VLDL exits from the endoplasmic reticulum in a specialized vesicle, the VLDL transport vesicle, in rat primary hepatocytes.
The movement of VLDL [very-LDL (low-density lipoprotein)] from the ER (endoplasmic reticulum) to the Golgi is required for its eventual secretion from hepatocytes and represents a potential target in controlling elevated concentrations of its metabolite LDL, the major determinant of atherosclerosis. To study this process, an in vitro ER-budding assay was developed to examine the generation of the VTV (VLDL transport vesicle) and PTV (protein transport vesicles) using ER isolated from [(14)C]TAG (triacylglycerol) and [(3)H]protein-labelled primary rat hepatocytes. VTVs do not contain albumin, as determined by immunoblots. VTVs were distributed in light-density fractions, whereas PTVs were mainly in the mid-portion of the sucrose gradient. Electron microscopy revealed that VTVs were larger ( approximately 100-120 nm) in size than PTVs ( approximately 55-70 nm). ER from 0.4 mM OA (oleic acid)-treated hepatocytes budded VTVs of a lighter density as compared with VTVs budded from ER of 0.1 mM or 0.004 mM OA-treated hepatocytes. The generation of VTVs from rat hepatic ER required cytosol, ATP, Sar1 (a GTPase) and incubation at 37 degrees C. Proteinase K treatment did not degrade the VTV cargo protein, apoB100 (apolipoprotein 100), indicating that VTVs were sealed. Immunoblots showed that VTV concentrated apoB100, Sar1 and rSec22b, and excluded albumin and calnexin. VTVs were shown to fuse with cis-Golgi and delivered their cargo to the Golgi lumen, as determined by in vitro fusion, and acquired endoglycosidase H resistance. These results suggest that a new ER-derived transport vesicle (VTV) has been identified and characterized which transports nascent VLDL from the hepatic ER to the Golgi.
Siddiqi
Shadab A
SA
The Division of Gastroenterology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA. ssiddiqi@utmem.edu
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008954:Models, Biological; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry; D014280:Triglycerides / Q000737:chemistry
D008079:Lipoproteins, VLDL; D011994:Recombinant Proteins; D014280:Triglycerides; D013395:Sucrose; D006026:Glycoside Hydrolases; D010450:Endopeptidases