Depletion of Sec13 causes a loss of peripheral ERES labelling, and an accumulation of COPII and cargo in the juxtanuclear region of cells. (A) Cells depleted of Sec13 for 72 hours using one or both of the siRNA duplexes (-1 or -2) were fixed and immunolabelled for Sec31A, Sec24C, GM130, Sec16 or ERGIC-53 as indicated. (B) Cells transfected with siRNA duplexes targeting both Sec31A and Sec31B were immunolabelled for Sec16 and ERGIC-53. Note the juxtanuclear accumulation of COPII (Sec16) and cargo (ERGIC-53) in both cases (arrows). (C) Lysates of cells depleted of Sec13 were immunoblotted with antibodies specific for ERGIC-53 and α-tubulin. Molecular-mass markers are shown in kDa. (D) Localization of Sec16A and ERGIC-53 at sub-saturating pixel intensities in lamin-A/C- or Sec13-suppressed cells. (E) Suppression of Sec13 expression causes a reduction in COPI labelling of both Golgi and peripheral punctae compared with lamin-A/C-suppressed controls. Sec31A labelling is used to show effective suppression of Sec13/31 expression. Scale bars: 10 μm.

Depletion of Sec13 causes an increase in the half life and immobile fraction of Sec23/24 at ERES. (A) Half life of recovery of YFP-Sec23A during photobleaching shows a statistically significant increase following depletion of Sec13 (P=0.0072; n=45 ERES from three independent experiments). (B) The immobile fraction of YFP-Sec23A at ERES shows a statistically significant increase following depletion of Sec13 (P=0.0097; n=45 ERES from three independent experiments).

Secretory transport of tsO45-G-YFP is not significantly inhibited following Sec13 depletion. Cells depleted of Sec13 or lamin A/C were infected with an adenovirus expressing tsO45-G-YFP followed by incubation at the restrictive temperature of 39.5°C for 18 hours. (A) Transport of tsO45-G-YFP through the secretory pathway in cells transfected with either lamin-A/C or Sec13 siRNA. Upper panels: YFP (total tsO45-G) fluorescence 60 minutes after the shift to 32°C shows indistinguishable Golgi localization and plasma-membrane delivery in either experiment; note the enhanced juxtanuclear clustering of the Golgi in Sec13-suppressed cells. Lower panels: shows only plasma-membrane-localized tsO45-G-YFP 90 minutes after the shift to 32°C. Scale bars: 20 μm. (B) Quantification of arrival of tsO45-G-YFP at the plasma membrane (monitored by immunolabelling paraformaldehyde-fixed but non-permeabilized cells with an antibody against an extracellular epitope); error bars show standard deviation (n=100 cells from three independent experiments). (C,D) Arrival of protein at the Golgi following release of cargo from the ER at 32°C for the times indicated was determined by monitoring acquisition of EndoH resistance and immunoblotting for YFP. Samples were also immunoblotted to confirm the efficacy of Sec13 depletion and α-tubulin was included as a loading control. Molecular-mass markers are shown in kDa.

Ultrastructure of ER bud sites in Sec13-suppressed cells. Electron micrographs of high-pressure-frozen and freeze-substituted HeLa cells. (A) In control (lamin-A/C depleted) cells, small 50 nm vesicles are in close proximity to the Golgi apparatus (G). These vesicles are presumptive ER-to-Golgi transport vesicles but could potentially be either COPI- or COPII-coated. Occasionally, a coated (COPII) budding profile can be observed emanating from the ER (arrowhead). (B,C) In Sec13-depleted cells, the small 50 nm vesicles can still be observed (arrows) but, in addition, dilated ER with several budding profiles can frequently be seen (B, inset in C shows magnification of indicated area, large white arrowhead). These budding profiles (arrowheads in C) appear to be in different states of fission and contain an electron-dense coat. Scale bar: 500 nm (A,B), 350 nm (C).

Suppression of Sec13 in primary human fibroblasts results in defective collagen secretion and deposition. Primary human dermal fibroblasts were transfected with siRNA duplexes to suppress Sec13 expression for 72 hours. Cells were then processed for immunofluorescence using antibodies to detect type-I collagen and Sec31A. (A) Cells were labelled with antibodies to detect the C-terminal telopeptide of collagen I (LF-67) and Sec31A. Scale bars: 20 μm. (B) Quantification of collagen deposition from three independent experiments shows a significant reduction (P<0.001) in deposition of fibrillar collagen. supplementary material Fig. S2 shows further detail and example images from this quantification. (C) Immunoblotting was used to verify suppression of Sec13 and concomitant loss of Sec31A in primary human dermal fibroblasts. Asterisks denote non-specific bands detected with the anti-Sec13 antibody; tubulin was included as a loading control. Molecular-mass markers are shown in kDa.