Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

Dixon, John R.; Chattapadhyay, Ranjan; Huima, Tellervo; Redman, Colvin M.; Banerjee, Dev

doi:10.1083/jcb.117.6.1161

Cited by 47 publications

(13 citation statements)

References 37 publications

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“…13 ' 18 A small amount of fulllength apoB-100 could, however, be detected after the proteolysis, suggesting that a portion of the tightly membrane-bound pool of apoB-100 is completely translocated to the luminal side of the microsomal membrane. An alternative explanation that we cannot exclude is that this small amount of the full-length apoB-100 represents an incomplete degradation of the membrane-bound apoB-100.…”

Section: Discussionmentioning

confidence: 99%

Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Borén

Rustaeus

Wettesten

et al. 1993

Arterioscler Thromb

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Apolipoprotein B-100 (apoB-100) appears in three forms in the endoplasmic reticulum of Hep G2 cells: (1) tightly bound to the membrane, ie, not extractable by sodium carbonate. This form is glycosylated but protease sensitive when present in intact microsomes, suggesting that it is only partially translocated to the microsomal lumen; (2) extractable by sodium carbonate and present on low-density lipoprotein-verylow-density lipoprotein (LDL-VLDL)-like particles. This form is glycosylated and secreted into the medium; and (3) extractable by sodium carbonate but having a higher density than the LDL-VLDL-like particles. This form, referred to as Fraction I, is glycosylated and protected against proteases when present in intact microsomal vesicles, indicating that it is completely translocated to the luminal side of the microsomal membrane. Fraction I is not secreted into the medium, but it disappears with time from the cell, suggesting that it is degraded. Oleic acid induced a 2.7-fold increase in the rate of the biosynthesis of triacylglycerol but not of phosphatidylcholine in Hep G2 cells. Incubation of the cells with oleic acid had no significant effect on the rate of initiation of the apoB-100-containing lipoproteins, nor did it influence the amount of apoB-100 that was associated with the membrane or the turnover of apoB-100 in the membrane. Instead, it increased the proportion of the nascent apoB polypeptides on initiated lipoproteins that was converted into full-length apoB-100 on LDL-VLDL-like particles, giving rise to an increased amount of these particles in the lumen of the secretory pathway. Pulse-chase experiments showed that incubation with oleic acid gave rise to an increased formation of LDL-VLDL-like particles on behalf of the formation of Fraction I. This effect of oleic acid could partially explain the protective effect of the fatty acid on apoB-100, preventing it from undergoing posttranslational degradation. (Arterioscler Thromb. 1993;13:1743-1754 KEY WORDS • apoB-100 • Hep G2 cells • triacylglycerol biosynthesis A polipoprotein (apo) B-100 occurs mainly on the / \ liver-derived lipoproteins, ie, very-low-density li-JL A . poproteins (VLDLs), intermediate-density lipoproteins, and low-density lipoproteins (LDLs). ApoB-100 is essential for the assembly of these lipoproteins.The interaction between apoB-100 and the lipids can occur cotranslationally and simultaneously with the translocation of the protein to the lumen of the secretory pathway.1 ApoB-100 could also be bound to the endoplasmic reticulum (ER) membrane to be consigned to posttranslational degradation. 12 The incorporation of apoB into lipoprotein particles is first detected when the nascent polypeptide has reached a size of 80 to 100 kDa. These nascent polypeptides form particles with a diameter and density of a high-density lipoprotein (HDL) particle. As the nascent polypeptide increases in length, the size and lipid load of the particle that is being formed increase. Thus, the size of the nascent polypep-

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Section: Discussionmentioning

confidence: 99%

Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Borén

Rustaeus

Wettesten

et al. 1993

Arterioscler Thromb

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“…Until the quality control surveillance machinery is satisfi ed with the status of these domains, it appears that apoB-100 must remain accountable to the ERAD pathway. ApoB-100 associates with the membrane during initial stages of assembly and then is released into the lumen during VLDL particle maturation ( 28,29 ), coinciding with conformational changes in apoB in the Golgi ( 30 ). Cytosol-exposed apoB-100 has been observed in post-ER compartments after translation has been completed but perhaps before the fi nal maturation step ( 31 ).…”

Section: Discussionmentioning

confidence: 99%

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Fisher

Khanna

McLeod

2011

Journal of Lipid Research

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This article is available online at http://www.jlr.org Apolipoprotein (apo) B-100 is the major protein component of VLDLs. Assembly of VLDL in the liver begins at the endoplasmic reticulum (ER) with the formation of a primordial lipoprotein. As apoB-100 enters the ER lumen cotranslationally, it must associate with suffi cient lipids for VLDL assembly to proceed. The microsomal triglyceride transfer protein (MTP) facilitates transfer of lipids onto nascent apoB-100 ( 1 ). ApoB-100 is somewhat unique in that its secretion can be regulated by degradation ( 2 ), whereas control of expression of most proteins is at the level of mRNA transcription or translation. During conditions that limit lipid supply, such as low MTP activity ( 3 ) or reduced lipid availability ( 4, 5 ), apoB-100 is delivered to and degraded by the cytosolic proteasome in a process termed ER-associated degradation (ERAD). ApoB-100 contains large hydrophobic regions that require lipidation during apoB-100 synthesis or the nascent protein is targeted to ERAD ( 6 ). In a process that remains poorly defi ned, apoB-100 can be secreted only if lipidation/assembly satisfi es the quality control surveillance system in the secretory pathway.The ERAD pathway removes malfolded proteins from the ER lumen or membrane [reviewed in ( 7 )]. ERAD helps reduce the burden on ER-resident chaperones and allows the cell to maintain ER homeostasis. The typical ERAD pathway for a protein in the secretory pathway consists of at least the following steps: substrate recognition, retrotranslocation from the ER into the cytosol and ubiquitination, followed by degradation in the proteasome.Abstract Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase 1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumulation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retr...

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“…Whereas VLDL particles have been readily isolated from the Golgi (9, 10), efforts to identify HDL particles along the secretory pathway have had little success (9). Although these findings provided little support for the intracellular assembly of nascent HDL, a number of other studies supported the concept of an intracellular apoA-I lipidation pathway (11)(12)(13)(14)(15)(16). With the discovery of the ABCA1 transporter, many of these concepts concerning nascent HDL assembly have been revised, and recent studies support the idea of both ABCA1-dependent and -independent mechanisms responsible for apoA-I lipidation (17,18).…”

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confidence: 99%

Quality control in the apoA-I secretory pathway

Bhat

Zabalawi

Shelness

et al. 2004

Journal of Lipid Research

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From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143-186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [ 35 S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that Ͼ 90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant ( ⌬ 6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated ⌬ 6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation. These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6. -Bhat, S., M. Zabalawi, M. C. Willingham, G. S. Shelness, M. J. Thomas, and M. G. Sorci-Thomas. Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions.

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Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

Cited by 47 publications

References 37 publications

Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Quality control in the apoA-I secretory pathway

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