Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae

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Abstract
The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated. Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115. Using in vitro translation and site‐directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI‐COOH at the C‐terminus of GM130, a region known to be required for its localization to the Golgi apparatus. The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189–201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains. Using green fluorescent protein (GFP)‐tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.