Abstract
Measurements of the lateral mobility of native and mutated membrane proteins, 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. (1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied 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 on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the "freezing" of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.
Highlights
Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells
To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails
The plasma membrane-coated pits are essentially immobile on the fluorescence photobleaching recovery (FPR) experimental time scale [46] and can retard the lateral mobility of membrane proteins destined for endocytosis
Summary
From the ‡Department of Biochemistry, The George S. In view of the large variety of internalization signals and the possible contribution of additional parameters, such as higher complex formation (aggregation) to the interactions with coated pits, it was desired to explore the relationships between the mode of these interactions (transient association/dissociation versus stable association) and internalization efficiency. To investigate this issue, we applied the lateral mobility-based approach to a series of influenza hemagglutinin (HA) mutants whose cytoplasmic domain contains different internalization signals. We have focused on HA mutants that were internalized at different rates [28] and varied in their tendency to form higher complexes
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