Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling

Abstract

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 ...

Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book

“…Because each tandem mass spectrum represents an independent and verifiable piece of data, this approach to database searching has the ability to identify proteins in mixtures, enabling a rapid and comprehensive approach for the analysis of protein complexes and other complicated mixtures of proteins 6,[8][9][10][11][12] . New biology has been discovered based on fast and accurate protein identification [13][14][15][16][17][18] . As tandem mass spectral protein identification has proliferated, it has become increasingly important to understand the rationale of individual database search algorithms, their relative strengths and weaknesses, and the mathematics used to match sequence to spectrum.…”

Highly Cooperative Control of Endocytosis by Clathrin

“…A recent proteomic analysis of clathrin-coated vesicles in neurons indicates that up to 200 different gene products are associated with this pathway (Blondeau et al, 2004). As with the entire genome it is likely that a large fraction of these constituents are devoted to regulation of clathrin function.…”

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

“…Recently, a novel 104-kDa coated vesicle-associated protein with a serine/threonine kinase homology domain (CVAK104) was discovered by mass spectroscopy analysis of purified rat brain CCVs and in isolated adaptor protein preparations derived from bovine brain (Blondeau et al, 2004;Conner and Schmid, 2005). CVAK104 directly binds to both clathrin and the plasma membrane adaptor AP2.…”