PMC

Transient Inter-Compartmental Contact Sites (TICCS) mediate lipid traffic between membrane compartments and lipid filled adiposomes. Here depicted is the movement of a lipid cargo such as membrane sterol (yellow) between the cell surface and lipid droplets using endosomal intermediates. Migration of the sterol is bidirectional and depends on the formation endosome-endosome as well as endosome-droplet TICCS. Recognition sites (red) for each compartment contain the molecular machinery necessary for the formation of TICCS. Compartmental specificity of TICCS formation is regulated by Rab-GTP.

Multiple Rab proteins are on the surface of lipid filled adiposomes. Purified droplets were processed either for immunoblotting (IB) or immunogold localization of the indicated Rab protein (Immunogold). For immunogold, samples were fixed and incubated in the presence of rabbit IgG (A), pAb α-Rab5 (B), pAb α-Rab11 (C), or pAb α-Rab18 (D). The samples were then incubated with the α-rabbit IgG conjugated to 10 nm gold. Gold particles (arrows) are distributed on the surface of the droplet. An asterisk indicates clusters of small droplets. Immunoblotting shows that each antibody recognizes a single band. Bar, 0.5 μm.

GDP-dependent removal of Rab from droplets by RabGDI. Purified droplets (50 μl, ∼20 μg protein/reaction) were incubated in the presence or absence of 10 μM RabGDI plus 2 mM of the indicated non-exchangeable nucleotide at 37°C for one hour. At the end of the reaction, the droplets were separated from reaction solution by flotation, washed 3 times, the proteins precipitated by acetone and processed for immunoblotting. A) RabGDI transfers Rab5 and 11 from droplets (Drop) to the reaction solution (Sol). The loss of ADRP occurs in buffer alone. Caveolin-1 is not removed. B) RabGDI cannot remove Rab5 when GTP_γs is in the reaction mixture. Ral B, another prenylated protein, and caveolin-1 are not removed under any condition.

Recruitment of EEA1 to droplets in vivo. We transfected HeLa cells with cDNAs coding for either the Myc-tagged Rab5^Q79L (A-F) or Myc-tagged Rab5^S34N (G-I) chimeric proteins. The cells were incubated for 15 hours in media containing 100 μM oleic acid to induce lipid droplets and then fixed and processed for indirect immunofluorescence localization of Myc. Both chimeric proteins efficiently targeted droplets (A, D, G). Rab5^Q79L (A, D) caused the recruitment of EEA1 (B, E) to distinct sites on the droplet (C, F). EEA1 was largely depleted from other sites within these cells. By contrast, in cells expressing Rab5^S34N (G) EEA1 had a normal punctate distribution (H) that was not associated with the droplet (I). The scale bar, 1 μm.

GTP-dependent recruitment of early endosomes (EE) to droplets in vitro. Purified droplets (50 μl, ∼20 μg of protein/reaction) were incubated with EE (50 μl, 5 μg of protein/reaction) at 37°C for 1 hr in the presence or absence of 2 mM GTP_γs, cytosol or ATP. At the end of the reaction, free EE was separated from bound EE by flotation of droplets, and the droplet fraction subsequently washed three times with buffer B by repeated centrifugation at 10,000 × g. Samples were processed for immunoblotting as described. A) GTP_γs–dependent recruitment of EE to droplets does not require the presence of cytosol. B) EE recruitment to droplets is dependent on concentration of EE (50 μl of droplets, plus 2 mM GTP_γs and the indicated amount of EE). All incubations were at 37°C for 1 hr. C) Pretreatment of droplets with GTP_γs supports EE recruitment. D) The Rabs on the EE are sufficient to recruit EE to droplets. Droplets were treated with 10 μM RabGDI for 1 hr to remove Rabs. These droplets were then mixed with EE and incubated in the presence of the indicated nucleotide for 1 hr at 37°C. Lane 9 is a sample of EE (1.25 μg). E) Rabs are necessary for recruitment of EE. Both droplets and EE were treated to remove Rabs with RabGDI. These fractions were then mixed in the presence of the indicated nucleotide (2 mM) and incubated for 1 hr at 37°C.

GTP-dependent recruitment of Rabs from cytosol to droplets (A-C) but not to liposomes (D). Rab proteins were released from droplets (A-C) with RabGDI as described in . Droplets (50 μl, ∼20 μg protein/reaction) were mixed with either 80 μg of cytosol (A) or varying amounts of cytosol (B-C) in the presence or absence of 2 mM GTP_γs. After incubation, droplets were washed three times and the proteins precipitated by acetone and processed for immunoblotting with an antibody against the indicated protein. A) GTP_γs pre-bound to droplet is not sufficient to recruit Rabs and EEA1 to droplets. Droplets were either not treated (,) or pretreated with GTP_γs and mixed with cytosol in the presence or absence of GTP_γs at 37°C for 1 hr. B) Rab recruitment is dependent on temperature and cytosol concentration. Incubations were carried out at 4°C and 37°C with either 0, 400 μg/ml or 800 μg/ml of cytosol (volume 100 μl). C) Recruitment of Rabs and EEA1 appears to be saturable. Incubation was carried out at 37°C with the indicated concentration of cytosol (volume 100 μl). D) Rabs are not recruited to liposomes. Liposomes were prepared using lipids extracted from total CHO K2 cell membranes and mixed with 80 μg/ml of cytosol and incubated at 37°C for the indicated time. A companion set of droplets that had not been treated with RabGDI were processed the exact same way.