ADP Ribosylation Factor and a 14-kD Polypeptide Are Associated with Heparan Sulfate-carrying Post-trans-Gol Network Secretory Vesicles in Rat Hepatocytes

heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with psS]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN-), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was ,,o100200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein B-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylatlon factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein