Identification of a redox-sensitive cysteine in GCP60 that regulates its interaction with golgin-160.

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.