A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a uniceilular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutate activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specifi to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins-were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.Previously we discovered a cytosolic phosphoprotein, parafusin, that plays a role in regulated exocytosis in the unicellular eukaryote Paramecium (1, 2) and that is evolutionarily conserved (3). Parafusih has been shown to be phosphorylated via a Ca2+-dependent protein kinase (4). Surprisingly, parafusin is also a phosphoglycoprotein in which a short chain ofmannose residues is 0-linked to serine. This chain is phosphoglucosylated by a glucose-1-phosphate phosphotransferase that uses UDP glucose (5). We have recently demonstrated that dephosphoglucosylation is catalyzed by a Ca2+-activated phosphodiesterase. Cells in which parafusin is normal but that are unable to release the content of their dense core secretory vesicles upon stimulation show inactive phosphodiesterase, suggesting that dephosphoglucosylation is a critical event in the pathway to exocytosis (4).Tryptic digests of parafusin purified as described earlier (6)

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.

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Background: An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.