IN-VITRO SYNTHESIS OF SULFATED GLYCOSAMINOGLYCANS COUPLED TO INTER-COMPARTMENTAL GOLGI TRANSPORT

We have used isolated rat liver Gels membranes to reconstitute the syn thesis of sulfated glycosaminoglycans (GAGs) onto the membrane-permeab le, external acceptor xyloside. Biosynthetic labeling of GAGs with [S- 35]sulfate in vitro is shown to have an absolute requirement for ATP a nd cytosolic proteins and is inhibited by dismantling the Golgi appara tus with okadaic acid or under mitotic conditions suggesting that inte rcompartmental transport between Golgi cisternae is a prerequisite for the successful completion of the initiation, polymerization, and sulf ation of GAGs, Accordingly, we show that in vitro synthesis of S-35-GA Gs utilizes the same machinery employed in Golgi transport events in t erms of vesicle budding (ADP-ribosylation factor and coatomer), dockin g (Rabs), targeting (SNAREs), and fusion (N-ethylmaleimide-sensitive f actor). This provides compelling evidence that GAGs synthesis is linke d to Gels membrane traffic and suggests that it can be used as a compl ementation-independent method to study membrane transport in Golgi pre parations from any source. We have applied this system to show that in tra-Golgi traffic requires the function of the Golgi target-SNARE, syn taxin 5.