Topography of sialoglycoproteins and sialyltransferases in mouse and rat liver Golgi.
We have studied the membrane topography of N-acetylneuraminic acid (NeuAc) in sialoglycoproteins of mouse and rat liver Golgi apparatus vesicles. Purified Golgi vesicles or Golgi vesicles present in microsomes were able to incorporate radiolabeled NeuAc from CMP-NeuAc into endogenous glycoproteins in vitro. The labeled glycoproteins showed a similar profile on sodium dodecyl sulfate gel electrophoresis to those isolated from Golgi fractions of animals injected with radiolabeled NeuAc. Using this labeling procedure in vitro, we found that only 5 to 20% of the incorporated NeuAc could be removed from intact vesicles upon treatment with neuraminidase or proteases. However, disruption of vesicles by detergents or mechanical means prior to the enzymatic treatments resulted in removal of labeled NeuAc; the extent of NeuAc removal could be correlated with that of vesicle disruption as measured by loss of latency of thiamine pyrophosphatase activity. This strongly suggests that NeuAc residues of glycoproteins are on the lumenal side of the Golgi apparatus membrane and that Golgi-derived vesicles have the same membrane orientation as in vivo. A similar approach was used to infer a lumenal orientation of sialyltransferases. Not all Golgi apparatus preparations routinely used may be suitable for membrane topography experiments of the kind described here.
D000818:Animals; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D006056:Golgi Apparatus / Q000032:analysis* / Q000201:enzymology; D008099:Liver / Q000032:analysis* / Q000201:enzymology; D051379:Mice; D008862:Microsomes, Liver / Q000032:analysis / Q000201:enzymology; D009439:Neuraminidase; D011402:Pronase; D051381:Rats; D012795:Sialoglycoproteins / Q000032:analysis*; D012799:Sialyltransferases / Q000032:analysis*; D014166:Transferases / Q000032:analysis*; D014357:Trypsin
D012795:Sialoglycoproteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D014166:Transferases; D012799:Sialyltransferases; D009439:Neuraminidase; D014357:Trypsin; D011402:Pronase