PARTITIONING OF PROTEINS INTO PLASMA-MEMBRANE MICRODOMAINS - CLUSTERING OF MUTANT INFLUENZA-VIRUS HEMAGGLUTININS INTO COATED PITS DEPENDS ON THE STRENGTH OF THE INTERNALIZATION SIGNAL

Internalization of membrane proteins involves their recruitment into p lasma membrane clathrin-coated pits, with which they are thought to in teract by binding to AP-2 adaptor protein complexes, To investigate th e interactions of membrane proteins with coated pits at the cell surfa ce, we applied image correlation spectroscopy to measure directly and quantitatively the clustering of influenza hemagglutinin (HA) protein mutants carrying specific cytoplasmic internalization signals. The HA system enables direct comparison between isolated internalization sign als, because HA itself is excluded from coated pits, The studies prese nted here provide, for the first time, a direct quantitative measure f or the degree of clustering of membrane proteins in coated pits at the cell surface, The degree of clustering depended on the strength of th e internalization signal and on the integrity of the clathrin lattices and correlated with the internalization rates of the mutants, The clu stering of the HA mutants fully correlated with their ability to co-pr ecipitate alpha-adaptin from whole cells, the first such demonstration for a membrane protein that is not a member of the epidermal growth f actor receptor family. Furthermore, both the clustering in coated pits and the co-precipitation with alpha-adaptin were dramatically reduced in the cold, suggesting that low temperature can interfere with the s orting of proteins into coated pits. In addition to the specific resul ts reported here, the general applicability of the image correlation s pectroscopy approach to study any process involving the clustering or oligomerization of membrane receptors at the cell surface is discussed .