The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis

Abstract

Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19) has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.

Figure 1. Tex19.1 interacts with Ubr2 in the testis.

Testicular protein extracts prepared from 20-day-old wild type and Tex19.1 ^−/− mice were used for co-immunoprecipitation (IP) experiments. (A) Identification of Tex19.1-associated proteins from testis by mass spectrometry. Tex19.1-associated proteins were co-immunoprecipitated from testicular extracts with affinity-purified antibody, and analyzed by SDS-PAGE and SYPRO Ruby staining. To confirm specificity, IP of proteins from Tex19.1^−/− testes was performed in parallel. A second differentially expressed band (lower mass, indicated by arrow) was identified as actin by mass spectrometry. (B) Co-IP of Ubr2 with Tex19.1 from testis. IP was performed with the anti-Tex19.1 antibody and probed with the anti-Ubr2 antibody. Note that Ubr2 was too low in abundance in the total testicular extract to be detected by western blot analysis. Myh11 (myosin heavy chain 11) served as a loading control. (C) Reciprocal Co-IP experiment. Tex19.1 was co-immunoprecipitated with anti-Ubr2 but not control antibody. Bands indicated by asterisks in the IP are likely to be antibody light/heavy chains or non-specific species. Molecular mass standards are shown in kilodaltons.

Figure 2. Binding of Ubr2 and Tex19.1 is arginylation-independent.

All co-transfections were performed in NIH3T3 cells. (A) Co-immunoprecipitation of Ubr2 with C2-Tex19.1 (wild type), C2G-Tex19.1, and C2V-Tex19.1 demonstrates no difference in interaction between wild-type and mutant proteins. (B) Ubr2 interacts with the evolutionarily conserved N-terminal half of Tex19 but not with the C-terminal half. Mouse Tex19.1, 351 aa, pI = 4.69; Tex19.1N, aa 1–163, pI = 4.15; Tex19.1C, aa 164–351, pI = 6.34. All Tex19 proteins were tagged with V5 epitope at the N-termini. The slow migration of both full-length Tex19.1 and Tex19N on SDS-PAGE was due to their low pI. Molecular mass standards are shown in kilodaltons.

Figure 3. Ubr2 interacts with Tex19.2.

Co-transfections were performed in NIH3T3 cells. Tex19.2 was tagged with the V5 epitope.

Figure 4. Meiotic defects in Tex19.1 ^−/− males.

(A) Schematic diagram of the Tex19.1 targeting strategy. Tex19.1 and Tex19.2 are located only 27 kb apart but are transcribed in opposite orientations. All three exons of Tex19.1 are drawn to scale as rectangles and are designated by numbers shown above. The entire Tex19.1 ORF is located in the last exon and is replaced by the rtTA2^S-M2-IRES-LacZ-PGK-Neo cassette in the mutant allele. PGK-Neo is flanked by loxP sites. The expression of rtTA (reverse tetracycline-controlled transactivator) and lacZ is expected to be driven by the Tex19.1 promoter. (B) Absence of Tex19.1 protein in Tex19.1 ^−/− testes. Western blot analysis was performed on 20 µg each of adult wild type, Tex19.1 ^+/−, Tex19.1 ^−/−, and XX^Y* testicular extracts. XX^Y* testes are depleted of germ cells and were used as controls. Molecular mass standards are shown in kilodaltons. (C) Sperm output correlates with testis weight. Five Tex19.1 ^−/− and four wild type mice of 2–3 months of age were plotted for testis weight and epididymal sperm count. (D, E) Histological analysis of testes from 3-month-old wild type (D) and Tex19.1 ^−/− (E) testes. Zyg/Pa, zygotene/pachytene spermatocytes; RS, round spermatids. Scale bar, 50 µm.

Figure 5. Depletion of the Tex19.1 protein in Ubr2-deficient testes.

(A) Detection of Tex19.1 transcript in Ubr2 ^−/− testis by RT-PCR. Sycp2 and β-actin served as germ cell-specific and ubiquitous expression controls . (B) Western blot analysis shows the absence of Tex19.1 protein in Ubr2 ^−/− testis. Sycp2 ^−/− testis exhibits meiotic arrest and serves as a control . (C) Loss of Tex19.1 protein in Ubr2 ^−/− germ cells. Testis sections from adult wild type and Ubr2 ^−/− mice were immunostained with anti-Tex19.1 and anti-Acrv1 antibodies. Acrv1 is a component of acrosomes and thus is only present in the haploid germ cells - spermatids. Note the abundance of round spermatids in the Ubr2-deficient testis. Scale bar, 25 µm.