Abstract 390: VLDL Exits From The Endoplasmic Reticulum In A Specialized VLDL-transporting-vesicle (VTV) In Rat Primary Hepatocytes.

Abstract

Transport of very low-density lipoprotein (VLDL) from its site of synthesis, the endoplasmic reticulum (ER), to the Golgi is required for its eventual secretion from hepatocytes. This step represents a potential therapeutic target in controlling VLDL export and thus control of its metabolic derivative, LDL, the major carrier of cholesterol and determinant of atherosclerosis. The present study was designed to understand how VLDL exits from the ER at the molecular level. We developed an in vitro ER-budding assay in which rat liver ER (500 ug) was pre-loaded with 14 C-triacylglycerol (TAG) to mark VLDL and 3 H-proteins to mark newly synthesized proteins. The ER was incubated with rat liver cytosol (1 mg), GTP, and ATP at 37 o C for 30 min. The reaction mix was fractionated on a continuous sucrose gradient and the distribution of 14 C-TAG and 3 H-protein across the gradient was determined. 14 C-TAG was found in the light density region of the gradient, the expected place for TAG-rich VLDL carrying vesicles whereas 3 H-proteins appeared in the mid portion, the expected place in the gradient for protein vesicles. We examined the distribution of apolipoprotein B100 (apoB100), a marker for VLDL and albumin (a typical liver secretory protein) across the same gradient by Western blotting. As expected, apoB100 was distributed in light fractions whereas albumin was mainly in the mid portion of the gradient. These data show that VLDL and albumin are transported in vesicles of differing density. We hypothesize that a specialized vesicle is utilized for VLDL transport, which we name the VLDL-transporting-vesicle (VTV). Our results show that the release of VTV from rat liver ER requires cytosol, GTP, Sar1 (a GTPase), ATP, and incubation at 37 o C. VTV was sealed as judged by apoB100 signal post proteinase K treatment. VTVs concentrate ApoB100, Sar1, and exclude ER-resident protein calnexin. VTV fuses with liver cis -Golgi and delivers its cargo, VLDL, to the Golgi lumen. 2D-gels and electron microscopy data reveal that VTVs are different in their protein composition and are larger in size when compared to albumin carrying vesicles. In conclusion, we have identified and characterized a new ER-derived vesicle, VTV, which transports nascent VLDL from the ER to the Golgi in primary hepatocytes.