Screening And Identification Of The RTN4-C Interactive Proteins With Yeast Two-Hybrid System

Reticulons (RTNs) are a relatively new eukaryotic gene family with unknown functions but broad expression and peculiar topological features. Relatively recently Reticulon (RTN) was described to code a family of proteins in higher vertebrates, which were characterized by two homologous large hydrophobic transmembrane domain and a potential C-terminal endoplasmic reticulum retrieval signal. All family members sharing homologies within the C-terminal region of about 200 amino acids, called the reticulon-homology domain (RHD).There are at least four RTN genes (RTN1, RTN2, RTN3, RTN4) in human and mouse. RTN4 plays a role in inhibition of axonal regeneration after an injury to the mammalian central nervous system (CNS), so RTN4 also called Nogo (neurite outgrowth inhibitor protein).Ectopic expressed RTN3 blocked the protein transport between the ER and Golgi, and could to cause the ER overload response, so in the result of the ER Ca²⁺ depletion triggered cell apoptosis.RTN4-C (Nogo-C) is the smallest protein of RTN4. Our previous study showed that overexpression of the RTN4-C might inhibit SMMC7721 cell growth and promote cell apoptosis. To investigate the mechanism of RTN4-C inhibition of cell growth and pro-apoptosis, the yeast two-hybrid method was adopted.1. Obtained RTN3 interacted with RTN4-C when using yeast two-hybrid system to screen mouse 7 days embryo library with RTN4-C as a bait.In this work we used the full length of RTN4-C as a bait to screen mouse 7 day embryo library, the results were checked, and sequenced the positive clones. At last we got three proteins interacted with RTN4-C, RIREN2310016E02, RTN3, Gpihbp1. Further we found RIREN2310016E02 bind RTN4-C is most strong, the next is RTN3, the last one is Gpihbp1. But there are few reports about RIREN2310016E02 and Gpihbp1. So we chose RTN3 for further research.2. RTN4-C can interact with RTN3 using co-immunoprecipitationIn this part we use co-immunoprecipitation to proof the interaction of RTN3 and RTN4-C. In the experiment RTN4-C was fused with Myc tag, and RTN3 was fused with HA tag, the fusion proteins were over expressed in HEK293 cell. First we used the Myc antibody to immunoprecipitation and detect with HA antibody. Next we used the HA antibody to immunoprecipitation and detect with Myc antibody. The results show that RTN4-C interacted with RTN3 in mammalian cells with overexpression.3. RTN4-C and RTN3 are co-localization in mammalian cellsThe proteins that can interacted usually co-localized in mammalian cells, in this work we also to detect the distribution of RTN4-C and RTN3. RTN3 and RTN4-C have the same distribution in cell with immunofluorescence, in order to confirm the result we construct fusion proteins in which RTN4-C fused with GFP and RTN3 fused with RFP. The fusion proteins expressed in Hela cells and find the proteins co-localized through fluorescence microscope.4. RTN4-C interacted with RTN3 through its hydrophobic regionIn the work we found which part of RTN4-C is important for the interaction with RTN3. We construct different deletion mutants, and co-immunopreciptation with RTN3 respectively. The results show that the hydrophobic region is essential for the interaction of RTN4-C and RTN3.5. The interaction of RTN4-C and RTN3 to suppress their pro-apoptosis abilityWe used FACS to detect the apoptosis of HEK293, in which RTN4-C and/or RTN3 overexpressed. The efficiency of apoptosis is lower when co-expressed RTN4 and RTN3 than they expressed respectively. In summary, our results show a direct interaction between RTN4-C and RTN3, and the hydrophobic transmembrane domains are important for the interaction, and RTN3 interacts with RTN4-C and inhibit its apoptotic activity.