Date Published: November 16, 2010
Publisher: Public Library of Science
Author(s): Fang Yang, Yong Cheng, Jee Young An, Yong Tae Kwon, Sigrid Eckardt, N. Adrian Leu, K. John McLaughlin, Peijing Jeremy Wang, Laszlo Orban. http://doi.org/10.1371/journal.pone.0014017
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.
Partial Text: The N-end rule pathway is a ubiquitin-dependent proteolytic system , . In this pathway, the stability of proteins is defined by their N-terminal amino acids that are distinguished into stabilizing and destabilizing residues. The latter constitute so-called N-degrons, which are signatures for degradation of short-lived proteins. Destabilizing residues include basic (Arg, Lys, and His) and bulky hydrophobic (Phe, Tyr, Trp, Leu, and Ile) residues. An N-degron can also be created by either endoproteolytic cleavage or modifications of a pre-N-degron (Cys, Asn, Asp, Gln, or Glu) through a series of N-terminal modifications . Cysteine at position 2 (after methionine) is a unique type of destabilizing residue in mammalian cells. If N-terminally exposed, Cys can be oxidized to Cys-O2(H) or Cys-O3(H) before being arginylated by the arginine (R)-transferase ATE1 –. The N-degron is recognized by a family of UBR box-containing E3 ligases . The mammalian genome encodes at least four UBR members (Ubr1, Ubr2, Ubr4 and Ubr5) characterized by the UBR box, a ∼70-residue zinc finger-like domain , .
Ubr2 is the recognition E3 component of the N-end rule pathway, in which an ubiquitin ligase recognizes a destabilizing N-terminal residue as an essential component of N-degron. It has been shown that Ubr2 plays a role in transcriptional silencing of meiotic chromosomes through ubiquitination of histone H2A , . Here, we provide several lines of evidence that Ubr2 plays a novel function outside the N-end rule pathway: protein stabilization. Firstly, we demonstrate that Ubr2 forms a stable complex with Tex19.1 in testis. If Tex19.1 were an enzymatic substrate of Ubr2 and thus destined for ubiquitin-dependent degradation, its association with Ubr2 might be too transient to be detected by co-IP. In fact, although Ubr2 protein was not detectable in the total testicular extracts by Western blot, it was readily co-immunoprecipitated with Tex19.1, suggesting the formation of a stable protein complex. Secondly, Tex19.1 bears an N-terminal cysteine. Therefore, Tex19.1 might be a substrate of ATE1-dependent arginylation and Ubr2-dependent ubiquitylation. However, our results show that the Ubr2-Tex19.1 interaction does not require the evolutionarily conserved Cys2 residue, a putative arginylation substrate. Thirdly, the Tex19.1 mouse mutant phenocopies the Ubr2 null mutant, underscoring the physiological relevance of the Tex19.1-Ubr2 interaction. Lastly, the Tex19.1 protein is absent rather than more abundant in Ubr2-deficient testes. As Ubr2 binds to Tex19.1, the most parsimonious explanation is that Ubr2 stabilizes Tex19.1 metabolically. However, we cannot exclude the possibility that Ubr2 might also play a role in the translation of the Tex19.1 mRNA.