Journal: bioRxiv

Article Title: Spartin-mediated lipid transfer facilitates lipid droplet turnover

doi: 10.1101/2023.11.29.569220

Figure Lengend Snippet: A : Domain diagrams of spartin proteins including Hs Spartin, Ce Spartin and Ct SpartinL. Hs Spartin and Ce Spartin have three domains: microtubule-interacting (MIT) domain, PH-like domain and senescence domain. Hs Spartin has a LC3A interacting region (LIR: 193-200) . Spartin-like protein from Chaetomium thermophilum ( Ct SpartinL) lacks the MIT domain. B: Left: SDS-PAGE of purified spartin. Right: Native blue PAGE analysis of same spartin proteins indicates that all three proteins likely trimerize. C : In vitro lipid binding assays show that Ce Spartin and Hs Spartin bind lipids. Ce Spartin and Hs Spartin were incubated with NBD-phospholipids or solvent (no lipids) and analyzed by native gel electrophoresis, where migration depends on charge-mass ratio of the protein-lipid complexes. D : Quantitative in vitro lipid binding assay estimates Ce Spartin binds one lipid/protein molecule. CeSpartin (4μM) and E-Syt2 (10, 5.0, 2.5 μM) were incubated with NBD-PS and analyzed as in C. (At 10 μM, E-Syt2 migrates as two species). The Coomassie density allows determination of the number of proteins in the sample, whereas the NBD fluorescence indicates quantity of lipids bound by this number of proteins. Because each Extended Synaptotagmin monomer is known to bind 2 lipids, we can extrapolate how many lipids the fluorescence signal associated with spartin represents. E : Lipidomic MS analysis of Ce Spartin purified from Expi293 cell shows that it binds neutral lipids (sterol esters, TAG and DAG) and glycerophospholipids. Percentage of co-purified phospholipids is plotted against phospholipids profile of Expi293 cells. F : Schematic of in vitro FRET-based lipid transfer assay. G : Lipid transfer assays show that spartin proteins transfer lipids between liposomes. NBD-PC/PE/PS substrates were tested for Ce Spartin, while only NBD-PC transfer assays were performed for Hs Spartin and Ct SpartinL. Proteins were purified from Expi293 cells and used at 0.25 μM (1:800 protein:lipid ratio). Each experiment was performed in triplicate; SD is indicated. In control experiments, to verify that the increase in fluorescence is not due to fusion of the donor and acceptor liposomes, we added dithionite following the assay. Dithionite reduces solvent exposed NBD and quenches its fluorescence but does not affect NBD-lipids in the liposome lumen. After dithionite addition, NBD-fluorescence is reduced equally whether spartin is present or not, indicating that only the outer leaflet of the liposomes changed in the course of the lipid transfer reaction, not both leaflets as would be expected had fusion taken place . H : In vitro lipid transfer assay shows that Hs Spartin (0.1 μM, 1:800 protein:lipid ratio) robustly transfers NBD-PC from artificial LDs to liposomes (n=3). Artificial LDs prepared as in were used as lipid donors instead of liposomes. See Fig. S1A for characterization of the artificial LDs by positive stain electron microscopy.

Article Snippet: From E. coli: The full length CeSpartin construct (pET28-6xhis-MBP-TEV cleavage site- Ce Spartin) was expressed in Nico21 cells (C2529H, New England Biolabs).

Techniques: SDS Page, Purification, In Vitro, Binding Assay, Incubation, Solvent, Nucleic Acid Electrophoresis, Migration, Fluorescence, Liposomes, Control, Staining, Electron Microscopy