Role of ADP Ribosylation Factor 1 in the Assembly and Secretion of ApoB-100–Containing Lipoproteins

Abstract

Objective-We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs). Methods and Results-The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15°C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously. Conclusions-Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids. Key Words: ADP ribosylation factor 1 Ⅲ apolipoprotein B Ⅲ coatamer 1 Ⅲ intracellular transport Ⅲ very-low-density lipoproteins A polipoprotein B (apoB)-containing very-low-density lipoproteins (VLDLs) are formed in a 2-step process. 1-3 In the first step, apoB forms a partially lipidated particle (a primordial lipoprotein or pre-VLDL) during its cotranslational translocation to the lumen of the endoplasmic reticulum (ER). This step involves the transfer of lipids to apoB catalyzed by the microsomal triglyceride transfer protein. In the absence of lipids or microsomal triglyceride transfer protein, the translocation of apoB is halted, and the protein is retracted through the translocon and degraded by proteasomes. 4 -8 The second step, in which the major amount of triglycerides is added to pre-VLDL, is less well-characterized. This step involves the formation of apoB-free lipid droplets in the smooth ER 9 that associate with apoB-containing pre-VLDL in a compartment that is separate from the rough ER. 9,10 Thus, transport and sorting processes involved in the transfer of proteins out of the rough ER may be important for the assembly of VLDL. Consistent with this possibility, the second step can be inhibited by brefeldin A 11 and is dependent on ADP ribosylation factor 1 (ARF1) and phospholipase D (PLD) activity. 12 ARF1, a member of the Ras superfamily of GTP-binding proteins, participates in the formation of coatamer I (COPI) secretory vesicles, which perform retrograde transport from the Golgi to the ER and within the Golgi stacks. 13 ARF1 and COPI are important for the anterograde transport from the ER to the Golgi apparatus 14 -16 , and ARF1 activates PLD1. [17][18][19] In...

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

“…Furthermore, the inhibition of ADP-ribosylation factor 1 (ARF1) by brefeldin A and expression of dominant negative mutants of ARF1 abolished HCV secretion (21). ARF1, a small GTPase protein is a modulator of trafficking to and from the Golgi, is also required for the assembly and secretion of VLDL particles (22)(23)(24). Together, these studies underscore the importance of VLDL egress through the Golgi secretory pathway for HCV virion secretion.…”

Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

“…McA-RH7777 cells expressing both human apoB-100 and apoC-III were cultured in DMEM containing 20% FBS for 1 h, and lysed in NP-40 buffer ( 37 ). The cell lysates were incubated with preimmune rabbit serum and Sepharose CL-4B beads for 2 h, followed by pelleting the beads by centrifugation (13,000 g , 10 min, 4°C).…”

“…Expression of aberrant ADP ribosylation factor 1 (ARF1 that activates PLD1) ( 37 ) or treatment with brefeldin A (inhibitor of ARF1) ( 31,33 ) effectively prevented TAG-rich VLDL secretion with little effect on the denser particles. Thus, maturation of VLDL probably not only requires suffi cient lipid substrate (mainly TAG) availability, but it also depends on the protein factors that effectively mediate the fusion of lipid droplets with apoB to form TAG-rich VLDL.…”

Role for ADP Ribosylation Factor 1 in the Regulation of Hepatitis C Virus Replication

“…Moreover, proteins that interact with Arf1 GEFs and that have been implicated in HCV RNA replication, such as Rab1 (64,65), have been identified by proteomic analyses as LD-associated proteins in coreexpressing cells (59) and other cells (80). Arf1 has been suggested to be involved in the biogenesis of VLDL (4,5). Given that LDs and VLDL function in the HCV life cycle, these observations prompted our hypothesis that LD-associated Arf1 plays an important role in HCV RNA synthesis and viral particle biogenesis.…”