Association between segments of zonula occludens proteins: live-cell FRET and mass spectrometric analysis.
The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells.
Leibniz-Institut für Molekulare Pharmakologie, Berlin-Buch, Germany.
Christine
C
Rueckert
Victor
V
Castro
Corinna
C
Gagell
Sebastian
S
Dabrowski
Michael
M
Schümann
Eberhard
E
Krause
Ingolf E
IE
Blasig
Reiner F
RF
Haseloff
D000818:Animals; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry / Q000379:methods*; D054731:PDZ Domains / Q000502:physiology*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D017434:Protein Structure, Tertiary; D019108:Tight Junctions / Q000378:metabolism* / Q000502:physiology; D014162:Transfection; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein