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Function of V‐ATPase as a putative endosomal pH‐senor: mapping the interaction sites between V‐ATPase a2‐isoform and ARNO.
Author(s) -
Merkulova Maria,
Marshansky Vladimir
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1156.3
Subject(s) - gene isoform , endosome , atpase , microbiology and biotechnology , biology , function (biology) , cytosol , biochemistry , chemistry , biophysics , enzyme , gene , intracellular
Previously we demonstrated that V‐ATPase a2‐isoform directly interacts with ARNO. The a2‐isoform is specifically targeted to early endosomes and a2/ARNO interaction is modulated by acidification of the endosomal lumen. This acidification‐dependent process is crucial for trafficking within the endosomal/lysosomal protein degradative pathway. We propose that the V‐ATPase a2‐isoform functions as an endosomal pH‐sensor. However, the molecular mechanism of this novel function of V‐ATPase and its interaction with small GTPases remain obscure. Here we mapped the interaction sites between a2‐isoform and ARNO. First, using recombinant constructs containing various ARNO domains we demonstrated that cytosolic N‐terminal tail of the a2‐isoform (a2N) specifically interacts with plekstrin‐homology domain of ARNO. Next, we synthesized 22 peptides which are covering the complete sequence of a2N. Peptide pull‐down assay demonstrated that ARNO strongly interacts with one (a2N‐03) and weekly interacts with three (a2N‐11, a2N‐12 and a2N‐18) peptides. Interaction of a2N‐03 with ARNO was specific since it does not occur with GST. Based on these data, the first model of the folding of V‐ATPase a2‐isoform is proposed. We also suggest the two‐step mechanism of interaction of a2‐isoform with ARNO during which some conformational changes are taking place. This study was supported by NIH DK038452 and BADERC DK057521 grants.