The tight junction protein ZO-2 has several functional nuclear export signals.
The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.
González-Mariscal
Lorenza
L
Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politécnico Nacional 2508, México, D.F., 07360, México. lorenza@fisio.cinvestav.mx
Ponce
Arturo
A
Alarcón
Lourdes
L
Jaramillo
Blanca Estela
BE
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D049790:Nuclear Export Signals / Q000235:genetics / Q000502:physiology*; D010047:Ovalbumin / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019108:Tight Junctions / Q000737:chemistry*; D014162:Transfection; D062827:Zonula Occludens-2 Protein
D008565:Membrane Proteins; D049790:Nuclear Export Signals; D011993:Recombinant Fusion Proteins; D062827:Zonula Occludens-2 Protein; D010047:Ovalbumin