ADP-RIBOSYLATION FACTOR AND A 14-KD POLYPEPTIDE ARE ASSOCIATED WITH HEPARAN SULFATE-CARRYING POST-TRANS-GOLGI NETWORK SECRETORY VESICLES INRAT HEPATOCYTES

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase expe riments with [S-35]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugatio n in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lys osomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was similar to 100-200 nm as determined by electron microsco py. A typical coat structure as described for intra-Golgi transport ve sicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml ). Other constitutively secreted proteins (rat serum albumin, apolipop rotein E, and fibrinogen) could not be detected in purified HSPG-carry ing vesicles, but banded in the denser fractions of the Nycodenz gradi ent. Moreover, during pulse-chase labeling with [S-35]methionine, labe led albumin did not appear in the post-TGN vesicle fraction carrying H SPGs. These findings indicate sorting of HSPGs and albumin into differ ent types of constitutive secretory vesicles in hepatocytes. Two prote ins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD periphe ral membrane protein (VAPP14). Concerning the secretory pathway, we co nclude from these results that ADP ribosylation factor proteins are no t only involved in vesicular transport from the ER via the Golgi to th e TGN, but also in vesicular transport from the TGN to the plasma memb rane.