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.
Highlights
Ilipin, adipophilin, and TIP-47) proteins, are thought to be key regulators of these processes by serving as scaffolds that organize and regulate the protein trafficking at lipid droplet surfaces [1,2,3]
We examined the binding of wild type and mutant Abhd5 to permeabilized COS-7 cells that were transfected with Atgl-EYFP and MldpECFP or with Atgl-EYFP alone (Fig. 7A)
The present study confirms that Mldp is highly expressed in cardiac myocytes and further shows that Abhd5 and Mldp are highly colocalized on individual lipid droplets
Summary
Antisera to glutathione S-transferase fusions of mouse Mldp and Abhd were generated in rabbits and affinity purified by Proteintech. The samples were washed extensively with PBS between antibody incubations. Under these conditions, there was no cross-reactivity between the two pairs of primary/secondary antibodies; control experiments demonstrated that omission of each primary antibody resulted in complete and specific elimination of fluorescent signals only in the corresponding channel. For double labeling of Abhd and Adrp, the specimens were incubated successively with the following antibodies: 1) affinity-purified rabbit antiAbhd (1:250) overnight at 4 °C, 2) Cy3-conjugated goat-anti-rabbit Fab (1:200) for 3 h at room temperature, 3) guinea pig anti-Adrp (1:200) for 2 h at room temperature, and 4) Alexa 488-conjugated goat-anti-guinea pig antibodies (1:500) for 2 h at room temperature
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