Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.