Fig 1: Palmitoyl-CoA Uptake and Shh Palmitoylation in Cells Expressing Hhat(A) COS-7 cells expressing EV, Hhat WT, Hhat H379A, or Hhat Y351A were incubated on ice with 65 μg/ml digitonin for 10 min to permeabilize the plasma membrane, then incubated for 60 min at room temperature with 8 μM NBD-palmitoyl-CoA. 30 min prior to imaging, 1 μM ER Tracker Red was added to the cells. Cells were washed and then imaged by confocal microscopy. Scale bar, 25 μm.(B) NBD-palmitoyl-CoA uptake was monitored in microsomal membranes from HEK293FT cells expressing EV, Hhat WT, Hhat H379A, or Hhat Y351A as described in Figure 2; n = 3.(C) Shh palmitoylation was monitored in COS-1 cells expressing Shh and either pcDNA, WT, or mutant (H379A, Y351A) Hhat. Cells were incubated with 15 μCi [125I] IodoPalmitate for 4 h, lysed, and Shh immunoprecipitated from cell lysates was analyzed by SDS-PAGE. [125I] CPM incorporated into Shh was quantified by phosphorimaging; n = 2.(D) Shh palmitoylation was monitored in microsomal membranes from (B). Membranes were incubated with biotinylated Shh peptide and [125I] IodoPalmitoyl-CoA for 60 min prior to the addition of streptavidin-agarose beads. Beads were washed, and [125I] CPM incorporated into the Shh peptide were determined in a gamma counter; n = 2. See also Figure S2.(E) Shh palmitoylation activity was assayed using purified Hhat as in (D); n = 2.
Fig 2: NBD-Palmitoyl-CoA Uptake Is Reduced in Membranes from Hhat−/− Cells(A–D) Microsomal membranes from control or Hhat−/− T47D cells (A and B) or Hhat−/− MEFs (C and D) were pretreated for 15 min with 10 μM TDI-3410, 10 μM Etomoxir, or both drugs, and the uptake of NBD-palmitoyl-CoA (A and C) or [125I] IodoPalmitoyl-CoA (B and D) was determined as described in Figure 2. (A) n = 4; (B) n = 2; (C) n = 3; and (D) n = 2.(E) Microsomal membranes from (C) were pretreated for 15 min with 10 μM TDI-3410, 10 μM Etomoxir, or both drugs, and the NBD-palmitoyl-CoA uptake assay was performed at room temperature or at 0°; n = 3.
Fig 3: Visualization of Palmitoyl-CoA Uptake by Liposomes Containing Purified, Reconstituted Hhat(A) RFU readings at 460/535 nm and 610/700 nm. Fluorescent intensity of liposomes containing Cy5.5-PE integrated into the membrane or NBD-palmitoyl-CoA alone at 460/535 nm and 610/700 nm was determined; n = 3. No signal inference between the signals for the Cy5.5 and NBD was observed.(B and C) Membrane-integrated Cy5.5-PE liposomes reconstituted with or without purified Hhat (WT or H379A) were pretreated with 10 μM TDI-3410 for 15 min followed by incubation with NBD-palmitoyl-CoA for 60 min at room temperature prior to quenching with Na2S2O42−.(B) RFU readings; n = 3.(C) Images of liposomes containing membrane integrated PE (red: Cy5.5-PE) and palmitoyl-CoA (green: NBD-palmitoyl-CoA) were acquired by confocal microscopy. Scale bar, 0.2 μm.(D) Line scan of fluorescence intensities of Z stacks from Video S1.
Fig 4: Quenching of Internalized NBD Fluorescence in Liposomes Containing Dithionite(A) Diagram of the modified NBD-palmitoyl-CoA uptake assay. Liposomes were reconstituted with or without purified Hhat (WT or H379A) in the presence of Na2S2O42−. Liposomes were re-isolated and then incubated with NBD-palmitoyl-CoA, and NBD fluorescence was quantified. The addition of 0.2% octyl glucoside reduced fluorescence levels to background.(B) Liposomes generated as in (A) were pretreated with 10 μM TDI-3410 for 15 min followed by incubation with NBD-palmitoyl-CoA for 60 min. RFU readings were taken before and after addition of 0.2% octyl glucoside; n = 3.(C) Liposomes were reconstituted with purified WT Hhat, Na2S2O42−, and WT or C24A Shh peptide. After the removal of the external quencher and peptide, NBD-palmitoyl-CoA was added, and the fluorescent signal was monitored for 120 min; n = 3.(D) Liposomes were reconstituted with purified WT Hhat, and uptake of [14C] palmitoyl-CoA was measured as in Figure 4C; n = 2.
Fig 5: NBD-Palmitoyl-CoA Uptake Assay(A) Microsomal membranes generated from HEK293FT cells transfected with pcDNA3.1 (empty vector; EV) or pcDNA3.1 encoding Hhat are incubated with NBD-palmitoyl-CoA, then treated with dithionite (Na2S2O42−), a membrane-impermeant reducing agent that quenches NBD fluorescence of molecules on the external side of the membrane. RFU readings at 535 nm reflect NBD-palmitoyl-CoA within the vesicle and protected from the quencher. The addition of 0.2% octyl glucoside permeabilizes the bilayer and allows access of the quencher to the interior of the vesicle.(B) Microsomal membranes from HEK293FT cells expressing pcDNA, Hhat WT, or Hhat H379A were incubated with NBD-palmitoyl-CoA for 60 min at room temperature prior to the addition of Na2S2O42−. NBD fluorescence in the interior of the vesicles was quantified; n = 3.(C) Microsomal membranes were pretreated for 15 min with 10 μM TDI-3410, then analyzed for NBD-palmitoyl-CoA; n = 3.
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