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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 7. — Structural integrity of tER–Golgi units requires an enzyme-active form of SMSr. (A) S2 cells were treated with ds SMSr #2 and transfected with hSMSr-V5 or hSMSr^D348E-V5. After 7 d, cells were double labeled for Golgi marker dGMAP and V5 to mark transfected cells. Confocal projections are presented. Note that cells expressing hSMSr (arrows) mostly show the typical wild-type Golgi organization of 17–25 large spots. In contrast, nontransfected cells or those expressing enzyme-dead hSMSr (arrowheads) typically have more numerous and smaller Golgi spots. (B) Mann II-RFP–expressing HeLa cells were treated with si hSMSr #2 and transfected with siRNA-resistant (res) hSMSr-V5 or hSMSr^D348E-V5. After 3 d, cells were labeled for V5 to mark transfected cells. Confocal projections are presented. Note that cells expressing hSMSr-V5 contain an intact Golgi (arrows). In contrast, cells expressing enzyme-dead hSMSr still have fragmented Golgi (arrowheads). (C) Quantitation of the rescue of the Golgi fragmentation phenotype in SMSr-depleted cells (HeLa/S2) after transfection of wild-type or enzyme-dead SMSr. SMSr-depleted cells are compared with control cells (si LA or ds GFP treated), and SMSr-depleted cells transfected with wild-type or enzyme-dead SMSr are compared with untransfected (untransf) SMSr-depleted cells. (D) Quantification of the rescue of the Golgi fragmentation phenotype in SMSr-depleted cells (Hela/S2) after transfection with hSMS1Nr-V5 or CERT-Flag or after treatment with 50 µg/ml fumonisin B1 (FB1) for 2 d (HeLa) or 5 h (S2) before fixation. SMSr-depleted cells expressing hSMS1Nr-V5 or CERT-Flag are compared with untransfected cells, and mock-treated cells are compared with FB1-treated cells. Errors bars indicate SD, n = 3. *, P < 0.001 by two-tailed unpaired Student's t test. Bars, 5 µm.