The Golgi-targeting sequence of the peripheral membrane protein p230

Vesicle transport requires the recruitment of cytosolic proteins to specifi c membrane compartments. We have previously characterised a brefeldin A-sen sitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cy tosol and the cytoplasmic face of buds/vesicles of trans-Golgi network memb ranes in a G protein-regulated manner. Identifying the mechanism responsibl e for Golgi targeting of p230 is important for the elucidation of its funct ion. By transfection of COS cells with deletion mutants of p230 we here dem onstrate that the C-terminal domain is necessary for targeting to the Golgi , Furthermore, the C-terminal 98 amino acid domain of p230 attached to the green fluorescent protein (GFP-p230-C98aa) was efficiently Golgi-localised in transfected COS cells. Deletion mutants of GFP-p230-C98aa together with alanine scanning mutagenesis identified a minimum stretch of 42 amino acids that is essential for Golgi targeting, suggesting that the conformation of the domain is critical for efficient targeting. In COS cells expressing hi gh levels of GFP-p230-C98aa fusion protein, endogenous p230 was no longer a ssociated with Golgi membranes, suggesting that the GFP fusion protein and endogenous p230 may compete for the same membrane target structures, The Go lgi binding of GFP-p230-C98aa is brefeldin A-sensitive and is regulated by G proteins. These studies have identified a minimal sequence responsible fo r specific targeting of p230 to the Golgi apparatus, which displays similar membrane binding characteristics to wild-type p230.