apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB

Abstract

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by ‫ف‬ 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion. -Gallagher, J. W., R. B. Weinberg, and G. S. Shelness. apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB.

ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins

“…Human apoA-IV and apoA-IV-KDEL expression plasmids were described previously ( 34 ). Plasmids containing the tetracycline regulator (pTet-On) and the tetracycline response element (pTRE2 hyg) were obtained from Clontech Laboratories, Inc. (Mountain View, CA).…”

“…For apoA-IV to facilitate the expansion of TG-rich lipoproteins, it must interact either directly with apoB or with the surface of apoBcontaining lipoproteins within the secretory pathway. In previous studies, we cotransfected apoA-IV, apoB, and MTP into COS cells and found that apoA-IV modifi ed with the C-terminal ER retention signal KDEL (apoA-IV-KDEL) inhibited the secretion of apoB constructs larger than apoB25, suggesting the existence of a protein-protein interaction between apoA-IV and specifi c sequences in the N-terminal region of the apoB molecule in the early stages of TG-rich lipoprotein assembly ( 34 ). Herein, we have extended these investigations to examine the effect of apoA-IV on endogenous apoB-traffi cking kinetics and the physical properties of secreted apoB-containing TG-rich lipoproteins.…”

“…After a 10 min pulse with [ 35 S]Met/Cys as described above, cells were washed and incubated with oleate-containing growth medium containing an additional 2.5 mM Met and 1 mM Cys for the indicated times. Cell lysate and media samples were prepared as described previously ( 34,37 ) and subjected to immunoprecipitation with rabbit anti-apoA-IV or goat anti-human apoB (Academy Biomedical, Houston, TX). Immune complexes were fractionated by SDS-PAGE, and dried gels were exposed to BioMax MS fi lm backed with a BioMax TransScreen-LE intensifying screen (Eastman Kodak, Rochester, NY) at Ϫ 70°C; band intensities were quantifi ed using a Molecular Dynamics 445 SI phosphorimager or a Fujifi lm BAS5000 phosphorimager.…”

Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini

“…ApoA-IV knock-out mice, under unstressed conditions, exhibit normal lipid absorption (3). However, when apoA-IV release from the endoplasmic reticulum is genetically disrupted, apoB and lipoprotein secretion is inhibited (4). On the other hand, overexpression of human apoA-IV in porcine intestinal cells enhances basolateral lipid secretion via production of larger chylomicron-like particles (5).…”

The Mechanism Underlying the Synergetic Hypocholesterolemic Effect of Sesamin and α-Tocopherol in Rats Fed a High-Cholesterol Diet

“…As co-expression of ApoB and ApoA4 modified with the carboxylterminal endoplasmic reticulum retention signal reduced the secretion of ApoB in COS cells, ApoA4 may physically interact with ApoB in the secretory pathway (37). This report suggests that down-regulation of ApoA4 gene expression reduced ApoB secretion.…”