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Using autoantibodies from a Sjögren's syndrome patient, we have previously identified a 230-kDa peripheral membrane protein associated with the cytosolic face of the trans-Golgi (Kooy, J., Toh, B. H., Pettitt, J. M., Erlich, R. and Gleeson, P. A. (1992) J. Biol. Chem. 267, 20255-20263). Here we report the molecular cloning and sequence analysis of human p230 and the localization of its gene to chromosome 6p12 22. Partial cDNA clones, isolated from a HeLa cell cDNA library using autoantibodies, were used to obtain additional cDNAs, which together span 7695 base pairs (bp). The p230 mRNA is approximately 7.7 kilobases. Two alternatively spliced mRNAs for p230 were detected. These differed by 21- and 63-bp insertions in the 3'-sequence, resulting in differences in amino acid sequence at the carboxyl terminus. The predicted 261-kDa protein is highly hydrophilic with 17-20% homology with many proteins containing coiled-coil domains. Apart from two proline-rich regions (amino acids 1-117 and 239-270), p230 contains a very high frequency of heptad repeats, characteristic of alpha-helices that form dimeric coiled-coil structures. p230 also includes the sequence ESLALEELEL (amino acids 538-546), a motif found in the granin family of acidic proteins present in secretory granules of neuroendocrine cells. This is the first report of a cytosolic Golgi protein containing a granin motif. The structural characteristics of p230 indicate that it may play a role in vesicular transport from the trans-Golgi.

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Vesicle transport requires the recruitment of cytosolic proteins to specific membrane compartments. We have previously characterised a brefeldin A-sensitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cytosol and the cytoplasmic face of buds/vesicles of trans-Golgi network membranes in a G protein-regulated manner. Identifying the mechanism responsible for Golgi targeting of p230 is important for the elucidation of its function. By transfection of COS cells with deletion mutants of p230 we here demonstrate 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 high levels of GFP-p230-C98aa fusion protein, endogenous p230 was no longer associated with Golgi membranes, suggesting that the GFP fusion protein and endogenous p230 may compete for the same membrane target structures. The Golgi binding of GFP-p230-C98aa is brefeldin A-sensitive and is regulated by G proteins. These studies have identified a minimal sequence responsible for specific targeting of p230 to the Golgi apparatus, which displays similar membrane binding characteristics to wild-type p230.

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