Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters

Accumulation of the GABA inhibitory neurotransmitter for rapid delivery into synapses can be accomplished by GAD65 dependent and independent membrane-targeting of GAD67

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters.

The inhibitory neurotransmitter gamma-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses

Using runaway replication to express polyhydroxyalkanoic acid (pha) genes from a novel marine bacterium in enteric bacteria: The influence of temperature and phasins on PHA accumulation

While plastics have revolutionized our world, plastic waste has serious environmental and economic impacts. Polyhydroxyalkanoic acid (PHA) is a bacterial carbon and energy reserve shown to be both biodegradable and biocompatible and could potentially replace conventional plastics. However, cost-effective mass production remains elusive. Bacteria often accumulate PHA as cytoplasmic granules. PHA synthase creates the PHA polymer from acetoacyl-CoA monomers, while phasins are small multifunctional proteins that are found in abundance on the granule surface. The PHA synthase gene from a novel marine isolate, Vibrio B-18 (or B-18), was placed in the presence or absence of an upstream phasin gene in a runaway replication plasmid using polymerase chain reaction (PCR) technology. Plasmid gene expression may be induced chemically or thermally. Overexpression of the PHA genes was demonstrated by SDS-PAGE analysis, and microscopy was used to detect PHA accumulation in three different enteric bacteria (Escherichia coli, Klebsiella aerogenes, and Shigella flexneri). While the B-18 genes were clearly overexpressed at 41°C, PHA accumulation occurred more readily at the lower (30°C) non-inducing temperature regardless of chemical induction if the phasin gene was present. A mutational analysis confirmed the identity of the start codon for the PHA synthase gene and provided evidence supporting the requirement for phasins to allow for PHA accumulation in the recombinant hosts. The findings described in this study confirm the conclusions obtained from related studies from other laboratories and lend support to the importance of including a phasin gene in addition to the basic genes needed for PHA synthesis and accumulation in recombinant enteric bacteria, such as Escherichia coli, Klebsiella aerogenes, and Shigella flexneri

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: Effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA–1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37°C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 μm with the mean value of 0.23 ± 0.06 μm