The Assembly and Secretion of Apolipoprotein B-48-containing Very Low Density Lipoproteins in McA-RH7777 Cells

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thesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 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 dependent 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 peptide (corresponding to Nterminal amino acids 2-17 of ARF-1) that displaces 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 assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

Section: Discussionsupporting

confidence: 81%

ADP-ribosylation Factor 1 and Its Activation of Phospholipase D Are Important for the Assembly of Very Low Density Lipoproteins

Asp¹,

Claesson²,

Borén³

et al. 2000

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“…For analysis by mass spectrometry, the gels were silver-stained as described (48). Protein bands were cut out, treated with trypsin, and analyzed by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as described (47), except that Mascot software (available at www.matrixscience.com) was used for protein assignment. If the spectra were not good enough, the samples were cleaned up and concentrated with a ZipTip containing C 18 material (Millipore) before the MS run.…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE and immunoblotting were carried out as described (43,47). For analysis by mass spectrometry, the gels were silver-stained as described (48).…”

Section: Methodsmentioning

confidence: 99%

A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System

Marchesan

Rutberg

Andersson

et al. 2003

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We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d < 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TGcontaining structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 ؋ g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.Within the cell, triglycerides (TG) 1 are stored in cytosolic lipid droplets. Little is known about how these structures are assembled. Several proteins have been identified on their surface, including adipocyte differentiation-related protein (ADRP or adipophilin), perilipins (reviewed in Refs. 1 and 2), and caveolin (see Refs. 3-5 and reviewed in Refs. 2 and 6), but their roles in lipid droplet assembly are unknown.ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16,18).Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (19 -22), either as an intracellular messenger or as a cone-shaped l...

“…Finally, studies by Stillemark et al (13) and Asp et al (14) provide details of the OA-induced conversion of an apoB-48 VLDL in McA RH7777 cells but did not clearly identify the site of the bulk addition of core lipid. Their studies suggest that the addition of bulk lipid required some vesicular transport from the rough ER to a more distal compartment, but they could not delineate among the smooth ER, the vesicular tubular structure (also known as the ER Golgi intermediate compartment) or the early cis-Golgi (4).…”

Section: Figmentioning

confidence: 99%

“…Recent studies suggest that transformation may require either lateral diffusion of apoB to a specialized compartment of the ER or vesicular transport of apoB from one secretory compartment to another (4,13,14), but a review of the literature indicates that significant uncertainty exists regarding this issue (2,5,(15)(16)(17)(18)(19)(20). In the studies presented here, using pharmacological and subcellular fractionation approaches we demonstrate that the oleic acid (OA)-induced conversion of apoB100 LDL/HL particles to VLDL in McA RH7777 cells occurs in the ER and not in the Golgi.…”

mentioning

confidence: 99%

The Conversion of ApoB100 Low Density Lipoprotein/High Density Lipoprotein Particles to ApoB100 Very Low Density Lipoproteins in Response to Oleic Acid Occurs in the Endoplasmic Reticulum and Not in the Golgi in McA RH7777 Cells

Yamaguchi

Gamble

Conlon

et al. 2003

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35 S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.Although the early, crucial steps in apolipoprotein B100 (apoB100)-lipoprotein assembly, including translation, translocation, and targeting of apoB100 for the ubiquitin-proteasomal degradation pathway have been well studied (1), much less is known about how the mature, triglyceride (TG) 1 -enriched lipoprotein is assembled. Thus, the exact process of bulk lipid addition and, in particular, the site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) to form mature, TG-enriched very low density lipoproteins (VLDL) has not been identified definitively. Early work by Hamilton and co-workers using electron microscopy (2), suggests that a lipid-poor form of apoB present in the rough endoplasmic reticulum (ER) could fuse with lipid droplets present in the smooth ER. That work first raised the possibility that VLDL was assembled in a two-step process. This scheme has been supported by studies from several laboratories (3-5), including our own (6). The two-step model includes the transformation of an "HDL-like" apoB-lipoprotein to a TG-enriched lipoprotein in the presence of adequate cell ...