The uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several alterations. It is then enclosed in distinct post-Golgi VLDL transport vesicles (PG-VTVs) and released into the blood. The data generated in our lab has proved the requirement of ATP for PG-VTV biogenesis however, ATP substitution with non-hydrolyzable ATP analogue (ATPγS) had no effect on this process. Therefore, the present study is based on the hypothesis that ATP mediated protein phosphorylation regulates PG-VTV biogenesis. First, hepatic subcellular organelles were isolated and their purity was determined by performing Western blot. A cell-free in vitro budding assay was performed in presence or absence of ATP, GTP and cytosol using 3[H]-TAG labelled hepatic Golgi to generate PG-VTVs. We performed Western blotting to confirm distinct protein phosphorylation at tyrosine residue during PG-VTV formation however, protein phosphorylation event did not occur when PG-VTV budding was blocked. Two-dimensional gel electrophoresis identified Syntaxin 17 (STX17) as the phosphorylated protein required for PG-VTV formation. ATP mediated phosphorylation of STX17 during biogenesis of PG-VTVs was confirmed by its presence on PG-VTVs. PG-VTV budding was found to be significantly reduced on performing budding assay using STX17 immunodepleted cytosol compared to positive control. RNAi mediated knockdown of STX17 in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3[H]-TAG liquid scintillation counter. Based on these results, it can be justified that STX17 plays a vital role in regulating PG-VTV budding and overall VLDL secretion from hepatocytes.
Master of Science (M.S.)
College of Medicine
Length of Campus-only Access
Masters Thesis (Open Access)
Saxena, Anika, "PG-VTV Biogenesis Requires ATP to Facilitate Phosphorylation of Syntaxin 17" (2018). Electronic Theses and Dissertations. 5907.