Fig. 2. TMEM24 N-terminal TM domain and C-terminal unstructured region are required for localization to ER-PM contacts.

(A to F) Comparative analysis of localization of full-length, ΔTM, and ΔCTerm TMEM24-EGFP when overexpressed in COS-7 [(A) to (C)] and INS-1 [(D) to (F)] cells monitored by confocal microscopy. mRFP-Sec61β was used as ER marker. Given the different shape of the two cell types (top drawings), the plasma membrane was observed “en face” in the thin lamellipodium of the COS-7 cells but in cross section in the INS-1 cell. Full-length TMEM24 [(A) and (D)] has a diffuse localization in the ER but clusters at hot spots that represent ER-PM contact sites. The reticular shape of the ER is optimally appreciated in the “en face” view of the COS-7 cell. The construct lacking the transmembrane region (ΔTM) [(B) and (E)] localizes primarily at the PM, where it has a diffuse distribution (as clear by the different views of the COS-7 cell and the INS-1 cell). The construct lacking the C-terminal region (ΔCTerm) [(C) and (F)] precisely colocalizes with the ER marker Sec61β. Scale bar is 2 μm. (G) Purified human TMEM24 lacking the transmembrane domain (residues 36 to 706) was incubated with liposomes containing 10% PE and either 20% PS or 5% other test lipid, with the remainder PC. Liposomes were sedimented and analyzed for associated protein. ΔTM-TMEM24 directly binds acidic membranes without specific lipid preference. PA and PI are less charged than the other phosphoinositides in the panel, and their charge, closer to the hydrophobic layer, may be more shielded by the head groups of neighboring phospholipids. (H) Purified SMP-C2 fragment of TMEM24 (residues 76 to 414) does not sediment with membranes in the presence of EDTA or calcium. Liposome compositions are as in (G).