Abstract
Golgin-160 is ubiquitously expressed in vertebrates. It localizes to the cytoplasmic side of the Golgi and has a large C-terminal coiled-coil domain. The noncoiled-coil N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal, and three caspase cleavage sites. Caspase cleavage of the golgin-160 head domain generates different fragments that can translocate to the nucleus by exposing the nuclear localization signal. We have previously shown that GCP60, a Golgi resident protein, interacts weakly with the golgin-160 head domain but has a strong interaction with one of the caspase-generated golgin-160 fragments (residues 140-311). This preferential interaction increases the Golgi retention of the golgin-160 fragment in cells overexpressing GCP60. Here we studied the interaction of golgin-160-(140-311) with GCP60 and identified a single cysteine residue in GCP60 (Cys-463) that is critical for the interaction of the two proteins. Mutation of the cysteine blocked the interaction in vitro and disrupted the ability to retain the golgin-160 fragment at the Golgi in cells. We also found that Cys-463 is redox-sensitive; in its reduced form, interaction with golgin-160 was diminished or abolished, whereas oxidation of the Cys-463 by hydrogen peroxide restored the interaction. In addition, incubation with a nitric oxide donor promoted this interaction in vitro. These findings suggest that nuclear translocation of golgin-160-(140-311) is a highly coordinated event regulated not only by cleavage of the golgin-160 head but also by the oxidation state of GCP60.
Highlights
528)C463S were lysed in detergent solution and the lysates Recently, we showed that GCP60 interacts better with golginwere incubated overnight with golgin-160-(140 –311) fused to 160-(140 –311) when compared with the 60 –311 fragment and Glutathione S-transferase (GST) or GST alone that had been previously rebound to gluta- the full-length head, 1–393 [17]
Golgin-160, initially identified from patients with autoimmune disease, localizes to the cytoplasmic side of the Golgi [27, 28]. It has the characteristic coiled-coil structure of golgin proteins in the C-terminal two-thirds of the protein, whereas the N-terminal portion, or head, contains a cryptic nuclear localization signal, Golgi targeting information, and three aspartates that can be cleaved by caspases during apoptosis [11, 15]
Cleavage of the golgin-160 head domain generates distinct fragments that can translocate to the nucleus after exposure of the cryptic nuclear localization signal (NLS) [15]
Summary
Oxidation Assay—HeLa cells transfected with plasmids of golgin-160 could be targeted by caspases-2, -3, and -7 during encoding GFP-GCP60-(328 –528) or GFP-GCP60-(328 – apoptosis, generating different fragments (Fig. 1A) [11]. The two tubes left generated by a single cleavage event would show a prewere incubated with 2 mM hydrogen peroxide (H2O2) (Sigma) ferred interaction with GCP60, we produced GST fusion profor 1 h at room temperature with rocking and washed three teins representing fragments cleaved once with caspase-2 at times with detergent solution, and one tube received sample Asp-59 (residues 60 –393), or at Asp-311 (residues 1–311), and buffer to elute the bound proteins, whereas the other tube was fragments cleaved once with caspase-3 at Asp-139
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