ANNEXIN-VI, A MARKER PROTEIN OF HEPATOCYTIC ENDOSOMES

Three highly purified endosomal fractions from rat liver were used to purify and characterize a major protein of endosomal membranes. Intrav enously injected ligands, which are taken up via receptor-mediated end ocytosis, accumulate first in the fraction of intermediate density, th e compartment of uncoupling of receptors and ligands. The high density membranous fraction is highly enriched in a receptor recycling compar tment. The endosomal fraction of lowest density is composed of multive sicular bodies, which appear to be the immediate prelysosomal compartm ent. The most prominent membrane protein of these endosomes is one of 68 kDa, as revealed by silver and Coomassie Brilliant Blue staining of SDS-gel electrophoretograms. This protein dominates profiles obtained from purified membranes of the compartment of uncoupling of receptors and ligands, multivesicular bodies, and receptor recycling compartmen t, but is greatly reduced in those obtained from plasma membranes and lysosomes. The 68-kDa protein was purified from endosomes and digested with trypsin, and cleavage products were analyzed by protein sequenci ng. The tryptic fragments of the endosomal 68-kDa protein share 96% id entity with corresponding sequences of mouse annexin VI and 91% identi ty with sequences of human annexin VI. Using immunoblots, high concent rations of annexin VI with an apparent molecular mass of 68 kDa were d etected in endosomal membranes by specific antiserum to annexin VI. Si gnificant amounts of annexin VI were also detected in Golgi membranes. Yet, the concentration was substantially lower than that of the three endosomal fractions. The association of annexin VI with endosomal mem branes is calcium-dependent, as revealed by the complete solubilizatio n from endosomal membranes by EGTA. Incubation of intact endosomes wit h Pronase leads to a complete degradation of annexin VI without any de tectable disintegration of proteins localized on the luminal surface o f endosomal membranes. Evidently, annexin VI is localized on the cytop lasmatic leaflet of the membrane of endosomes and may be of significan ce for their intracellular trafficking.