ADP-ribosylation factor 1 and its activation of phospholipase D are important for the assembly of very low density lipoproteins

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low d ensity lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells, Transient expression of ARF-1(T31N), a GDP-re strictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) V LDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the se cond step of VLDL assembly) and without altering total protein production o r biosynthesis of transferrin, phosphatidylcholine, or triglycerides, These effects were confirmed in stable inducible transfectants. In contrast, exp ression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, d id not affect VLDL assembly, Thus, active ARF-1 is needed for the second st ep of the process. To further explore these observations, we developed a ce ll-free system based on the postnuclear supernatant isolated from McA-RH777 7 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly d ependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL , Brefeldin A inhibited VLDL assembly in this system, as did a synthetic pe ptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displace s ARF-1 from the membrane, Thus, active ARF-1 is also needed for cell-free assembly of VLDL, Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL a ssembly in this system, indicating that the process requires ongoing hydrol ysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid ( PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas a-butanol, which does not inhibit PA formation, failed to do so. T hus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholi ne is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our result s indicate that ARF-1 participates in the second step of VLDL assembly thro ugh a process that involves activation of PLD and production of PA.