Effects of ARF1 and phospholipase D on the assembly and secretion of apoB-100 containing lipoproteins

Abstract

Hydrophobic insoluble lipids such as triglycerides and cholesterol ester are transported in spherical, water-soluble molecular complexes called lipoproteins. Lipoproteins are secreted mainly from the liver and intestine into the plasma for distribution to peripheral tissues. In this thesis, the assembly and secretion of one form of these lipoproteins have been studied: very low density lipoproteins (VLDL). Apolipoprotein (apo) B-100 is the major structural protein of VLDL and is essential for assembly of the particle. The assembly of VLDL has been suggested to involve two relatively well-defined steps. In the first step, apoB-100 is co-translationally translocated through the ER membrane forming pre-VLDL with a density corresponding to high density lipoproteins (HDL) or low density lipoproteins (LDL). The second step is thought to include the formation of a apoB- free lipid droplets in the ER lumen, which are fused en bloc with pre-VLDL forming VLDL. The results presented show that apoB-100 is sorted to degradation at different levels in the secretory pathway. Thus, membrane-associated apoB-100 was degraded via an ATP-dependent pathway, most likely corresponding to the recently discovered proteasomal pathway. ApoB-100 present on HDL and LDL particles in the secretory pathway was degraded by an ALLN-sensitive degradation pathway. The GTP-binding protein ADP ribosylation factor 1 (ARF1) inhibited the non-proteasomal degradation of apoB-100. ARF1 was also essential for the second step in the assembly of apoB-100 VLDL, giving a molecular explanation for the inhibitory effect of brefeldin A on the process. Phospholipase D (PLD) activity stimulated the assembly of apoB-100 VLDL in a cell-free system reconstituting the assembly process. Moreover, the results indicate that ARF1 mediates its effect on the assembly of VLDL by activating PLD. The activity of PLD was also essential for the formation of small cytosolic lipid droplets in a cell-free system. Thus, the dependence on PLD for both of these processes may suggest a common mechanism for the formation of small lipid droplets, either they are sorted to the cytosol or sorted to the ER lumen and secreted as VLDL.