Purchase this article with an account.
J Fan, AK Donovan, DR Ledee, PS Zelenka, AB Chepelinsky; Identification of Proteins Interacting with the Lens Major Intrinsic Protein (MIP)/Aquaporin 0 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4639.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Major Intrinsic Protein (MIP)/Aquaporin 0, which is required for lens transparency, is specifically expressed in the lens fiber membranes. Our study sought to identify proteins that interact with MIP, using the yeast two-hybrid system, and to characterize these interacting proteins with respect to MIP functions. Methods: MIP cDNA fragments encoding either the N-terminal 47 amino acids or the C-terminal 74 amino acids were used as baits to screen a rat lens cDNA yeast two-hybrid library. The library was constructed with the HybriZAP-2.1 XR Library Construction Kit (Stratagene) and contains cDNAs isolated from 18-day rat embryo lenses. The cDNA inserts from positive clones that grew on His-lacking medium and expressed LacZ (blue colonies) were sequenced with an automated Beckman-Coulter DNA sequencer. The identities of the cDNA inserts were determined by BLAST searches of the GenBank. Results: The yeast two-hybrid screening using the MIP N-terminal or C-terminal fragment as the bait led to eleven independent positive clones. BLAST searches of DNA and protein databases showed that three of these positive clones encode a protein that is in the correct reading frame. Two of the three in-frame clones have 100% identity to the rat ribosomal protein L4; the other one corresponds to the rat gamma-E-crystallin. Conclusion: Identification of proteins that interact with lens MIP may provide insight into the biological functions of MIP. Experiments are underway to demonstrate whether the positive clones encode proteins that interact specifically with MIP in vivo. Further experiments will allow us to confirm whether these protein-protein interactions have any physiological relevance to MIP functions.
This PDF is available to Subscribers Only