Abstract
A protein of Mr 100,000 (MVP100) is highly enriched in the electromotor system of electric rays. Biochemical analysis indicates that MVP100 is contained in the cholinergic nerve terminals of Torpedo electric organ as part of a large cytosolic complex. On sucrose density gradient centrifugation MVP100 comigrates with synaptic vesicles or synaptosomes. It can be partially separated from synaptic vesicles by gel filtration or glycerol velocity gradient centrifugation. Within the complex MVP100 behaves like a hydrophobic protein and is protected against proteolytic attack. MVP100 can be immunodetected by an antibody against phosphotyrosine, and it becomes phosphorylated on incubation with [gamma-32P]ATP. By screening an electric ray electric lobe cDNA library the primary structure of MVP100 was analyzed. MVP100 is highly homologous to the major vault proteins of slime mold and rat and to the human lung resistance-related protein. Compared with non-neural tissues the expression of MVP100 is highest in brain and enriched in the electric lobe that contains the somata of the electromotor neurons. Immunoelectron microscopic analysis reveals a close association of MVP100 and synaptic vesicles in the nerve terminals of the electric organ.
Highlights
Vaults are ubiquitous, evolutionarily conserved cytoplasmic ribonucleoprotein particles of unknown function [1]
Copurification of Synaptic Vesicles and MVP100 —We initially identified MVP100 as a protein copurifying with cholinergic synaptic vesicles
The resulting microsomal fraction enriched in synaptic vesicles is fractionated further by discontinuous sucrose density gradient centrifugation, and the fractions obtained are analyzed for various markers (Fig. 1)
Summary
Subcellular Fractionation—All purification steps were performed at 4 °C. Synaptic vesicles from frozen electric organ of Torpedo marmorata (120 –140 g, wet weight) were extracted in 0.4 M NaCl (3.5 mM EGTA, 10 mM HEPES/NaOH, pH 7.4) and purified further using a discontinuous sucrose gradient as described previously [14]. Chemical Treatment of Intact Synaptic Vesicles—Synaptic vesicles derived from 20 g of tissue obtained after discontinuous sucrose gradient centrifugation and high speed centrifugation were resuspended in isotonic saline solution (1 ml of 0.4 M NaCl, containing 10 mM HEPES/ NaOH, pH 7.4). They were incubated (4 °C for 30 min with gentle rotation) at acidic or basic pH (pH 3.0 or 11.5) or in media containing 0.4 M KCl, 2 M KCl, or 1% Triton X-100 in addition to sodium chloride. Stripes were embedded in Epon and processed further as for tissue sections
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