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. 2009 Jun 15;185(6):1013–1027. doi: 10.1083/jcb.200903152

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© 2009 Vacaru et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — SMSr-depleted cells accumulate ceramides in the ER. (A) HeLa cells treated with si LA, si hSMSr, or siRNA-targeting hSMS1 (si hSMS1) for 3 d were subjected to lipid extraction. Lipid classes were quantified by MS/MS analyses and expressed in mole percent of total lipid analyzed. Data shown are representative of three independent experiments. (B) HeLa cells expressing hSMS1-V5 were subjected to subcellular fractionation, yielding a PNS and fractions enriched for mitochondria (mito)/Golgi, PM/early endosomes (EE), or ER. Equal amounts of protein from each fraction were analyzed by immunoblotting. Note that the ER fraction was virtually free of other organelles except for a minor contamination with PM (<10%). (C) MS/MS analysis of ceramide and PE levels in ER fractions prepared from HeLa cells treated with si LA or si hSMSr for 3 d. (D) HeLa cells transfected with hSMS1-V5 or an hSMS1 swap mutant carrying the N-terminal tail of hSMSr, hSMS1Nr-V5, were double labeled for V5 and cis-Golgi marker GM130. (E) S2 cells cotransfected with hSMS1-V5 or hSMS1Nr-V5 and ER marker PEMT-GFP were labeled for V5. The white box marks a cell captured from a different field. Note that hSMS1Nr-V5 localizes to the ER and nuclear envelope (D and E, arrows). (F) S2 cells transfected with hSMS1-V5 or hSMS1Nr-V5 were labeled with [¹⁴C]choline for 5 h and subjected to lipid extraction, TLC, and autoradiography. (G) dSMSr-depleted S2 cells were transfected with hSMS1-V5 or hSMS1Nr-V5 and labeled for 5 h with [¹⁴C]serine. Levels of radiolabeled ceramides were determined by TLC and autoradiography and expressed as a percent of mock-treated cells. (H) SMSr-depleted cells (S2/HeLa) were transfected with hSMSr-V5 or hSMSr^D348E-V5 and labeled for 5 h with [¹⁴C]serine. Levels of radiolabeled ceramides were determined as in G. SMSr depletions were with ds dSMSr #2 (S2) or si hSMSr #2 (HeLa). Transfection of HeLa cells was with siRNA-resistant (res) hSMSr constructs. Untransf, untransfected. Error bars: (G) SD, n = 3; (C and H) range, n = 2.