PH-INDEPENDENT RETROGRADE TARGETING OF GLYCOLIPIDS TO THE GOLGI-COMPLEX

A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understo od. In the present work, we used bacterial toxins to study the retrogr ade pathway in Vero cells. The recombinant B subunit of verotoxin 1B ( VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to t he glycolipid globotriaosyl ceramide, entered endosomes by both clathr in-dependent and -independent pathways, reaching the Golgi complex. On ce internalized, the toxin-receptor complex did not recycle back to th e plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another gly colipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograd e transport of VT1B and CTB was unaffected by addition of weak bases i n combination with concanamycin, a vacuolar-type ATPase inhibitor. Rat io imaging confirmed that these agents neutralized the luminal pH of t he compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments ind icated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid ''rafts'' and that internalizat ion is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of t he secretory system and that assembly of F-actin is not required for t his process.