Intracellular interactions between protein 4.1 and glycophorin C on transport vesicles, as determined by fluorescence correlation spectroscopy

Interaction of protein 4.1 (4.1R) with the transmembrane protein glycophorin C (GPC) regulates the functions of erythrocyte membrane. Fluorescence correlation spectroscopy (FCS) was used to define the interaction of EGFP-4.1R with DsRed-GPC on transport vesicles (TVs) by measuring their fluctuation in living cells. DsRed-GPC expressed in HeLa cells was delivered to the plasma membrane through slow vesicle transport. EGFP-4.1R, which freely diffused in the cytosol when expressed alone, diffused slowly when co-expressed with DsRed-GPC, indicating association of EGFP-4.1R with TVs. Fluorescence cross-correlation spectroscopy (FCCS) showed direct interaction of EGFP-4.1R with DsRed-GPC on TVs. The present study demonstrates that 4.1R binds to GPC on TVs in living cells.Structured summary of protein interactionsGPC and 4.1R30physically interact by fluorescence correlation spectroscopy (View interaction)

► Interaction of protein 4.1R with glycophorin C (GPC) stabilizes erythrocyte membranes.
► Fluorescence correlation spectroscopy was adopted to clarify the completion order.
► 4.1R diffuses very slowly only in the cytosol of GPC co-expressing HeLa cells.
► Specific interaction of 4.1R with GPC on transporting vesicle (TV) was also proved.
► These data suggested that 4.1R-GPC interaction actually started on TV in living cells.