ERES associate with microtubules and GFP–p150^Glued. (a) Vero cells expressing Sec23Ap–YFP were microinjected with rhodaminetubulin and imaged after at least 1 h of incubation at 37 °C. The four images show different time points of a time-lapse sequence, in which ERES become rapidly associated with a growing microtubule. The tip of the growing microtubule is highlighted with red arrowheads. ERES that become associated with it are highlighted with white arrowheads. Scale bar, 10 μm; see Supplementary Information, Movie 1. (b) Magnification of images at various time points of the time-lapse shown in a. Scale bar, 3 μm. (c) HeLa cells expressing GFP–p150^Glued and GFP–Sec23Ap were imaged at 37 °C. GFP–p150^Glued predominantly labels the growing tips of microtubules (arrows), which can be seen to track through the cytoplasm directly through GFP–Sec23Ap-labelled ERES (arrowheads); see Supplementary Information, Movies 2 and 3. (d) Maximum intensity projections of all frames from the time-lapse sequence shown in c. ERES are pseudo-coloured with green circles; continuous tracks of GFP–p150^Glued reveal the paths taken by polymerizing microtubules, which can be seen to grow directly through ERES (arrowheads).

Interaction of Sec23Ap and p150^Glued. (a) Two-hybrid screening reveals a specific interaction between Sec23Ap and the C terminus of p150^Glued (amino acids 938–1254). All colonies grow on double dropout medium (DDO) but only those showing a positive interaction grow on quadruple dropout (QDO). p150^Glued (amino acids 938–1254) interacts specifically with Sec23Ap and Sec24Dp but not with lamin, Sec13p or Sec31Ap. (b) Surface plasmon resonance demonstrates a direct interaction between Sec23Ap and p150^Glued. One hundred resonance units (RUs) of either GST or GST–CT^Glued were immobilized. His₆-hSec23Ap shows no binding to GST (grey) but binds GST–CT^Glued (black). Note the large increase in RUs immediately following injection of His₆-hSec23Ap (t = 0 s) is due to slight differences in refractive indices of the buffers; RUs increase as His₆-hSec23Ap flows across the surface loaded with GST–CT^Glued but not that loaded with GST. At t = 60 s His₆-hSec23Ap is exchanged for PBS. Fast dissociation of His₆-hSec23Ap (t = 60 s) is followed by slow dissociation of a more tightly bound component (60–120 s). (c) Histogram showing binding of His₆-hSec23Ap to GST or GST–CT^Glued from six separate experiments. Error bars show ± 1 standard deviation. (d) Direct binding of purified His₆-Sec23Ap to GST–CT^Glued. 5 μg of immobilized GST (lane 1) or GST–CT^Glued (lane 2) were incubated with His₆-Sec23Ap for 2 h at 4 °C. After washing, proteins were separated and immunoblotted with anti-Sec23Ap. The inset panel shows 5% of the input His₆-Sec23Ap. (e) Co-immunoprecipitation of Sec23Ap and p150^Glued from HeLa cell lysates. No interaction was detected following immunoprecipitation (IP) with mouse IgG (lane 1) or rabbit IgG (not shown). Interaction was detected following immunoprecipitation of p150^Glued and immunoblotting of Sec23Ap (lane 2). Lane 3 shows 10% of the lysate's input. (f) p150^Glued is immunoprecipitated with antibodies specific for Sar1p and Sec23Ap, as well as p50^dynamitin and p150^Glued itself. Co-immunoprecipitation is not seen with antibodies specific to β′-COP or Sec31Ap. Efficacy of antibodies in immunoprecipitation was confirmed by immunoblotting with the same antibody (lower panel). Asterisk: specific immunoprecipitation of p150^Glued is shown in the top panel. (g) Expression of CFP–CT^Glued reduces the amount of p150^Glued that can be co-immunoprecipitated with Sec23Ap (left-hand panels) but does not affect co-immunoprecipitation of p150^Glued with p50^dynamitin (right-hand panels).

Expression of the C-terminal 317 amino acids of p150^Glued (CT^Glued) inhibits cargo export from the ER. (a) Cells were transfected with ts045-G–GFP and either CFP or CFP–CT^Glued and grown at 39.5 °C for 16 h followed by incubation at 32 °C. The first panels show the expression of CFP and CFP–CT^Glued. Subsequent panels show stills from the associated time-lapse movie (see Supplementary Information, Movie 4) taken at the times indicated. Note the substantial localization of ts045-G–YFP to a juxtanuclear (Golgi) region at 300 s in control cells (arrowheads), which does not occur until 900 s in CFP–CT^Glued-expressing cells. (b) Enlargements of the boxed regions (600 s) in a showing localization of ts045-G–YFP at time points indicated). Analysis of the first 3 min of these time-lapse movies shows that the formation of VTCs is delayed in cells expressing CFP–CT^Glued (see also Movies 4 and 5, available as Supplementary Information). Later time points reveal the delay in formation of punctate structures (ERES and VTCs, arrowheads). VTCs do form in CFP–CT^Glued-transfected cells but their appearance is significantly delayed. (c) Quantitative analysis of delivery of ts045-G–YFP to the plasma membrane shows a significant lag in transport in cells co-expressing CFP–CT^Glued. The cell surface fluorescence intensity was measured using immunofluorescence of fixed, but nonpermeabilized, cells and normalized to the amount of ts045-G–YFP at the cell surface at 90 min (a measure of the total ts045-G–YFP). Error bars show ±1 s.d.; P < 0.05; n = 3 experiments, total 30 cells per experiment. (d) Velocities of individual VTCs were tracked in cells between 0 and 600 s after shift to 32 °C. The histogram shows the average velocities along with the scatter of individual particle velocities (>100 clearly definable VTCs; at least 20 from each of 5 different cells).

ERES associate with growing microtubules. (a) Association of ERES with newly polymerized microtubules (green) following nocodazole washout. Vero cells expressing YFP–Sec23Ap (red) were microinjected with 1 mg ml−1 rabbit IgG or 1 mg ml−1 anti-EAGE, each with a co-injection marker of 0.03 mg ml−1 rhodamine-dextran to identify injected cells. Sar1p(H79G) was expressed by co-transfection from a plasmid. Depolymerization of microtubules, nocodazole washout, immunostaining of microtubules and determination of the association of COPII-labelled ERES with microtubules was performed as described in Methods. Arrows point to ERES that were considered not to be associated with microtubules, and arrowheads point to ERES that were associated. Scale bar (all panels), 20 μm. (b) Quantification of the fraction of microtubules associated with at least one COPII-labelled ERES. (c) Quantification of the fraction of COPII-labelled ERES associated with at least one microtubule. Error bars represent s.e.m.

Colocalization of COPII and p150^Glued. (a) HeLa cells labelled with antibodies directed against Sec23Ap (top left and green in merge) and two different monoclonal antibodies directed against p150^Glued (bottom left and red in merge), followed by DAPI counterstaining of DNA (blue in merge). Scale bar, 5 μm. (b) Enlargements of a showing (arrowheads) colocalization of Sec23Ap (green) and p150^Glued (red) in HeLa cells at steady state. Regions shown are 10 × 10 μm. (c) After either a 10 min or 30 min incubation with nocodazole, cells were fixed and processed for immunofluorescence with antibodies directed against Sec23Ap and either α-tubulin or p150^Glued. (d) Enlargements of the boxed regions in the merge images from c highlighting colocalization of Sec23Ap and p150^Glued (arrowheads). Quantification of these experiments shows no difference in the degree of colocalization of ERES with p150^Glued, either in the presence or absence of microtubules.