BD Bioscience; Bedford, MA) and growth continued at 30°C for 3–5 hours with shaking at 230 rpm until the cultures reached OD600 0.5–0.8. 1.5 ml of each culture was transferred to eppendorf tubes, centrifuged, and resuspended in approximately one fifth the original volume with Z buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, pH 7.0, 0.01 M KCl, 0.001 M MgSO4). Cells were broken by repeated freeze/thaw and the reaction started by addition of 0.7 ml Z buffer with 0.27% β-mercaptoethanol and 160 μl of ONPG (4 mg/ml O-nitrophenyl B-D-galactopyranoside, in Z buffer, Sigma; Atlanta, GA), followed by incubation at 30°C until reactions became yellow, and terminated with 0.4 ml of 1 M Na2CO3. The absorbance of each supernatant at OD420 was determined and the activity calculated according to the following equation: , where T is the incubation time in minutes, V is 0.1 ml × concentration factor, and OD600 is the A600 of 1 ml of culture. The activity of each co-transfectant was assayed in triplicate. The yeast strain AH109 (Clontech; Palo Alto, CA) cotransformed with pGADT7-T and pGBKT7-53 were used as positive control for protein-protein interaction while cells containing pGADT7 and pGBKT7-Lam, expressing proteins that do not interact, were negative controls for these experiments.
A normalized multiple tissue Northern blot (Clontech; Palo Alto, CA) was used to identify TLRR transcripts in different mouse tissues using the TLRRA fragment described above as probe (Figure 3A). Total RNA from various tissues was also isolated using the Trizol reagent (Invitrogen; Carlsbad, CA), separated, blotted to Nytran (Schleicher and Schull; Keene, NH), and probed with the TLRRA fragment giving identical results (data not shown). The DNA probe was labeled by mixed primer labeling using the High Prime kit from Roche Diagnostics (Indianapolis, IN) and [α32P]dATP. Blots were hybridized to probe in ExpressHyb solution (Clontech; Palo Alto, CA) or Church and Gilbert solution  overnight at 68°C. After hybridization, blots were washed at high stringency and exposed to X-ray film with a intensifying screens for 1 hour.
Antibody Preparation and Western Blot
The TLRR sequence was scanned for antigenic peptides using the EMBOSS site  and a unique peptide, FSDRNIDSIDDLSLC, was chosen to immunize rabbits (Harlan Bioproducts for Science; Indianapolis, IN). The resultant sera were affinity purified against the corresponding peptide using the Sulfolink kit (Pierce; Rockford, IL) according to the manufacturer's instructions. Protein samples containing equal protein prepared from mouse tissue extracts, or affinity purification fractions, were separated by polyacrylamide gel electrophoresis (PAGE) through 10% acrylamide gels or precast 8–16% acrylamide gels (Invitrogen; Carlsbad, CA), equilibrated in and electrophoretically transferred from the gel matrix to PVDF membrane (BioRad Laboratories; Hercules CA) in Towbin transfer buffer. Proteins were detected on the membrane with affinity purified TLRR antibody. Immune complexes bound to the membrane were detected with horseradish peroxidase-conjugated donkey secondary antibody (Jackson ImmunoResearch Inc.; West Grove, PA) diluted 1:40,000 in TTBS (100 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween20) and developed with enhanced chemiluminescent reagents as described by the manufacturer (Amersham Pharmacia Biotech; Piscataway, NJ).
All use of animals was approved and conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Mouse testis extract was prepared as previously described . Briefly, decapsulated testes from adult mice were homogenized in buffer (10 mM MES, pH 7.65, 1 mM EGTA, 0.5 mM MgCl2, 30% glycerol, 0.1% NP40) containing protease inhibitors (10 μM benzamidine, 0.1 mg/ml leupeptin, 0.1 mg/ml aprotinin, 0.1 mg/ml TAME, 3 μM PMSF) and centrifuged two times, first at 100,000 × g and then 130,000 × g, to remove cellular debris generating a high speed supernatant fraction. Protein concentration in tissue lysates was determined by the Coomassie brilliant blue method (Biorad; Hercules, CA).
An affinity purification column was prepared by attaching the KIFC1 targeting peptide identified previously (GKAASGASGASGRAAAIAGRAD; ) to a methacrylate resin (UNC Microprotein Sequencing and Peptide Synthesis Facility, Department of Microbiology, University of North Carolina at Chapel Hill) . The 2 ml column was equilibrated in bead binding buffer (25 mM Potassium Phosphate, pH 7.5, 150 mM KCl, 1 mM MgCl2) supplemented with protease inhibitors. Approximately 20–50 mg testis lysate in 1.0 ml binding buffer was loaded onto the column and incubated, with gentle mixing, overnight at 4°C. The column was then washed with a total of 10–12 ml buffer and the flow through collected until the OD280 had returned to baseline. Bound complexes were eluted from the column in 200 μl fractions first with IgG elution buffer (Pierce; Rockford, IL) followed by IgG elution buffer with 250 mM added NaCl.
Proteins in eluted fraction were precipitated by addition of 1 μl 2% Na Deoxycholate and 5 μl 10% TCA, vortexed for several seconds, and the samples incubated on ice for 30 minutes. Precipitated proteins were pelleted by centrifugation at 20,000 × g for 10 minutes at room temperature and the supernatant removed. Any residual TCA was then removed from the pellet by incubation with 200 μl acetone, followed by incubation at room temperature for about 10 minutes and centrifugation at 20,000 × g, 10 min at room temperature. The supernatant was removed, the acetone wash repeated and the samples air-dried, resuspended in loading buffer and resolved by PAGE. The presence of the TLRR protein in the column fractions was determined by western blot as described above.
TLRR was detected in tissue sections essentially as previously described . Testes were obtained from sexually mature mice (29–31 g, CD-1; Charles River, Wilmington, MA) and immersion fixed overnight in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, pH 7.4) after piercing of the capsule. The organs were then incubated overnight in 0.5 M sucrose in PBS, placed in cryoprotectant, cut into 10 μm sections, transferred to Vectabond coated slides (Vector Laboratories; Burlingame, CA), quickly dipped in -20°C acetone, and allowed to dry. The sections were washed with PBS and treated with 0.3% Triton X-100 for 15 minutes at room temperature. The tissue was blocked in 2% BSA in TBST (20 mM Tris, pH 7.5, 154 mM NaCl, 2 mM EGTA, 2 mM MgCl2, 0.1% Triton X-100) and incubated with TLRR polyclonal antibody diluted 1:50 in TBST (approximately 100 μg/ml).
The TLRR polyclonal antibody was detected with a Texas Red conjugated donkey anti-rabbit IgG secondary antibody (1:100 dilution; Jackson ImmunoResearch Laboratories; West Grove, PA). DNA was stained with DAPI (Invitrogen; Carlsbad, CA) incorporated into Vectashield mounting media (Vector Laboratories; Burlingame, CA). In some experiments, the acrosome was stained with FITC conjugated peanut agglutinin (Sigma-Aldrich; St. Louis, MO) by inclusion in the secondary antibody incubation at a concentration of 20 μg/ml. The intracellular localization of proteins was observed with a Nikon E600 fluorescence microscope fit with appropriate filters and images captured with an Orca II CCD camera (Hamamatsu, Bridgewater, NJ) and analyzed with Metamorph image analysis and acquisition software (Universal Imaging Corporation, Downingtown, PA).