Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging.
We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length ;long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the ;short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23.
Crane
Jonathan M
JM
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
Verkman
Alan S
AS
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1 / Q000737:chemistry; D051401:Aquaporin 4 / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004058:Diffusion; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion
D051401:Aquaporin 4; D050505:Mutant Proteins; D020033:Protein Isoforms; D051398:Aquaporin 1