Technical Reference #287

287.

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum Andreas Girod; Brian Storrie; Jeremy C. Simpson; Ludger Johannes; Bruno Goud; Lynne M. Roberts; J. Michael Lord; Tommy Nilsson and Rainer Pepperkok, EMBL Heidelberg, Nature Cell Biology, 1(287), (1999)
Link To Paper

Abstract:
The cytosolic coat-protein complex COP-I interacts with cytoplasmic ‘retrieval’ signals present in membraneproteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex and is required for bothanterograde and retrograde transport i

Materials & Methods:
Material. Tissue-culture media fetal calf serum (FCS) trypsin-EDTA penicillin and streptomycin were from Life Technologies (Karlsruhe Germany); L-glutamine fluorescein isothiocyanate (FITC)-conjugated dextran diphtheria toxin Pseudomonas exotoxin chicken ovalbumin bovine serum albumin and rabbit IgG were from Sigma; BFA and HEPES were from AppliChem GmbH (Darmstadt Germany); saponin and mowiol were from Calbiochem; [35S]methionine was from NEN Life Science Products; and Cascade blue bovine serum albumin (BSA) and Cascade blue dextran were from Molecular Probes (Eugene OR). BSA was conjugated with Alexa-350 (Molecular Probes) according to the manufacturer’s instructions. The following primary antibodies have been described previously: monoclonal anti-KDEL-receptor antibody47 rabbit Fab fragments of anti-EAGE (anti b-COP)31 rabbit Fab fragments of anti-KDELreceptor18 anti-ERGIC-53 (ref. 48) anti-p63 (ref. 33) anti-Myc (9E10)23 anti-VSV-G (rabbit or mouse monoclonal antibody P5D4)49 anti-EEA1 (ref. 41) anti-GM130 (ref. 38) and CM1A10 (ref. 50). The following secondary antibodies were from Molecular Probes (Leiden Netherlands): goat anti-mouse IgG (Alexa-488- or Alexa-568-conjugated) goat anti-rabbit IgG (Alexa-488- or Alexa-568-conjugated) and goat anti-human IgG (Alexa-488-conjugated). Donkey anti-mouse or anti-rabbit IgG (Cy3-conjugated) was from Dianova (Hamburg Germany). Construction of Rab6-GDP and Rab6-GTP expression plasmids. The polymerase chain reaction (PCR) was used to introduce flanking BamHI sites and a Kozak consensus sequence using Rab6-GDP or Rab6-GTP complementary DNA as a template40. Primers used were: forward 5¢-TGATCCGGATCCGCCATGTCCACGGGCGGAGAC; reverse 5¢-TGATCCGGATCCTTAGCAGGAACAGCCTCCTTC. Obtained PCR fragments were purified digested with BamHI and cloned into the mammalian expression vector pCMUIV4. The sequences of cloned inserts were confirmed. Cell culture and metabolic labelling. Cells were routinely cultured in medium (HeLa cells in Dulbecco’s modified eagle’s medium (DMEM) plus 10% FCS; Vero cells in minimum essential medium (MEM) plus 5% FCS) supplemented with 10 mM L-glutamine penicillin (100 Uml–1) and streptomycin (100 mg ml–1) together with either 300 mg ml–1 G418 (HeLa transfectants) or 10 mM HEPES pH 7.2 (Vero cells). Cells were grown in a humidified incubator at 37 °C and 5% CO2.. Medium with 10 mM HEPES pH7.2 used during the experiments was always pre-equilibrated with 5% CO2. Metabolic labelling and determination of toxicity was carried out essentially as described18. Protein purification. Recombinant wild-type or KDEL-tagged Shiga toxin B-chain and recombinant Sar1dn were purified as described1525. Purified B-chain was labelled with Cy3 according to the manufacturer’s instructions (Amersham) as in previous studies16. Aliquots of Sar1dn were stored at a concentration of 1.8 mg ml–1 at –80 °C and diluted to a concentration of 0.75 mg ml–1 for microinjection. Microinjection and visualization of ST-B–Cy3 and ST-B–KDEL–Cy3 transport. For microinjection31 cells were grown for 1–2 days in 3.5-cm glass-bottom dishes (MatTek Corporation Ashland MA) or on glass coverslips. Phenol-free medium was used for incubation outside an incubator containing low carbonate (0.85 g l–1) supplemented with penicillin streptomycin glutamine and 30 mM HEPES pH 7.2. At 30 min before toxin treatment cells were incubated in binding medium (FCS-free culture medium containing 0.1% BSA or ovalbumin) for 5 min and for the remaining 25 min in the same medium supplemented with labelled toxin fragment. After removal of unbound toxin by washing for 5 min on ice with cold PBS cells were incubated in MEM at 37 °C. The localization of toxin fragments was imaged in either living or fixed cells at the indicated time points. Toxin experiments were done in Vero cells as a continuation of our previous work18. Microinjection of Sar1dn. For anti-EAGE Fab microinjection experiments cells were microinjected with anti-EAGE in the presence of Sar1dn protein and Alexa-350-conjugated BSA as a co-injection marker. Protein injections were done in the presence of 5 mg ml–1 emetine and cells were subsequently incubated in the presence of emetine. For experiments involving Arf-1(Q71L) cells were co-injected with plasmids encoding Arf-1(Q71L) and Sar1dn together with Cascade-blue-conjugated BSA as co-injection marker. In the positive control experiment expression of the Arf-1(Q71L) produced the expected accumulation of COP-I with vesicles30. In experiments involving Rab6 expression the nuclei of all cells in a defined area were microinjected with pCMUIV plasmids encoding either Rab6-GDP or Rab6-GTP mixed with Cascade blue dextran as a co-injection marker. After a 6-h expression period the same HeLa cells were cytoplasmically injected with Sar1dn protein in the presence of Alexa-350-conjugated BSA as a coinjection marker. Emetine was used to inhibit protein synthesis. Immunofluorescence analysis. For indirect immunofluorescence cells were grown on glass coverslips. After fixation and permeabilization with ice-cold methanol 3.5% paraformaldehyde plus 0.1% Triton-X100 or 0.1% saponin cells were treated with an appropriate dilution of primary antibody for 30 min. Cells were then washed three times with PBS and incubated with the secondary antibodies for 30 min. After three more washes with PBS the coverslips were mounted on microscope slides using mowiol.

Microscopic Technique
Fluroescence Microscopy

Cell Type(s)
HeLa