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. 2000 Feb;11(2):721-34.
doi: 10.1091/mbc.11.2.721.

Palmitoylation of apolipoprotein B is required for proper intracellular sorting and transport of cholesteroyl esters and triglycerides

Y Zhao  1 , J B McCabe, J Vance, L G Berthiaume
Affiliations
Free PMC article

Palmitoylation of apolipoprotein B is required for proper intracellular sorting and transport of cholesteroyl esters and triglycerides

Y Zhao et al. Mol Biol Cell. 2000 Feb.
Free PMC article

Abstract

Apolipoprotein B (apoB) is an essential component of chylomicrons, very low density lipoproteins, and low density lipoproteins. ApoB is a palmitoylated protein. To investigate the role of palmitoylation in lipoprotein function, a palmitoylation site was mapped to Cys-1085 and removed by mutagenesis. Secreted lipoprotein particles formed by nonpalmitoylated apoB were smaller and denser and failed to assemble a proper hydrophobic core. Indeed, the relative concentrations of nonpolar lipids were three to four times lower in lipoprotein particles containing mutant apoB compared with those containing wild-type apoB, whereas levels of polar lipids isolated from wild-type or mutant apoB lipoprotein particles appeared identical. Palmitoylation localized apoB to large vesicular structures corresponding to a subcompartment of the endoplasmic reticulum, where addition of neutral lipids was postulated to occur. In contrast, nonpalmitoylated apoB was concentrated in a dense perinuclear area corresponding to the Golgi compartment. The involvement of palmitoylation as a structural requirement for proper assembly of the hydrophobic core of the lipoprotein particle and its intracellular sorting represent novel roles for this posttranslational modification.

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Figures

Figure 1
Figure 1
Incorporation of [125I]iodopalmitate into secreted apoBs: apoB-29, apoB-31, apoB-37, and apoB-100 incorporate [125I]iodopalmitate, whereas apoB-18 does not. Distribution of cysteine residues in apoB-100 (A) and various apoB truncated constructs (B). Free cysteine residues are represented by long ticks, and short ticks represent cysteine residues found in disulfide linkages. (C) Western blot analysis of various secreted [125I]iodopalmitate-labeled apoB constructs with the use of the 1D1 anti-human apoB mouse mAb demonstrates the presence of the various apoBs on the PVDF membrane. (D) Incorporation of [125I]iodopalmitate into corresponding secreted apoB proteins visualized by autoradiography of the PVDF membrane. Exposure time was 3 d with the use of Molecular Dynamics phosphorimager cassettes.
Figure 2
Figure 2
ApoB-29 is palmitoylated on cysteine residue 1085 via a hydroxylamine-sensitive thioester bond. (A) Autoradiogram of [125I]iodopalmitate-labeled secreted WT apoB-29 starting material (lanes 1 and 2) blotted onto a PVDF membrane before hydrolytic treatment. (B) Autoradiogram of a PVDF membrane after a 72-h treatment with either 1 M Tris-HCl, pH 7.0, or 1.0 M hydroxylamine-HCl, pH 7.0. (C) TLC analysis of [125I]iodopalmitate standard and hydrolyzed radiolabel extracted from [125I]iodopalmitate-labeled apoB-29. Exposure time was 14 d on film with an intensifying screen. (D) Western blot analysis of WT apoB-29 and Cys1085Ser apoB-29 secreted from corresponding [125I]iodopalmitate-labeled McArdle-RH7777 stable cell lines. (E) Autoradiogram of WT apoB-29 and Cys1085Ser apoB-29 secreted from corresponding [125I]iodopalmitate-labeled McArdle-RH7777 stable cell lines. Exposure time was 3 d with the use of Molecular Dynamics phosphorimager cassettes.
Figure 3
Figure 3
Palmitoylation of apoB-29 is required for secretion of larger and lower-density lipoprotein particles. (A) ApoB-29 particles secreted from McArdle-RH7777 cells were separated on 3–10% nondenaturing gradient gel electrophoresis and analyzed by Western blot analysis. Typical gel migration patterns of WT palmitoylated apoB-29 and nonpalmitoylated Cys1085Ser apoB-29 are shown. (B) Isopycnic density gradient analyses of lipoprotein particles secreted from McArdle-RH7777 cells. Fractions collected from the bottom to the top of the gradient were numbered 1–20. Densities (grams per milliliter) of fractions 1, 5, 10, 15, and 20 are shown above the corresponding fraction numbers. Typical distributions of Cys1085Ser mutant apoB-29, WT apoB-29, and endogenous rat apoB-100 lipoproteins throughout the KBr gradient as revealed by Western blot analysis are shown. (C) Graphical representation of the average relative distribution of WT (□) and Cys1085Ser mutant (♦) apoB-29s in the KBr gradient as a function of fraction number. Relative amounts of apoB-29s were calculated as a percentage of the peak value in fractions 1–14 of each gradient. Average peak densities (grams per milliliter) of particles containing WT (n = 7) or Cys1085Ser mutant (n = 6) apoB-29s are shown above the corresponding curves.
Figure 4
Figure 4
Palmitoylation of apoB-29 is required for assembly of neutral lipids into the hydrophobic core of the lipoprotein particle. (A) TLC analysis of lipid content corresponding to individual density gradient fractions 1–7 (depicted in Figure 3) is shown for WT apoB-29 (WT) or nonpalmitoylated mutant apoB-29 (Mut). (B) An aliquot of the lipid extract corresponding to one-tenth of pooled gradient fractions 11–20 isolated from McArdle-RH7777 cells secreting either WT apoB-29 (WT) or Cys1085Ser mutant apoB-29 (Mut). Positions of lipid standards are indicated on the left for CE, TG, free fatty acid (FFA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). The TLC plate was exposed to film for 16 h.
Figure 5
Figure 5
Palmitoylation of apoB-29 confers localization to large vesicular structures containing endogenous apoB-100 in McArdle-RH7777 cells. Confocal microscope images of the intracellular distribution of various apoBs obtained by indirect immunofluorescence. (A) Typical localization of WT human apoB-29 (a and d), nonpalmitoylated Cys1085Ser human apoB-29 (b and e), and endogenous rat apoB-100 (c and f) in different McArdle-RH7777 cells visualized with the use of appropriate secondary antibody conjugated to TR. (B) Double-labeling immunofluorescence was carried out to detect WT human apoB-29 and endogenous rat apoB-100 in McArdle-RH7777 cells stably expressing WT apoB-29 (MH-WT apoB-29). WT apoB-29 or endogenous apoB-100 was visualized with the use of a TR-conjugated secondary antibody (red) in the left panel or an FITC-conjugated secondary antibody (green) in the middle panel, respectively. The merged image is shown in the right panel.
Figure 6
Figure 6
Palmitoylation of apoB-29 is required for localization to a subcompartment of the ER. Colocalization studies of various apoBs with the ER marker protein PDI. Various human apoBs were detected with the 1D1 mouse mAb and are shown in red with the use of anti-mouse IgG-TR–conjugated secondary antibody (left panels). PDI was detected with the use of the rabbit polyclonal anti-PDI antibody and is shown in green with the use of anti-rabbit IgG-FITC–conjugated secondary antibody (middle panels). The merged images are shown in the right panels. Staining of McArdle-RH7777 cells stably expressing WT apoB-29, Cys1085Ser apoB-29, or apoB-18 and nontransfected McArdle-RH7777 cells is shown and identified as follows: MH-WT apoB-29, MH-apoB-29(Cys1085Ser), MH-apoB-18, and MH, respectively.
Figure 7
Figure 7
Nonpalmitoylated apoB-29 is concentrated in the Golgi compartment. Colocalization of various apoBs with the Golgi marker protein α-mannosidase II. Various apoBs are visualized in red (left panels) with the use of the 1D1 anti-human (anti-h) apoB or the SY anti-rat (anti-r) apoB primary antibody with appropriate TR-conjugated secondary antibody. α-Mannosidase II staining was detected with the use of rabbit polyclonal anti-α-mannosidase II and is shown in the middle panels in green with the use of appropriate FITC-conjugated secondary antibody. The merged images are shown in the right panels. Abbreviations for the various McArdle-RH7777 cells are as described for Figure 6.
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