In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.
We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-b, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-a. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-b exon in the nervous system. Synaptotagmin I C2B-b showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-a, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-b with a higher affinity than synaptotagmin I C2B-a. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.
Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that is important for the kinetics of both exocytosis and endocytosis, and is thus a candidate molecule to link these two processes. Although the tandem Ca 2+ -binding C2 domains of Syt1 have important roles in exocytosis and endocytosis, the function of the conserved juxtamembrane ( jxm) linker region has yet to be determined. We now demonstrate that the jxm region of Syt1 interacts directly with the pleckstrin homology (PH) domain of the endocytic protein dynamin 1. By using cell-attached capacitance recordings with millisecond time resolution to monitor clathrin-mediated endocytosis of single vesicles in neuroendocrine chromaffin cells, we find that loss of this interaction prolongs the lifetime of the fission pore leading to defects in the dynamics of vesicle fission. These results indicate a previously undescribed interaction between two major regulatory proteins in the secretory vesicle cycle and that this interaction regulates endocytosis.
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