Two distinct binding interactions in the p97/p47 complex. (A) Each GST-tagged deletion mutant of p47 was incubated with p97 in a buffer containing 0.1% Triton X-100 on ice, then isolated on glutathione beads, and bound proteins were fractionated by SDS-PAGE. Blots were probed with anti-p97 antibodies. (B) GST-tagged p97 (1.5 μg) was incubated with p47full (0.5 μg) either alone or in combination with a deletion mutant of p47 (4 μg) on ice. Blots were probed with antibodies to the His-tag on p47 fragments. (C) GST-tagged deletion mutants of p97 were incubated with each of the p47 fragments containing the p97-binding sites on ice. Blots were probed with antibodies to the His tag on the p47 fragments. (D) p97 was preincubated with an excess amount of p47 fragments containing the p97-binding sites and purified by gel filtration followed by EM observation.

Identification of VCIP135. (A) A working model of how a p97/p47-interacting protein (protein X) forms an unstable ternary complex with p97 and p47. (B) Purification of VCIP135 from rat liver cytosol. Each small p47 fragment with a p97-binding site was immobilized on beads and incubated with rat liver cytosol. Bound proteins were eluted by a 1 M KCl wash, TCA-precipitated, fractionated, and silver stained. VCIP135 was isolated on p47(171–270) beads (asterisk).

VCIP135 binds to and dissociates the p97/p47 complex. (A) Rat liver cytosol (3.5 mg total protein) was incubated with preimmune serum or anti-VCIP135 antibodies. The immunoprecipitates were fractionated by SDS-PAGE followed by staining with Coomassie blue (CBB, left) and Western blotting with antibodies to VCIP135, p97, and p47 (WB, right). (B) Rat liver cytosol was size fractionated by 5–30% sucrose sedimentation, and the fractions were assayed by Western blotting with antibodies to VCIP135 and p97. (C) His-tagged deletion mutants of VCIP135 were incubated with p97 in a buffer containing 0.1% Triton X-100 on ice and then precipitated by anti–His tag antibodies. Blots were probed with anti-p97 antibodies. (D) The sequence alignment between VCIP135(744–859) and p47(270–370). Identical (asterisk) and conserved residues (□) are marked. Arrows and black bars show β-strands and α-helices, respectively. The three surface residues identified as potential p97-interacting residues in p47 are boxed (Yuan et al., 2001) and are conserved in VCIP135. (E) Rat liver cytosol (3.5 mg total protein) was cross-linked with SAND and incubated with preimmune serum or anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE followed by Western blotting with antibodies to VCIP135, p97, p47, and Ufd1p (a negative control). (F) p97 was incubated with an excess amount of p47, and the p97/p47 complex was purified using gel filtration in the absence of nucleotides. The purified p97/p47 complex (1.2 μg p97) was incubated in a buffer (50 μl) containing 1 mM nucleotide on ice for 30 min. VCIP135 (0.8 or 1.6 μg) was added into the mixture and incubated on ice for 1 h, and p97 that formed a complex with p47 was coimmunoprecipitated with anti p47 antibodies. Blots were probed with antibodies to p97 and p47.

The localization of VCIP135. Double immunofluorescence staining of VCIP135 and either GalT, a Golgi marker, or PDI, an ER marker, shows that VCIP135 localizes to Golgi and ER. NRK cells were fixed by methanol at −20°C for 4 min, stained with antibodies, and observed by confocal microscopy. Bars: (top) 10 μm; (bottom) 2 μm.

VCIP135 binds to syntaxin5 and dissociates the p97/p47/syntaxin5 complex via p97 catalyzed ATP hydrolysis. (A) His-tagged VCIP135 (0.4 μg) was incubated with either GST, GST–syn5ΔTM, or GST–syn1ΔTM (0.5 μg each) in a buffer (50 μl) containing 0.1% deoxycholate on ice and isolated on glutathione beads. Blots were probed with antibodies to VCIP135. (B) 1 M KCl-washed Golgi membranes were incubated with His-tagged VCIP135 on ice for 1 h. The membranes were isolated from unbound VCIP135 by centrifugation and solubilized in a buffer containing 1% CHAPS. VCIP135 and its interacting proteins were immunoprecipitated by mAbs to the His tag on VCIP135 and fractionated by SDS-PAGE followed by Western blotting with antibodies to syntaxin5 peptide, VCIP135, and GS15 (a negative control). The bottom band of VCIP135 is a degradative product. (C) GST–syn5ΔTM (0.5 μg) or GST (0.5 μg) was incubated with p97 (2 μg), p47 (1 μg), VCIP135 (3 μg), or their combinations in 50 μl of buffer containing 0.1% deoxycholate, but no nucleotide on ice, and isolated on glutathione beads. Blots were probed with antibodies to VCIP135, p97, and p47. (D) GST–syn5ΔTM (0.5 μg) was incubated with VCIP135 (0.35 or 0.7 μg) on ice for 30 min, immobilized on glutathione beads, and washed to remove unbound VCIP135. The nucleotide-free p97/p47 (2 μg p97) was preincubated in buffer containing 1 mM nucleotides on ice for 30 min and added into the beads. After 3-h incubation at 4°C, the beads were washed and subjected to Western blotting with antibodies to VCIP135, p97, and p47. (E) p97/p47/GST–syn5ΔTM-beads and VCIP135/p97/p47/GST–syn5ΔTM-beads were prepared as in C in the presence of 1 mM AMP–PNP. Then, the beads were washed with buffer containing 5 mM AMP–PNP or ATP and incubated at 4°C for 3 h. (F) VCIP135/GST–syn5ΔTM beads were prepared as in C. p97 was added into the mixture and incubated at 4°C for 3 h. Blots were probed with antibodies to VCIP135. (G) VCIP135 (1 or 2 μg) and α-SNAP (0.1 μg) were incubated with GST–syn5ΔTM (0.4 μg) or GST (0.4 μg) on ice for 1 h and then isolated on glutathione beads. Blots were probed with antibodies to VCIP135 and α-SNAP. See also Figs. S3 and S4 available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1.

VCIP135 is essential for p97/p47-mediated membrane fusion. (A) Mitotic Golgi membranes were incubated with the indicated components at 37°C for 60 min. The membranes were fixed, processed, and the percentage of membrane in cisterna was determined. An antibody to VCIP135 was added together with the p97/p47 complex (50 μg p97/ml) to mitotic Golgi membranes. The same antibody was quenched by a VCIP135 fragment and added. Mean ± SD (n = 4); 0% represents the buffer (21.2% in cisternal membranes) and 100% represents p97/p47 alone (41.8% in cisternal membranes). (B) Antibodies to VCIP135 were added to mitotic Golgi membranes together with the components of the NSF pathway: NSF (100 μg/ml), α-SNAP (25 μg/ml), and γ-SNAP (25 μg/ml), p115 (8 μg/ml). Mean ± SD (n = 3); 0% represents the buffer (22.5% in cisternal membranes) and 100% represents NSF/SNAPs/p115 alone (40.4% in cisternal membranes). (C) The top panels show the amounts of VCIP135 in mitotic Golgi membranes and 1 M KCl-washed membranes. 1 M KCl-washed or nonwashed mitotic Golgi membranes were incubated with several components or their combinations; p97/p47 (50 μg p97/ml), VCIP135 (15 μg/ml), p97 (50 μg/ml). Mean ± SD (n = 4); 0% represents the buffer (23.2% in cisternal membranes) and 100% represents non salt-washed membranes with p97/p47 alone (43.4% in cisternal membranes). (D) 1 M KCl-washed mitotic Golgi membranes were incubated with p97/p47 (50 μg p97/ml) and several doses of VCIP135. Results are the averaged percentage (n = 3); 0% represents VCIP135/p97 = 0 (21.2% in cisternal membranes) and 100% represents VCIP135/p97 = 1/4 (44.1% in cisternal membranes).

VCIP135 is required for Golgi assembly in vivo. (A) NRK cells at prophase (or early prometaphase) were injected with antibodies and fixed after they exited mitosis. Golgi (green, top), injected antibodies (red, top), and chromatin (bottom) were stained by monoclonal anti-GalT antibodies, anti–rabbit IgG antibodies, and Hoechst33342, respectively. Bars, 10 μm. (B) Representative EM images of Golgi in daughter cells after antibodies or quenched antibodies were microinjected. Bars, 0.5 μm. (C) Immunolabeling EM images of Golgi area in the NRK cells microinjected with anti-VCIP135 antibodies. For immunolabeling of Golgi marker proteins, monoclonal antibodies to either GalT (left) or GM130 (right) were used. Arrows show immunolabeled vesicles and tubules. Bars, 0.5 μm. (D) Golgi membranes were classified and counted as described previously (Shorter and Warren, 1999; Seemann et al., 2000) in microinjected cells. Mean ± SD (n = 7–9).

VCIP135 is required for ER network formation in vivo. (A) Antibodies were microinjected into CHO cells expressing GFP-tagged HSP47. Since ER structures were easily damaged and lost after fixation, they were observed in living cells without fixation by confocal microscopy. Representative images of ER in daughter cells after the injection of antibodies are shown. N, nucleus. Bars, 5 μm. (B) The number of three-way junctions was counted by confocal microscopy as a parameter of ER network formation. Mean ± SD (n = 9).