Identification of a Ubiquitin-Binding Structure in the S-Locus F-Box Protein Controlling S-RNase-Based Self-Incompatibility

In flowering plants, self-incompatibility （SI） serves as an important intraspecific reproductive barrier to promote outbreeding. In species from the Solanaceae, Plantaginaceae and Rosaceae, S-RNase and SLF （S-locus F-box） proteins have been shown to control the female and male specificity of SI, respectively. However, little is known about structure features of the SLF protein apart from its conserved F-box domain. Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain （UBD） structure conserved among the SLF protein family. By using an ex vivo system of Nicotiana benthamiana, we found that the UBD mediates the SLF protein turnover by the ubiquitin-proteasome pathway. Furthermore, we detected that the SLF protein was directly involved in S-RNase degradation. Taken together, our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.

Diversified Application of Barcoded PLATO(PLATO-BC) Platform for Identification of Protein Interactions

Proteins usually associate with other molecules physically to execute their functions.Identifying these interactions is important for the functional analysis of proteins.Previously,we reported the parallel analysis of translated ORFs(PLATO)to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the “bait”molecules,followed by the deep sequencing analysis of mRNA.However,the sample processing,from extraction of precipitated mRNA to generation of DNA libraries,includes numerous steps,which is tedious and may cause the loss of materials.Barcoded PLATO(PLATO-BC),an improved platform was further developed to test its application for protein interaction discovery.In this report,we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis(IBM).Tripartite motif containing 21(TRIM21)was identified as a potentially new IBM autoantigen.We also expanded the application of PLATO-BC to identify protein interactions for JQ1,single ubiquitin peptide,and NS5 protein of Zika virus.From PLATO-BC analyses,we identified new protein interactions for these “bait”molecules.We demonstrate that Ewing sarcoma breakpoint region 1(EWSR1)binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4.RIO kinase 3(RIOK3),a newly identified ubiquitin-binding protein,is preferentially associated with K63-ubiquitin chain.We also find that Zika NS5 protein interacts with two previously unreported host proteins,par-3 family cell polarity regulator(PARD3)and chromosome 19 open reading frame 53(C19orf53),whose attenuated expression benefits the replication of Zika virus.These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of “bait”molecules.

Structural Basis for Ubiquitin Recognition by a Novel Domain from Human Phospholipase A2 Activating Protein

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A2 activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3.