Internalization-competent Influenza Hemagglutinin Mutants Form Complexes with Clathrin-deficient Multivalent AP-2 Oligomers in Live Cells

Abstract

Most membrane proteins are endocytosed through clathrin-coated pits via AP-2 adaptor complexes. However, little is known about the interaction of internalization signals with AP-2 in live cells in the absence of clathrin lattices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order complexes via binding to AP-2, we exploited the inability of HAs from different influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails mutated to include internalization signals. Using antibody-mediated immunofluorescence co-patching on live cells, we demonstrate that internalization-competent HA mutants form higher order complexes and that this clustering depends on the strength of the internalization signal. The clustering persisted in cells treated with hypertonic medium to disperse the clathrin lattices, as validated by co-immunoprecipitation experiments. The clustering of HAs bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of α-adaptin with these HAs, even in hypertonically treated cells; (ii) the co-localization (after hypertonic treatment) of AP-2 with antibody-mediated patches of these mutants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. These results suggest that even in the absence of clathrin lattices, AP-2 exists in multivalent complexes capable of simultaneously binding several internalization signals from the same family.