In vitro synthesis of sulfated glycosaminoglycans coupled to inter-compartmental Golgi transport.
We have used isolated rat liver Golgi membranes to reconstitute the synthesis of sulfated glycosaminoglycans (GAGs) onto the membrane-permeable, external acceptor xyloside. Biosynthetic labeling of GAGs with [35S]sulfate in vitro is shown to have an absolute requirement for ATP and cytosolic proteins and is inhibited by dismantling the Golgi apparatus with okadaic acid or under mitotic conditions suggesting that inter-compartmental transport between Golgi cisternae is a prerequisite for the successful completion of the initiation, polymerization, and sulfation of GAGs. Accordingly, we show that in vitro synthesis of 35S-GAGs utilizes the same machinery employed in Golgi transport events in terms of vesicle budding (ADP-ribosylation factor and coatomer), docking (Rabs), targeting (SNAREs), and fusion (N-ethylmaleimide-sensitive factor). This provides compelling evidence that GAGs synthesis is linked to Golgi membrane traffic and suggests that it can be used as a complementation-independent method to study membrane transport in Golgi preparations from any source. We have applied this system to show that intra-Golgi traffic requires the function of the Golgi target-SNARE, syntaxin 5.
Imperial Cancer Research Fund, Cell Biology Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
C J
CJ
Fernández
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G
Warren
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006027:Glycosides / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D015739:Nocodazole / Q000494:pharmacology; D050765:Qa-SNARE Proteins; D051381:Rats
D000970:Antineoplastic Agents; D006025:Glycosaminoglycans; D006027:Glycosides; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; C010807:xylosides; C013786:A73025; D015739:Nocodazole