We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Mammalian cells concentrate Golgi membranes around the centrosome in a microtubule-dependent manner. The mechanisms involved in generating a single Golgi ribbon in the periphery of the centrosome remain unknown. Here we show that GMAP-210, a cis-Golgi microtubule binding protein, recruits gamma-tubulin-containing complexes to Golgi membranes even in conditions where microtubule polymerization is prevented and independently of Golgi apparatus localization within the cell. Under overexpression conditions, very short microtubules, or tubulin oligomers, are stabilized on Golgi membranes. GMAP-210 depletion by RNA interference results in extensive fragmentation of the Golgi apparatus, supporting a role for GMAP-210 in Golgi ribbon formation. Targeting of GMAP-210 or its C terminus to mitochondria induces the recruitment of gamma-tubulin to their surface and redistribution of mitochondria to a pericentrosomal location. All our experiments suggest that GMAP-210 displays microtubule anchoring and membrane fusion activities, thus contributing to the assembly and maintenance of the Golgi ribbon around the centrosome.

The serum from a patient with Sjö gren's syndrome (RM serum) was used to screen a human testis cDNA expression library. A cDNA of 865 base pairs containing the entire coding sequence for a novel protein was isolated. The 14-kDa predicted protein contains an acidic domain (amino acids 6 -80) with a high frequency of heptad repeats characteristic of ␣-helices that form dimeric coiled-coil structures and an alkaline carboxylterminal domain (amino acids 81-119). It seems to be widely expressed, but its expression level varies depending on tissues. A protein of apparent molecular mass of 14 kDa was immunoprecipitated from cell lysates by the autoimmune serum, and it was recognized by rabbit antibodies raised to a recombinant bacterial fusion protein generated from the cDNA clone. Conventional and confocal immunofluorescence microscopy on HeLa and 3T3 cells transiently transfected with a tagged form of the protein showed numerous punctate structures scattered throughout the nucleus. This novel protein has been termed NA14 for Nuclear Autoantigen of 14 kDa.