Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy. T he sorting of receptors and other cell-surface proteins from the plasma membrane via clathrin-mediated endocytosis is the basis for a range of essential cellular processes, including the uptake of nutrient and signaling receptors, the control of cell and serum homeostasis through the internalization of plasma membrane pumps, and a contribution to learning and memory through the regulation of surface expression of neurotransmitter receptors (1). Until recently, it was thought that clathrin assembly into progressively curved lattices provided the driving force for the formation of clathrin-coated pits (CCPs) and vesicles (CCVs), and that the adaptor protein 2 (AP-2) complex was solely responsible for recruiting clathrin to the membrane and for binding to endocytic cargo, concentrating the cargo in CCPs (1, 2). However, clathrin assembly may not be sufficient to drive membrane curvature (3), and the previously accepted obligatory role for AP-2 in coat assembly and cargo recruitment has been recently questioned (4-6).In neuronal tissues, CCVs are postulated to be responsible for the recycling of synaptic vesicles (SVs) during neurotransmission (7). As such, CCVs retrieve SV membranes from the plasma membrane after SV collapse, concomitant with neurotransmitter release. Many of the components of the endocytic machinery are concentrated in the presynaptic compartment (8), and disruption of these proteins affects neurotransmission (9). Moreover, a number of SV proteins have been identified as components of isolated CCVs (10, 11). Synaptic transmission involving intermittent fusion of SVs without complete collapse (12, 13) has also been demonstrated. The prevalence of such a ''kiss-and-run'' mechanism with the alternative model of full fusion is uncertain (14). Even in the membrane retrieval model via CCVs, it remains unclear whether SVs are generated directly from CCVs (15, 16) or whether they require an additional sorting step through endosomal membranes localized in the presynaptic compartment (7, 17). Here, using ...

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.

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Background: AGR2 is a novel ER protein for which the molecular and cellular functions remain uncharacterized. Results: AGR2 associates to nascent chains in the ER, and its silencing impacts UPR and ERAD and sensitizes cells to autophagy. Conclusion: AGR2 plays an important role in the maintenance of ER homeostasis. Significance: AGR2-mediated control of ER homeostasis could be of importance for cancer development.