Mapping the functional domains of the Golgi stacking factor GRASP65

Abstract

The Golgi reassembly stacking protein (GRASP) family has been implicated in the stacking of Golgi cisternae and the regulation of Golgi disassembly/reassembly during mitosis in mammalian cells. GRASP65 is a dimer that can directly link adjacent surfaces through trans-oligomerization in a mitotically regulated manner. Here we show that the N-terminal GRASP domain (amino acids 1-201) is both necessary and sufficient for dimerization and trans-oligomerization but is not mitotically regulated. The C-terminal serine/proline-rich domain (amino acids 202-446) cannot dimerize nor can it link adjacent surfaces. It does, however, confer mitotic regulation on the GRASP domain through multiple sites phosphorylated by the mitotic kinases, cdc2/B1, and the polo-like kinase. Transient expression corroborated these results by showing that the GRASP domain alone inhibited mitotic fragmentation of the Golgi apparatus.

Fig. 1. The mitotic phosphorylation sites of GRASP65 are in the C-terminal SPR domain

A, GRASP65 structure and constructs. The upper schematic shows the domain structure of GRASP65, which comprises an N-terminal GRASP domain (myr, myristoylated N-terminal glycine) followed by a short GM130 binding region-(197–211), and a SPR domain at the C terminus. Short vertical lines above the schematic mark potential phosphorylation sites. Longer vertical lines below the schematic mark caspase-3 cleavage sites. Lower schematics(details at left) represent constructs used for phosphorylation as well as native GRASP65 (bottom). B, phosphorylation of GRASP65 and its constructs. Native GRASP65 and purified recombinant constructs were incubated with MC or the indicated kinases and immunoblotted for GRASP65. Note that the combination of cdc2/B1 and plk kinases phosphorylated GRASP65 to a similar extent as mitotic cytosol. The increases in MW were measured relative to the untreated sample.

Fig. 2. The N-terminal GRASP domain is both necessary and sufficient for dimerization

GRASP65 (His-tagged) was co-expressed with A, GRASP65 (full-length, MBP-tagged); B, GRASP domain (1–201, GST-tagged); C, ΔN15-GRASP domain (16–201, GST-tagged);or D, SPR domain (202–446, GST tagged) in Escherichia coli and purified sequentially on amylose or glutathione columns followed by a nickel column (Left panels in each pair), or the reverse (Right panels in each pair). Equal amounts of protein from each purification step were analyzed by immunoblotting for GRASP65 or the tag. Note that, in A–C, the ratio of the two proteins in the final isolated complex is ~1:1, irrespective of the expression levels or the order in which the columns were used for purification.

Fig. 3. The GRASP domain is necessary and sufficient for oligomerization but is not mitotically regulated

A, GRASP65 (full-length, His-tagged) was ncubated with either GRASP65 (full-length, MBP-tagged), the GRASP domain (1–201, GST-tagged), or the SPR domain (202–446, GST-tagged), in the presence of interphase (IC, lanes 1–3) or mitotic cytosol (MC, lanes 4–6). The protein complex was then isolated using nickel beads. Equal portions of the input (I), unbound (U), or bound (B) fractions were analyzed by immunoblotting for GRASP65. Note the presence of the GRASP domain in the bound complexes pre-incubated with either interphase or mitotic cytosol (lanes 3 and 6). B, GRASP65, the GRASP domain, and the SPR domain were each purified and incubated in the presence or absence of cdc2/B1, sedimented in glycerol gradients, and fractions were analyzed by immunoblotting. Note that the GRASP domain was necessary for rapid sedimentation and, when present alone (without the SPR domain), was unaffected by treatment with mitotic kinase. C, GRASP65 or the GRASP domain were sedimented in glycerol gradients as in B, and the indicated fractions from each gradient were pooled, dialyzed, concentrated, and reloaded onto a similar gradient. Note that the GRASP domain, but not full-length GRASP65, was stable to recentrifugation.

Fig. 4. The GRASP domain is necessary and sufficient for trans-oligomerization (bead aggregation) but is not mitotically regulated

A, purified GRASP65 constructs were covalently coupled to Dynal beads and incubated with IC (upper panels) or MC (lower panels). After incubation the beads were transferred to glass slides, and random fields were photographed. A representative image of each condition is shown. B, quantitation of (A) and of beads coated with other GRASP65 constructs. Results are shown as the mean ± S.E. from three independent experiments. Note that aggregation requires the GRASP domain, but mitotic regulation requires the SPR domain. C, Dynal beads coated with His-GRASP65 were incubated with interphase cytosol in the presence of the indicated GRASP65 constructs. Representative images are shown. Bar, 500 µm. D, quantitation of (C). Note that aggregation is only inhibited by constructs containing the GRASP domain.

Fig. 5. Mitotic phosphorylation sites in the SPR domain of GRASP65

A, potential sites (serines and threonines indicated by lines) were mutated to alanines (left panel), and the purified proteins incubated with MC or the indicated kinases followed by SDS-PAGE and immunoblotting for GRASP65 (middle panel). Calculated shifts in molecular weight are shown at right. The shifts were inversely correlated to the number of sites mutated. B, phosphorylation mutants of GRASP65 (lines above schematics at left) were incubated with interphase cytosol (middle panel) or the mitotic kinase cdc2/B1 (right panel), sedimented in glycerol gradients, and blotted for GRASP65. Note the increase in MW after kinase treatment in the right panel. L, 10% input; P, pellet. C, Dynal beads coated with different GRASP65 phosphorylation mutants (lines above schematics at left) were incubated with IC or MC, or a sequence of cytosols (IC->MC or MC->IC). Bar, 500 µm. Quantitation of the results is shown at right. Note that mutation of all seven sites dramatically inhibited disaggregation by MC.

Fig. 6. GRASP65 is involved in stacking in vivo

A, HeLa cells were stably transfected with the constructs schematized beneath the images. Representative fluorescence images of interphase (upper panels) and mitotic (lower panels) cells double-labeled with the Golgi marker, GM130. Note that the N-terminal myristoylation signal is needed for Golgi localization. Bar, 10 µm. B–G, representative EM images of mitotic (B–D) and interphase (E) cells and quantitation (F, G). Results expressed as the mean ± S.E. from three independent experiments (n = number of cell profiles counted). Note that the GRASP domain inhibits fragmentation during mitosis but only when localized to the Golgi by the N-terminal myristoylation signal. Bar, 0.25 µm.