Fig. 7: Identification of the N-terminal 4-helix bundle as another LD-binding region in HSL.

a, b Evaluation of the ALD-binding abilities of the H-motif and the N-terminal 4-helix bundle using the flotation assay. MBP-HSL(489-538) and MBP-HSL(1-136) refer to the H-motif and the N-terminal 4-helix bundle (residues 1−136) of HSL fused with an MBP to its N-terminus, respectively. c The AlphaFold2 predicted structure of the amphipathic loop containing residues 97 to 103 (RRSIFFR) in the N-terminal 4-helix bundle. d, e Evaluation of the ALD-binding abilities of HSL and its variants using the flotation assay. HSL(FL, 3A) is the full-length HSL carrying the F519A/W526A/F530A triple mutations. HSL(FL, 5A) carries two additional mutations, I100A and F101A, compare to HSL(FL, 3A). f Subcellular localization of human HSL and its variants in 3T3-L1 preadipocytes. The wild-type HSL, HSL(I100A, F101A), and HSL(FL,5A), each with an EGFP fused to its C-terminus, were transiently expressed in 3T3-L1 preadipocytes. Then the cells were imaged as described in Fig. 3e. g Quantification of the EGFP signal of human HSL and its variants on lipid droplets in 3T3-L1 preadipocytes (a total of 15 cells were analyzed from three independent measurements). The data in a, d, and f, each are the results of a representative experiment out of three independent experiments. The data in b, e, and g represent the mean ± SD of three independent measurements. The data in b, e, and g were analyzed using the unpaired t-test in Prism to calculate the two-tailed P-values. Source data are provided as a Source Data_Fig. 7 file.