Phospholipase D2 is required for efficient endocytic recycling of transferrin receptors.

RNA interference-mediated depletion of phospholipase D2 (PLD2), but not PLD1, inhibited recycling of transferrin receptors in HeLa cells, whereas the internalization rate was unaffected by depletion of either PLD. Although reduction of both PLD isoforms inhibits PLD activity stimulated by phorbol 12-myristic 13-acetate, only depletion of PLD2 decreased nonstimulated activity. Cells with reduced PLD2 accumulated a greater fraction of transferrin receptors in a perinuclear compartment that was positive for Rab11, a marker of recycling endosomes. EFA6, an exchange factor for Arf6, has been proposed to stimulate the recycling of transferrin receptors. Thus, one consequence of EFA6 overexpression would be a reduction of the internal pool of receptors. We confirmed this observation in control HeLa cells; however, overexpression of EFA6 failed to decrease the internal pool of transferrin receptors that accumulate in cells previously depleted of PLD2. These observations suggest that either PLD2 is required for a constitutive Arf6-mediated recycling pathway or in the absence of PLD2 transferrin receptors accumulate in recycling endosomes that are not responsive to overexpression of EFA6.

Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts.

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein-raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein-raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains.

Features of influenza HA required for apical sorting differ from those required for association with DRMs or MAL.

The influenza virus hemagglutinin (HA) is sorted to the apical membrane in polarized epithelial cells and associates with detergent-resistant membranes (DRMs). By systematic mutagenesis of the transmembrane residues, we show that hemagglutinin requires 10 contiguous transmembrane amino acids to enter detergent-resistant membranes and that the surface of the trimeric hemagglutinin transmembrane domain facing the lipid environment as well as that facing the interior of the trimer is important for stable association with detergent-resistant membranes. However, association with detergent-resistant membranes was not required for apical sorting. MAL/VIP17 is a protein that is required for apical transport and a small fraction of hemagglutinin co-precipitates with MAL. Mutations that prevented HA from being isolated in detergent-resistant membranes decreased co-precipitation with MAL. The hemagglutinin and MAL that co-precipitated were contained in a detergent-resistant vesicle. However, most of the co-precipitation of newly synthesized hemagglutinin with MAL occurred only after the majority of hemagglutinin reached the cell surface. Both the timing and the limited extent of co-precipitation suggest that the majority of vesicles containing hemagglutinin and MAL are not the detergent-resistant membrane transport intermediates carrying hemagglutinin from the TGN to the apical surface.