Reggies/flotillins interact with Rab11a and SNX4 at the tubulo-vesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

Abstract

The lipid raft proteins reggie-1 and -2 (flotillins) were implicated in membrane protein trafficking but exactly how has remained elusive. We discovered that reggie-1 and -2 associate with the Rab11a, SNX4 and EHD1 decorated tubulo-vesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. shRNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduced the association of Rab11a with tubular structures and impaired the recycling of the transferrin (Tf)-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutive-active Rab11a rescued TfR recycling in reggie-deficient HeLa cells. Likewise, in a Ca2+ switch assay in reggie-depleted A431 cells, internalized E-cadherin was not efficiently recycled to the plasma membrane upon Ca2+ repletion. However, E-cadherin recycling was rescued by overexpression of constitutive-active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Interestingly, impaired recycling in reggie-deficient cells led to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway which is -like reggies- evolutionarily conserved.