Luminal Ca²⁺ regulates size of rbet1-positive pre-Golgi structures. (A) NRK cells were either mock treated, incubated at 15°C for 30 min, or treated with CPA to deplete luminal Ca²⁺ (see Materials and Methods), and then fixed and immunostained for rbet1 and the Golgi marker GM130. Shown are single focal planes from deconvolved widefield image stacks. (B) Cytosolic Ca²⁺ dynamics during the CPA regimen to deplete luminal Ca²⁺. Fluorescence ratios are calibrated to free Ca²⁺ values in Supplemental Figure S1A. (C) Luminal ER Ca²⁺ dynamics during the CPA regimen. Fluorescence ratios are calibrated to free Ca²⁺ values in Supplemental Figure S1B. (D–F) Quantitation of peripheral rbet1-positive objects in experiments such as described in A. Values represent per cell means derived from at least 20 randomly chosen cells. Error bars display SE. CX, cycloheximide; Tg, thapsigargin. In F, only objects that fall within the size bins indicated above each plot are included in each panel. Selected p values from two-tailed Student's t test are included. Areas of objects were calculated assuming that one image pixel width calibrates to 224 nm in the cell. Single-pixel objects (with calculated area 0.05 μm²) are subresolution but were not eliminated from the size analysis; they did not contribute to the 0.2–0.45 μm² and >0.45 μm² size bins in F.

BAPTA, but not EGTA, stimulates COPII vesicle fusion. (A) Homotypic COPII vesicle fusion measured using the in vitro VSV-G heterotrimer cargo mixing assay. The no myc control includes radioactive VSV-G* vesicles, but not VSV-G-myc vesicles, to control for the specificity of the anti-myc immunoprecipitation. BAPTA and EGTA were included in fusion reactions at the indicated concentrations. The on ice reactions had the indicated chelators added after the fusion incubation, before detergent solubilization, to control for possible nonfusion-related effects of the chelators. (B) Titration of fusion reactions with BAPTA in the presence and absence of excess free Ca²⁺. Because the BAPTA stimulation curve is pushed to the right, it seems that only free BAPTA, and not the Ca²⁺-bound chelator, has a stimulatory effect on heterotrimer formation. (C) Structures of EGTA (black) and BAPTA (black plus red). (D) Effects of aminomethoxy (/AM) derivatives of BAPTA and EGTA on homotypic COPII vesicle fusion. Both /AM esters were able to partially mimic the effects of BAPTA, suggesting that the selective effects of BAPTA were due to its ability to rapidly chelate escaping luminal Ca²⁺. Fusion assay data are presented as means of duplicate determinations with error bars representing SE where larger than symbol size.

ALG-2 regulates in vitro COPII vesicle homotypic fusion in a Ca²⁺-dependent manner. (A) Homotypic COPII vesicle fusion measured using the in vitro VSV-G heterotrimer cargo mixing assay. Purified GST, GST-ALG-2 wild-type, or GST-ALG-2 E47,114A mutant proteins were included in fusion incubations at the indicated concentrations. The ice control includes radioactive VSV-G* vesicles and VSV-G-myc vesicles, but the low temperature prevents a specific fusion signal. (B) PAGE gel on purified proteins used in A and C, stained with Coomassie Blue. Proteins were used in fusion assays in the same proportions as on the gel. (C) Fusion experiment using partially inhibitory doses of the proteins indicated along the bottom. The left panel experiment was conducted in the absence of BAPTA. The right panel experiment was conducted, in the same experiment, in the presence of 2 mM BAPTA. Fusion values for both panels are normalized to the positive control signal in the absence of BAPTA (set to 100%). BAPTA negates the specific inhibitory effect of GST-ALG-2 but not that of anti-syntaxin 5 antibody. Fusion assay data are presented as means of duplicate determinations, with error bars representing SE where they are larger than symbol size.

Luminal Ca²⁺ regulates the size of ERGIC but not ERES structures. (A) NRK cells were either mock treated or treated with CPA to deplete luminal Ca²⁺ (see Materials and Methods), then fixed and immunostained for p24 and the Golgi marker mannosidase II. Shown are single focal planes from deconvolved widefield image stacks. (B) Immunolabeling for rbet1 and sec16 under the same experimental conditions as described in A. (C) Quantitation of numbers of peripheral p24-positive objects in three size bins under the experimental conditions shown in A. (D) Quantitation of peripheral objects positive for rbet1 and sec16 from the experiment shown in B. All size bins were included in one analysis. The rbet1 and sec16 object data are from precisely the same set of double-labeled cells. Values represent means derived from at least 20 randomly chosen cells. Error bars display SE. Selected p values from two-tailed Student's t test are included.

Luminal Ca²⁺ and ALG-2 regulate the retention of select coat subunits on pre-Golgi fusion intermediates. (A) BAPTA specifically extracts select COPI and COPII subunits from forming pre-Golgi intermediates. Homotypic fusion in vitro assays were conducted in the absence or presence of 2 mM BAPTA or EGTA (indicated above), and immunoisolated using anti-myc antibodies. Vesicles were generated from VSV-G-myc transfected cells (+) or nontransfected cells (−) to demonstrate specificity of isolation. In addition, 1 μM purified sar1 T39N was included during the budding stage to demonstrate that the isolated intermediates are COPII derived. Immunoblotted proteins are indicated along left edge. IP3R3, a resident ER membrane protein, additionally demonstrates specificity of budding. (B) Quantitation of a similar immunoisolation experiment (see blot in Supplemental Figure S3). In this experiment, it can be seen that peripheral membrane protein ALG-2 is also sensitive to BAPTA, but p115 is not. (C) Similar immunoisolation experiment where purified ALG-2 is present during the fusion experiment. ALG-2 caused a dramatic retention of outer shell component sec31 but not the COPI component β-COP. The absence of ER resident luminal protein PDI demonstrates specificity of the intermediates isolated. A star indicates the position of cross-reactive antibody heavy chain bands. GMP-PNP shows an estimate of components present on a fully coated vesicle as opposed to components remaining on tethered/fused pre-Golgi intermediates (D) Quantitation of experiment from C. For quantitations (B and D), band intensities were normalized to the recovery of cargo VSV-G-myc in each condition before plotting them on a relative scale with the highest recovery set to 1. Differences in band intensities seen in the input cells lanes (A) were caused by using slightly denser cell suspensions for the nontransfected cell controls.

ALG-2–dependent coat retention primarily inhibits via a prefusion mechanism. (A) Outline of experiment to distinguish between pre- or postfusion mechanisms by which ALG-2 inhibits heterotrimer assay (see text for explanation). Cargos VSV-G-myc and radioactive VSV-G* are depicted in green and red, respectively. COPII coat is represented by thick black lines. (B) Heterotrimer data from experiment depicted in A. Heterotrimers are normalized with 100% representing the amount produced during the first incubation in the absence of recombinant ALG-2, before addition of BAPTA and second incubation. BAPTA was omitted from the conditions plotted at time 0; BAPTA was added to all other conditions, while on ice, just before beginning the second incubation at 32°C. ALG-2 added during the first incubation had a dramatic consequence on the subsequent heterotrimer signal (red vs. blue lines), but ALG-2 added only during the second incubation had no influence (green square). Plotted are means of duplicate reactions, with error bars to represent SE where they exceeded symbol size.