ABSTRACT
A familial form of Amyotrophic lateral sclerosis (ALS8) is caused by a point mutation (P56S) in the vesicle-associated membrane protein associated protein B (VapB). Human VapB and Drosophila Vap-33-1 (Vap) are homologous type II transmembrane proteins that are localized to the ER. However, the precise consequences of the defects associated with the P56S mutation in the endoplasmic reticulum (ER) and its role in the pathology of ALS are not well understood. Here we show that Vap is required for ER protein quality control (ERQC). Loss of Vap in flies shows various ERQC associated defects, including protein accumulation, ER expansion, and ER stress. We also show that wild type Vap, but not the ALS8 mutant Vap, interacts with a lipid-binding protein, Oxysterol binding protein (Osbp), and that Vap is required for the proper localization of Osbp to the ER. Restoring the expression of Osbp in the ER suppresses the defects associated with loss of Vap and the ALS8 mutant Vap. Hence, we propose that the ALS8 mutation impairs the interaction of Vap with Osbp, resulting in hypomorphic defects that might contribute to the pathology of ALS8.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Animals, Genetically Modified/metabolism , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/metabolism , Receptors, Steroid/metabolism , Transgenes/physiology , Vesicular Transport Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , Animals, Genetically Modified/genetics , Blotting, Western , Cell Proliferation , Cells, Cultured , Drosophila melanogaster/genetics , Endoplasmic Reticulum Stress/physiology , Female , Humans , Immunoprecipitation , Male , Mutation/genetics , Quality Control , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Steroid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Two-Hybrid System Techniques , Ubiquitin/metabolism , Vesicular Transport Proteins/geneticsABSTRACT
Synaptic vesicle endocytosis is critical for maintaining synaptic communication during intense stimulation. Here we describe Tweek, a conserved protein that is required for synaptic vesicle recycling. tweek mutants show reduced FM1-43 uptake, cannot maintain release during intense stimulation, and harbor larger than normal synaptic vesicles, implicating it in vesicle recycling at the synapse. Interestingly, the levels of a fluorescent PI(4,5)P(2) reporter are reduced at tweek mutant synapses, and the probe is aberrantly localized during stimulation. In addition, various endocytic adaptors known to bind PI(4,5)P(2) are mislocalized and the defects in FM1-43 dye uptake and adaptor localization are partially suppressed by removing one copy of the phosphoinositide phosphatase synaptojanin, suggesting a role for Tweek in maintaining proper phosphoinositide levels at synapses. Our data implicate Tweek in regulating synaptic vesicle recycling via an action mediated at least in part by the regulation of PI(4,5)P(2) levels or availability at the synapse.
Subject(s)
Drosophila Proteins/physiology , Endocytosis/physiology , Nerve Tissue Proteins/physiology , Neurons/metabolism , Phosphatidylinositol Phosphates/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Animals , Blotting, Western , DNA, Complementary , Diptera , Endocytosis/genetics , Eye Abnormalities/genetics , Immunohistochemistry , Microscopy, Electron, Transmission , Molecular Sequence Data , Mutation , Neurons/ultrastructure , Pyridinium Compounds/metabolism , Quaternary Ammonium Compounds/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Synapses/genetics , Synapses/ultrastructure , Synaptic Transmission/genetics , Synaptic Vesicles/genetics , Synaptic Vesicles/ultrastructureSubject(s)
Antiviral Agents/adverse effects , Hepatitis C, Chronic/drug therapy , Interferon-alpha/adverse effects , Ribavirin/adverse effects , Thyrotoxicosis/chemically induced , Thyrotoxicosis/physiopathology , Adrenergic beta-Antagonists/therapeutic use , Antithyroid Agents/therapeutic use , Antiviral Agents/therapeutic use , Drug Therapy, Combination , Female , Humans , Interferon alpha-2 , Interferon-alpha/therapeutic use , Middle Aged , Patient Compliance , Polyethylene Glycols , Propranolol/therapeutic use , Propylthiouracil/therapeutic use , Recombinant Proteins , Retreatment , Ribavirin/therapeutic use , Severity of Illness Index , Thyrotoxicosis/drug therapy , Thyrotoxicosis/psychologyABSTRACT
Secretion of noradrenaline from large dense-core vesicles in chromaffin cells involves both rapid and slow components of exocytosis which are differentially sensitive to changes in external calcium, osmotic pressure and interruption of the interacting SNARE proteins. Electrical signs of secretion of ATP from sympathetic nerve terminals of mouse vas deferens, the excitatory junctional currents (EJCs), also indicate both rapid and slow mechanisms of exocytosis, which might also show such differential sensitivity. We report here that the large and fast EJCs are highly sensitive to changes in extracellular calcium ions whereas the small and slow EJCs are not. Furthermore, the frequency of fast EJCs is accelerated by hypotonic solutions whereas the slow EJCs are accelerated by hypertonic solution. Fast EJCs, but not slow EJCs, are blocked by peptide fragments of alpha-SNAP and syntaxin whereas slow EJCs are not. These observations point to two classes of exocytosis from sympathetic nerve terminals that parallel those of exocytosis from chromaffin cells.
