Probing the cis-arrangement of prototype tight junction proteins claudin-1 and claudin-3

Claudins form a large family of tight junction (TJ) proteins featuring four transmembrane segments (TM1 to 4), two extra- and one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the TJ-free cell line HEK 293. By Förster/fluorescence resonance energy transfer (FRET) analysis, the proximity of claudin N- and C-termini integrated in homopolymeric strands composed of claudin-3 or of heteropolymeric strands composed of claudin-1 and claudin-3 were determined. Main results are that (i) within homo- and hetero-polymers the average distance between the cytoplasmic ends of the TM1s of cis-interacting claudin molecules is shorter than the average distance between their TM4s and (ii) TM1 segments of neighbouring claudins are oriented towards each other as the cytoplasmic end of TM1 is in close proximity to more other TM1 segments than TM4 to other TM4 segments. The results indicate at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers. The data provide novel ins...

Source: BJ Cell - April 7, 2015 Category: Biochemistry Authors: S Milatz, J Piontek, J Schulzke, I E. Blasig, M Fromm, D Günzel Tags: BJ Biomolecules Source Type: research