Intracellular processes associated with glycoprotein transport and processing.
The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.
Slomiany
A
A
Research Center, University of Medicine and Dentistry of New Jersey, Newark 07103-2400.
Grzelinska
E
E
Grabska
M
M
Yamaki
K
K
Tamura
S
S
Kasinathan
C
C
Slomiany
B L
BL
D000818:Animals; D001693:Biological Transport, Active; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008854:Microscopy, Electron; D009077:Mucins / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D013270:Stomach
D008562:Membrane Glycoproteins; D008563:Membrane Lipids; D009077:Mucins; D010713:Phosphatidylcholines