DYNAMIC OR STABLE INTERACTIONS OF INFLUENZA HEMAGGLUTININ MUTANTS WITH COATED PITS - DEPENDENCE ON THE INTERNALIZATION SIGNAL BUT NOT ON AGGREGATION

Measurements of the lateral mobility of native and mutated membrane pr oteins, combined with treatments that alter clathrin lattice structure , are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I. (19 91) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state o f the protein, we have extended this approach to investigate the inter actions between coated pits and several influenza hemagglutinin (HA) m utants, which differ in the internalization signals in their short cyt oplasmic tails. The lack of internalization signals in the trimeric wi ld-type HA enables a direct comparison between specific internalizatio n signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and va ried in their tendency to aggregate into complexes larger than trimers . Our results indicate that the mode of interaction with coated pits ( transient association-dissociation versus stable entrapment) depends o n the internalization signal and affects the internalization efficienc y. Mutants that contain a strong internalization signal and undergo fa st endocytosis were entrapped in coated pits for the entire duration o f the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal inte rnalization signal, which was internalized 10-fold slower, exhibited t ransient interactions with coated pits. Both types of interactions dis appeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following th e ''freezing'' of coated pits by cytosol acidification. Unlike the dep endence on the cytoplasmic internalization signal, the interactions wi th coated pits did not depend on the aggregation state (measured by su crose gradient centrifugation after solubilization in n-octylglucoside ) of the mutants.