Abstract
Golgin-160, a ubiquitous protein in vertebrates, localizes to the cytoplasmic face of the Golgi complex. Golgin-160 has a large coiled-coil C-terminal domain and a non-coiled-coil N-terminal ("head") domain. The head domain contains important motifs, including a nuclear localization signal, a Golgi targeting domain, and three aspartates that are recognized by caspases during apoptosis. Some of the caspase cleavage products accumulate in the nucleus when overexpressed. Expression of a non-cleavable form of golgin-160 impairs apoptosis induced by some pro-apoptotic stimuli; thus cleavage of golgin-160 appears to play a role in apoptotic signaling. We used a yeast two-hybrid assay to screen for interactors of the golgin-160 head and identified GCP60 (Golgi complex-associated protein of 60 kDa). Further analysis demonstrated that GCP60 interacts preferentially with one of the golgin-160 caspase cleavage fragments (residues 140-311). This strong interaction prevented the golgin-160 fragment from accumulating in the nucleus when this fragment and GCP60 were overexpressed. In addition, cells overexpressing GCP60 were more sensitive to apoptosis induced by staurosporine, suggesting that nuclear-localized golgin-160-(140-311) might promote cell survival. Our results suggest a potential mechanism for regulating the nuclear translocation and potential functions of golgin-160 fragments.
Highlights
The structure of the Golgi is very dynamic
We found that the Golgi complex-associated protein of 60 amino acids (GCP60) binds golgin-160 and, interestingly, shows preferential binding for a fragment of golgin-160 that is generated by caspase cleavage
Golgin-160 Interacts with GCP60 in Vitro—We previously showed that the N-terminal “head” domain of golgin-160 encodes important motifs (9, 14)
Summary
The structure of the Golgi is very dynamic. During mitosis the Golgi reversibly disassembles into small vesicular and tubular compartments, which contain some structural components of the Golgi. We found that the Golgi complex-associated protein of 60 amino acids (GCP60) binds golgin-160 and, interestingly, shows preferential binding for a fragment of golgin-160 that is generated by caspase cleavage. To confirm the interaction of golgin-160 with GCP60 we performed an in vitro binding assay using GST fused to golgin-160-(1–393) or GST alone and lysates of HeLa cells expressing GFP-GCP60.
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