Concepción Fedriani

GMAP-210 Recruits γ-Tubulin Complexes to cis-Golgi Membranes and Is Required for Golgi Ribbon Formation

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.

NA14 is a novel nuclear autoantigen with a coiled-coil domain.

The serum from a patient with Sjögren’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 α-helices that form dimeric coiled-coil structures and an alkaline carboxyl-terminal 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 NuclearAutoantigen of 14 kDa.

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network

GMAP-210 (Golgi-microtubule-associated protein of 210 kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83--98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105--196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

Solubilization and electrophoretic studies of cyst wall proteins of a hypotrichous ciliate

A method for induction of synchronous encystment in a hypotrichous ciliate, Paraurostyla sp. is described. Cyst walls, isolated by shaking with glass beads, were analyzed by SDS‐polyacrylamide gel electrophoresis. To test optimal conditions of solubilization of cyst wall proteins, different treatments using Triton X‐100, EDTA, EGTA, urea, SDS and 2‐mercaptoethanol were carried out. At least, 15 different proteins were identified as specific to the cyst wall. Four low molecular weight polypeptides (40, 27–26, 20 and 18 kDa represented aproximately 70% of the cyst wall proteins. The 170, 135 and 40‐kDa bands exhibited a PAS‐positive reaction. Hydrogen and disulphide bonds were shown to be the most important interactions involving cyst wall proteins. Amino acid composition of cyst wall proteins was also investigated by HPLC. High amounts of glycine, cystine and proline were detected.