Functional interactions between Mldp (LSDP5) and Abhd5 in the control of intracellular lipid accumulation - PubMed (original) (raw)

Functional interactions between Mldp (LSDP5) and Abhd5 in the control of intracellular lipid accumulation

James G Granneman et al. J Biol Chem. 2009.

Abstract

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.

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Figures

FIGURE 1.

FIGURE 1.

Colocalization of Mldp, Adrp and Abhd5 in mouse cardiomyocytes. A, permeabilized microdissected cardiac muscle fibers were subjected to double-label immunofluorescence for the indicated antigens. Confocal images are shown on the left, and representative line scans of those images are shown on the right. Peaks in the line scan graphs represent individual lipid droplets. RFU, relative fluorescence units.B, average coefficients of determination (Pearson's_r_2) indicating the magnitude of colocalization from 4–5 samples. Values are the means ± S.E.

FIGURE 2.

FIGURE 2.

Mldp directs Abhd5 to lipid droplets. 3T3-L1 fibroblasts were transfected with Mldp-EYFP and ECFP-Abhd5 singly and in combination, and lipid was loaded overnight and imaged live by confocal microscopy. Cells were subsequently counterstained with nile red to identify lipid droplets (B and D). When singly transfected, Mldp-EYFP (A) was highly targeted to clusters of lipid droplets (B). Singly transfected ECFP-Abhd5 (C) accumulated in the perinuclear region and was poorly colocalized with lipid droplets (D), which were diffusely distributed. When co-transfected (E and F), ECFP-Abhd5 (E), and Mldp-EYFP (F) were highly colocalized on lipid droplet clusters.

FIGURE 3.

FIGURE 3.

ECFP-Abhd5 binds specifically to lipid droplets containing Mldp-EYFP. 3T3-L1 fibroblasts were transfected with Mldp (top row) or G. princeps luciferase-EYFP control (bottom row) and loaded with lipid. Arrows indicated positions of untransfected cells. Permeabilized cells were incubated with recombinant ECFP-Abhd5 prepared from 293T cells, and bound ECFP-Abhd5 was imaged by confocal microscopy. Capture parameters are identical for both ECFP-Abhd5 images. ECFP-Abhd5 bound MLDP-EYFP containing lipid droplets in proportion to Mldp concentration and did not bind significantly to nontransfected cells (arrows) or cells transfected with control vector. ECFP-Abhd5 did not significantly bind lipid droplets, indicated by nile red staining, in the absence of Mldp expression. Bar = 10 μm.

FIGURE 4.

FIGURE 4.

G. princeps luciferase complementation between Mldp and Abhd5. A, 293T cells were transfected with Abhd5, full-length Mldp1, or translationally truncated Mldp2 fused to the N or C terminus of luciferase and similar fusions made with Rab18 (negative control) or Plin (positive reference). Cells were incubated for 24 h in media containing oleic acid to facilitate lipid droplet formation. Strong luciferase activity was reconstituted by co-expression of complementary fusions of Mldp and Abhd5. Values are the means ± S.E. normalized to Plin/Abhd5 for each experiment. Results are from six independent experiments performed in quadruplicate. B, complementation of luciferase activity using split fragment fusions synthesized in vitro by bacterial-based in vitro transcription/translation. Values are from three independent experiments performed in triplicate.

FIGURE 5.

FIGURE 5.

Effects of lipid loading on protein interactions with Abhd5. A, oleic acid increases luciferase complementation between Abhd5 and Mdlp but not between Mldp oligomers. Values are normalized to Abhd5/Mldp without oleic acid for each of six experiments performed in quadruplicate. Transfected 293T cells were incubated overnight in normal media, then exposed to OA complexed to BSA or BSA alone for 1 h. Oleic acid treatment increased the interaction of Abhd5 and Mldp by 2–3-fold (**, p < 0.01) but did not influence homotypic interaction of Mldp. Minimal luciferase activity was observed in cells cotransfected with Abhd5 and Adrp or Rab18. OA slightly increased luciferase activity in cells expressing N-Abhd5 and C-Adrp, but these values were only ∼10% that seen with Abhd5 and Mldp.B, Triacsin-C treatment blocks the effects of OA on Abhd5-Mldp protein complementation. Transfected 293T cells were treated with BSA-OA or BSA alone with Triacsin-C or DMSO vehicle for 2 h. OA treatment increased the interaction of Abhd5 and Mldp, and this increase was significantly suppressed by Triacsin-C. Values are the means ± S.E. of five independent experiments performed in quadruplicate.

FIGURE 6.

FIGURE 6.

E262K mutation of Abhd5 disrupts its interaction with Mldp. A, COS-7 cells were transfected with Mldp-EYFP and treated with oleic acid overnight. Cells were fixed and permeabilized and incubated with 293T lysates containing equal concentrations of wild type or E262K Abhd5-Cherry. Binding of wild type Abhd5 was in direct proportion to Mldp concentration, as determined by line scan analysis of fluorescence (right panel). No specific binding to Mldp was detected for the E262K mutant. B, the interaction of wild type and mutant Abhd5 with Mldp and Atgl was assessed by luciferase complementation assay. E262K mutation disrupted binding to Mldp but not to Atgl. Shown is a representative experiment performed in quadruplicate. The experiment was performed three times with similar results.

FIGURE 7.

FIGURE 7.

Expression of Mldp increases the colocalization and interaction of wild type, but not mutant Abhd5 and Atgl. A, COS-7 cells were transfected with Atgl-EYFP with Mldp-ECFP or Atgl alone, permeabilized, and incubated with equal concentrations of wild type or E262K Abhd5-Cherry. Colocalization of bound Abhd5 with Atgl was determined by line-scan analysis (right column) of individual cells, identified by the_asterisk_ in Atgl images. Wild type Abhd5-bound lipid droplets containing Mldp and Atgl (top row). No specific binding of wild type Abhd5 was observed in cells expressing Atgl alone (middle row). Mutant Abhd5 failed to bind lipid droplets containing Mldp and Atgl (bottom row). AFU, arbitrary fluorescence units._r_2, coefficient of determination for the linear association. b, linear regression slope indicating amount of Abhd5 bound per unit Atgl. B, expression of Mldp-ECFP increases the interaction of wild type, but not mutant Abhd5 with Atgl in protein complementation assay. PC, protein complementation. Values are the means ± S.E. for eight independent experiments.

FIGURE 8.

FIGURE 8.

E262K mutation of Abhd5 prevents its activation of Atgl in the presence of Mldp. A, COS-7 cells were transfected with fluorescently tagged proteins as indicated and incubated with oleic acid overnight. Cells were scored as to the presence or absence of lipid droplets. #, p < 0.001 versus control; *, p < 0.001_versus_ lipase-dead (LD) Atgl. Values are means ± S.E. from 5–6 experiments. B, COS-7 cells were treated as above, and neutral lipid accumulation was measured using Lipidtox fluorescent stain as detailed under “Experimental Procedures.” *, p < 0.05 versus control; ***, p < 0.001_versus_ control. Values are the means ± S.E. for three experiments.

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