Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking

Samata Tiwari,Shaila Siddiqi,Olga Zhelyabovska,Shadab A Siddiqi

doi:10.1074/jbc.m115.705269

Samata Tiwari, Shaila Siddiqi + Show 2 more

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https://doi.org/10.1074/jbc.m115.705269

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Abstract

The transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.

Highlights

very low density lipoprotein (VLDL) secretion from the liver as evident by both in vitro and in vivo studies [1,2,3]
small VCP-interacting protein (SVIP) Is Localized to the Hepatic endoplasmic reticulum (ER), Cytosol, Golgi, and VLDL transport vesicle (VTV) in Primary Hepatocytes—Because VLDL is synthesized in the ER and transported first to the Golgi and to the plasma membrane for its eventual secretion, we wished to determine the distribution of SVIP in subcellular organelles associated with its synthesis and intracellular trafficking in primary rat hepatocytes
We observed a significant increase in TAG secretion in a concentration-dependent manner from myristic acid-treated hepatocytes as compared with control hepatocytes, which were treated with bovine serum albumin (BSA) alone (Fig. 4A)

Summary

SVIP Facilitates ER Exit of Nascent VLDL

Because the VTV carries a distinct large cargo, nascent VLDL, we expect that proteins in addition to COPII components would be involved in VTV biogenesis and VLDL packaging. SVIP inhibits endoplasmic reticulum-associated degradation (ERAD) either by blocking the formation of a trimeric complex (composed of VCP/p97, derlin, and Grp78), which mediates ERAD, or by dissociating the trimeric complex [26]. This inhibitory function of SVIP indicates its role in nascent protein transport from the ER to the Golgi. Because SVIP blocks the interaction between Grp and other proteins of ERAD, it might protect apoB100 from degradation and facilitate VLDL secretion. Our data indicate a functional role of SVIP in the formation of the nascent VLDL-containing vesicle, VTV, from the ER membranes. Our results suggest that silencing of SVIP using siRNA reduces the VLDL secretion from the hepatocyte

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