Lennart Asp

The assembly and secretion of apolipoprotein B-containing lipoproteins

The assembly of lipoproteins containing apolipoprotein B is a complex process that occurs in the lumen of the secretory pathway. The process consists of two relatively well-identified steps. In the first step, two VLDL precursors are formed simultaneously and independently: an apolipoprotein B-containing VLDL precursor (a partially lipidated apolipoprotein B) and a VLDL-sized lipid droplet that lacks apolipoprotein B. In the second step, these two precursors fuse to form a mature VLDL particle. The apolipoprotein B-containing VLDL precursor is formed during the translation and concomitant translocation of the protein to the lumen of the endoplasmic reticulum. The VLDL precursor is completed shortly after the protein is fully synthesized. The process is dependent on the microsomal triglyceride transfer protein (MTP). Although the mechanism by which the lipid droplets are formed is unknown, recent observations indicate that the process is dependent on MTP. The fusion of the two precursors is not dependent on MTP, but the mechanism remains to be elucidated. The conversion of the apolipoprotein B-containing precursor to VLDL seems to be dependent on the ADP ribosylation factor 1 (ARF 1) and its activation of phospholipase D. During their assembly, nascent apolipoprotein B chains undergo quality control and are sorted to degradation. Such sorting, which occurs cotranslationally during the formation of the apolipoprotein B-containing precursor, involves cytosolic chaperons and ubiquitination that targets apolipoprotein B to proteasomal degradation. Other levels of sorting occur in the secretory pathway. Thus, lysosomal enzymes are involved as well as the LDL receptor.

PLD1 and ERK2 regulate cytosolic lipid droplet formation

We have previously uncovered roles for phospholipase D (PLD) and an unknown cytosolic protein in the formation of cytosolic lipid droplets using a cell-free system. In this report, PLD1 has been identified as the relevant isoform, and extracellular signal-regulated kinase 2 (ERK2) as the cytosolic protein. Increased expression of PLD1 increased lipid droplet formation whereas knockdown of PLD1 using siRNA was inhibitory. A role for ERK2 in basal lipid droplet formation was revealed by overexpression or microinjection, and ablation by siRNA knockdown or pharmacological inhibition. Similar manipulations of other Map kinases such as ERK1, JNK1 or JNK2 and p38α or p38β were without effect. Insulin stimulated the formation of lipid droplets and this stimulation was inhibited by knockdown of PLD1 (by siRNA) and by inhibition or knockdown (by siRNA) of ERK2. Inhibition of ERK2 eliminated the effect of PLD1 on lipid droplet formation without affecting PLD1 activity, suggesting that PLD1 functions upstream of ERK2. ERK2 increased the phosphorylation of dynein which increased the amount of the protein on ADRP-containing lipid droplets. Microinjection of antibodies to dynein strongly inhibited the formation of lipid droplets, demonstrating that dynein has a central role in this formation. Thus dynein is a possible target for ERK2.

Adipocyte Differentiation-Related Protein Promotes Fatty Acid Storage in Cytosolic Triglycerides and Inhibits Secretion of Very Low–Density Lipoproteins

Objective—We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low–density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. Methods and Results—An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into &bgr;-oxidation. Conclusions—ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to &bgr;-oxidation and promotes the secretion of apoB-48 VLDL1.

Intracellular assembly of VLDL: two major steps in separate cell compartments.

The assembly of very low-density lipoproteins (VLDL) occurs in two major steps. The first step is the co-and post-translational lipidation of apoB, forming pre-VLDL in the rough endoplasmic reticulum. The microsomal triglyceride transfer protein catalyzes this step. In the second step pre-VLDL is converted to bona fide VLDL in a smooth membrane compartment. This step depends on ADP-ribosylation factor 1 and its activation of phospholipase D.

Early Stages of Golgi Vesicle and Tubule Formation Require Diacylglycerol

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.

Assembly of Very Low Density Lipoprotein: A Two-Step Process of Apolipoprotein B Core Lipidation

The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically.

Intracellular Assembly and Degradation of Apolipoprotein B-100-containing Lipoproteins in Digitonin-permeabilized HEP G2 Cells

Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells. Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein. Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway. Neither albumin nor α1-antitrypsin showed any significant posttranslational intracellular degradation under the same condition. The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetyl-leucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein. Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100. This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100. The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse-labeled apoB-100 from the luminal lipoprotein-associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded. The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense (“high density lipoprotein-like”) particles. This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis. Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane- associated form of apoB-100. These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.

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

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.

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5′-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.