Ca2+-dependent activator protein for secretion 1 is critical for constitutive and regulated exocytosis but not for loading of transmitters into dense core vesicles

Although CAPS1 was originally identified as a soluble factor that reconstitutes Ca(2+)-dependent secretion from permeabilized neuroendocrine cells, its exact function in intact mammalian cells remains controversial. Here we investigate the role for CAPS1 by generating stable cell lines in which CAPS1 is strongly down-regulated. In these cells, Ca(2+)-dependent secretion was strongly reduced not only of catecholamine but also of a transfected neuropeptide. These secretion defects were rescued by infusion of CAPS1-containing brain cytosol or by transfection-mediated expression of CAPS1. Whole cell patch clamp recording revealed significant reductions in slow burst and sustained release components of exocytosis in the knockdown cells. Unexpectedly, they also accumulated higher amounts of endogenous and exogenous transmitters, which were attributable to reductions in constitutive secretion. Electron microscopy did not reveal abnormalities in the number or docking of dense core vesicles. Our results indicate that CAPS1 plays critical roles not only in Ca(2+)-dependent, regulated exocytosis but also in constitutive exocytosis downstream of vesicle docking. However, they do not support the role for CAPS1 in loading transmitters into dense core vesicles.     

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter

The β-glucosidase, which hydrolyzes the β(1–4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2–4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5–6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

     

Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (“closed” vs. “open”) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of “closed” syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1–depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the “open” syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between “closed” syntaxin-1A and Munc18-1, whereas the same mutation in the “open” syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.