Vesicular lumen acidification

The transition from coated vesicle to early endosome is accompanied by acidification of the vesicular lumen that continues into the late endosomal and lysosomal compartments, reaching a final pH in the perinuclear lysosome of approximately 4.5. Such acidification associated with endosome maturation provides the means by which certain viruses gain access to the cytosol. Acid-induced conformational changes in the viral proteins trigger translocation across the endosomal membrane via a fusion process. By taking advantage of the endosomal acidification, pH-sensitive liposomes, adenovirus and endosomolytic peptides have been used to facilitate the release of plasmids into the cytoplasm prior to lysosomal degradation. 

A typical presynaptic bouton is a specialized portion of the axon. It is characterized by an active zone, a region where the presynaptic plasma membrane comes into close contact with the postsynaptic plasma membrane and an associated cluster of vesicles (De Camilli et al., 2001). A few synaptic vesicles are adjacent to the active zone and are referred to as docked vesicles. Vesicle exocytosis occurs at the active zone subsequent endocytic retrieval of vesicular components may occur both at the active zone and in the peri-active zone area (Roos and Kelly, 1999). Vesicle maturation involves acidification of the lumen, loading with the neurotransmitter, association with peripheral membrane proteins needed for exocytosis, and recapture into a vesicle cluster. The vesicle cluster is embedded in an ac tin-rich area and is generally located next to mitochondria, which provides the energy required for the vesicle cycle and neurotransmitter dynamics and to the endoplasmic reticulum whose function includes the regulation of local cytosolic calcium. 

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synapto-somes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 p.1 Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992 Hartinger and Jahn, 1993). To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. 

Fig. 2. Acidification of small synaptic vesicles by glutamate and chloride in synap-tosomes. The acidification assay was performed as described in section 3.2 of this chapter. Two representative experiments with intact (upper trace) or SLO-permeabilized (lower trace) synaptosomes are shown. The ordinate gives the changes of absorbance obtained (A 492-530). Final concentrations of potassium glutamate (Glut), KCl, and ammonium sulfate (NH/) were 10 mM, 45 mM and 30 mM, respectively. The uptake of glutamate and chloride result in an acidification of the lumen of small synaptic vesicles, which increases the vesicular uptake of acridine orange, resulting in a decrease in the amount of extravesicular dye. This acidification can be only observed when the plasma membrane is permeabilized...

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