A role for giantin in docking COPI vesicles to Golgi membranes - PubMed (original) (raw)

A role for giantin in docking COPI vesicles to Golgi membranes

B Sönnichsen et al. J Cell Biol. 1998.

Abstract

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH2-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.

PubMed Disclaimer

Figures

Figure 1

Figure 1

p115 binds to giantin. (A) Rat liver Golgi membranes were solubilized with buffer containing 0.5% Triton X-100, the extract cleared by centrifugation, and then incubated with biotin-p115 immobilized on streptavidin beads, or control beads. The clarified Golgi extract (lanes 1 and 4; 20% of total) and proteins bound to p115 beads (lanes 2 and 5; 50% of total) or control beads (lanes 3 and 6; 50% of total) were fractionated by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose and probed for GM130 and giantin, using specific antibodies. Molecular weight markers are shown in kD. (B) The clarified Triton X-100 extract of Golgi membranes was either preincubated with preimmune serum (PI, lane 1) or with anti- giantin antibodies (lane 2) before incubation with p115 beads. Bound proteins were fractionated by SDS-PAGE and visualized by Coomassie staining. (C) Golgi membranes were incubated for 10 min at 37°C with interphase HeLa cytosol (lane 1), mitotic HeLa cytosol (lane 2), or mitotic HeLa cytosol inactivated by pretreatment with staurosporine (lane 3). After solubilization of the reisolated membranes with Triton X-100, the cleared supernatants were incubated with p115 beads. Bound proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, and then probed for GM130 and giantin.

Figure 1

Figure 1

p115 binds to giantin. (A) Rat liver Golgi membranes were solubilized with buffer containing 0.5% Triton X-100, the extract cleared by centrifugation, and then incubated with biotin-p115 immobilized on streptavidin beads, or control beads. The clarified Golgi extract (lanes 1 and 4; 20% of total) and proteins bound to p115 beads (lanes 2 and 5; 50% of total) or control beads (lanes 3 and 6; 50% of total) were fractionated by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose and probed for GM130 and giantin, using specific antibodies. Molecular weight markers are shown in kD. (B) The clarified Triton X-100 extract of Golgi membranes was either preincubated with preimmune serum (PI, lane 1) or with anti- giantin antibodies (lane 2) before incubation with p115 beads. Bound proteins were fractionated by SDS-PAGE and visualized by Coomassie staining. (C) Golgi membranes were incubated for 10 min at 37°C with interphase HeLa cytosol (lane 1), mitotic HeLa cytosol (lane 2), or mitotic HeLa cytosol inactivated by pretreatment with staurosporine (lane 3). After solubilization of the reisolated membranes with Triton X-100, the cleared supernatants were incubated with p115 beads. Bound proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, and then probed for GM130 and giantin.

Figure 1

Figure 1

p115 binds to giantin. (A) Rat liver Golgi membranes were solubilized with buffer containing 0.5% Triton X-100, the extract cleared by centrifugation, and then incubated with biotin-p115 immobilized on streptavidin beads, or control beads. The clarified Golgi extract (lanes 1 and 4; 20% of total) and proteins bound to p115 beads (lanes 2 and 5; 50% of total) or control beads (lanes 3 and 6; 50% of total) were fractionated by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose and probed for GM130 and giantin, using specific antibodies. Molecular weight markers are shown in kD. (B) The clarified Triton X-100 extract of Golgi membranes was either preincubated with preimmune serum (PI, lane 1) or with anti- giantin antibodies (lane 2) before incubation with p115 beads. Bound proteins were fractionated by SDS-PAGE and visualized by Coomassie staining. (C) Golgi membranes were incubated for 10 min at 37°C with interphase HeLa cytosol (lane 1), mitotic HeLa cytosol (lane 2), or mitotic HeLa cytosol inactivated by pretreatment with staurosporine (lane 3). After solubilization of the reisolated membranes with Triton X-100, the cleared supernatants were incubated with p115 beads. Bound proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, and then probed for GM130 and giantin.

Figure 2

Figure 2

GM130, but not giantin, is depleted from COPI vesicles. Golgi membranes were incubated for 60 min at 37°C with interphase HeLa cytosol in the presence of GTPγS to accumulate COPI vesicles. After addition of salt to release the vesicles from Golgi remnants, samples were centrifuged to equilibrium on a 30–50% sucrose gradient. Membranes were reisolated by centrifugation, fractionated by SDS-PAGE, Western blotted, and then quantitated. (A) GM130 and giantin. (B) β-COP. The fractions containing COPI vesicles and Golgi remnants are indicated.

Figure 3

Figure 3

Purified COPI vesicles contain giantin by immunoelectron microscopy. COPI vesicles generated as described in the legend to Fig. 2 were fixed, cryosectioned, and labeled with antibodies to giantin followed by protein A coupled to 10-nm gold. Bar, 200 nm.

Figure 4

Figure 4

Biotinylated p115 binds to giantin in COPI vesicles. (A) Triton X-100 extracts of rat liver Golgi membranes or purified COPI vesicles from interphase incubations were incubated with biotinylated p115 immobilized on streptavidin beads, or control beads. Starting extracts (30% of total), and proteins bound to p115 beads or control beads (100% of total) were fractionated by SDS-PAGE, transferred to nitrocellulose, and then probed with antibodies against giantin and GM130. (B) Purified COPI vesicles were incubated with or without biotinylated p115 (bio-p115) for 15 min on ice, the membranes reisolated on a sucrose flotation gradient, and then incubated with or without the cross-linker, DTSSP. After quenching the cross-linker, proteins were extracted with Triton X-100, 0.5 M KCl, and then the extracts were incubated with streptavidin beads. The cross-linker was cleaved by boiling the samples in sample buffer with 50 mM DTT. Bound and unbound fractions were subjected to SDS-PAGE and analyzed by Western blotting, probing with antibodies against giantin, p115, and β-COP. Note that the streptavidin beads quantitatively depleted biotinylated p115 from the extracts.

Figure 5

Figure 5

Binding of p115 to COPI vesicles is saturable. Purified COPI vesicles (8–10 μg) were incubated with increasing amounts of p115 for 15 min on ice. Membranes were reisolated by centrifugation through a 35% sucrose cushion, the pellets fractionated by SDS-PAGE and the p115 quantitated after Western blotting (inset shows representative experiment). Vesicle recovery was reproducibly 80–90% as assessed by probing with β-COP antibodies. Results are expressed as the mean of three experiments ± SEM.

Figure 6

Figure 6

p115 binds to giantin on COPI vesicles and GM130 on Golgi remnants. Golgi membranes were incubated at 37°C with interphase (30 min) or mitotic cytosol (20 min) in the presence of GTPγS, salt was added, and samples were fractionated to separate COPI vesicles from Golgi remnants (see Materials and Methods). Samples (8–10 μg) were incubated for 15 min on ice with 100 ng p115. Antibodies to GM130 and giantin were used to pretreat the membranes for 15 min on ice before addition of p115. The GM130 peptide was added together with the p115 at a 70-fold molar excess. Membranes were reisolated, fractionated by SDS-PAGE, the amount of bound p115 quantitated by Western blotting and normalized for β-COP (COPI vesicles) or GM130 (Golgi remnants). Membrane recovery was 80–90% for COPI vesicles and 70–80% for Golgi remnants. Results are expressed as a percentage of the interphase vesicles or remnants ± SEM (n = 3–5, depending on the experiment). The 100% values were 88 ± 5.8 ng p115/10 μg interphase vesicles and 112 ± 18.3 ng/10 μg interphase remnants. (A) Binding to COPI vesicles. (B) Binding to Golgi remnants.

Figure 7

Figure 7

The GM130 peptide releases COPI vesicles from Golgi membranes. Salt-washed Golgi membranes were incubated with interphase cytosol for 30 min at 37°C in the presence of GTPγS. In some experiments, the cytosol was preincubated for 15 min on ice with the GM130 peptide at the indicated molar ratios over cytosolic p115. At the end of the incubation, those samples using untreated cytosol were either left untreated (−salt; GM130 peptide) or treated with KCl at a final concentration of 250 mM to release COPI vesicles (+salt). Golgi remnants were sedimented in a microfuge and released COPI vesicles in the supernatant were recovered by high speed centrifugation through a 35% sucrose cushion. Remnants and vesicles were fractionated by SDS-PAGE and quantitated by Western blotting using antibodies to β-COP and p115. (A) Release of COPI vesicles is expressed as a percentage of the +salt control ± SEM (n = 3). (B) p115 bound to Golgi remnants is expressed as a percentage of the −salt control ± SEM (n = 3).

Figure 8

Figure 8

Docking of COPI vesicles to Golgi membranes is facilitated by p115. Purified COPI vesicles (8–10 μg) were incubated with salt-washed Golgi membranes (8 μg) at 30°C for (A) increasing times ± 75 ng p115 or (B) increasing amounts of p115 for 10 min. Docked vesicles were isolated by medium-speed centrifugation, and free vesicles were recovered from the supernatant by high speed centrifugation. Results are expressed as the amount of β-COP in the medium speed pellet as a percentage of that in the combined medium and high speed pellets ± SEM (n = 3). Insets show the β-COP signal in the medium speed pellets of representative binding experiments. Recovery of COPI vesicles was reproducibly 80–90% of the input vesicles.

Figure 9

Figure 9

Docking requires giantin in the COPI vesicles and GM130 on Golgi membranes. Purified COPI vesicles (8–10 μg) were incubated with p115 (75 ng) in the absence or presence of salt-washed Golgi membranes (8 μg) for 10 min at 30°C. Where indicated the vesicles or Golgi membranes were pretreated with antibodies to giantin or GM130. Samples were processed as in Fig. 8 and vesicle docking is expressed as a percentage of the control without pretreatment ± SEM (n = 3).

Similar articles

Cited by

References

    1. Allan VJ, Kreis TE. A microtubule-binding protein associated with membranes of the Golgi apparatus. J Cell Biol. 1986;103:2229–2239. - PMC - PubMed
    1. Barlowe C. COP II - a membrane coat that forms endoplasmic reticulum-derived vesicles. FEBS (Fed Eur Biochem Soc) Lett. 1995;369:93–96. - PubMed
    1. Barroso M, Nelson DS, Sztul E. Transcytosis-associated protein (TAP)/p115 is a general fusion factor required for binding of vesicles to acceptor membranes. Proc Natl Acad Sci USA. 1995;92:527–531. - PMC - PubMed
    1. Bednarek SY, Ravazzola M, Hosobuchi M, Amherdt M, Perrelet A, Schekman R, Orci L. COP I-coated and COP II-coated vesicles bud directly from the endoplasmic-reticulum in yeast. Cell. 1995;83:1183–1196. - PubMed
    1. Campbell JL, Schekman R. Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles. Proc Natl Acad Sci USA. 1997;94:837–842. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources