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Tandem MS Analysis of Brain Clathrin-Coated Vesicles Reveals Their Critical Involvement in Synaptic Vesicle Recycling

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Tandem MS Analysis of Brain Clathrin-Coated Vesicles Reveals Their Critical Involvement in Synaptic Vesicle Recycling Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling Francois Blondeau*, Brigitte Ritter*, Patrick D. Allaire*, Sylwia Wasiak*, Martine Girard*, Natasha K. Hussain*, Annie Angers*, Valerie Legendre-Guillemin*, Line Roy†, Daniel Boismenu†, Robert E. Kearney†, Alexander W. Bell†, John J. M. Bergeron†‡, and Peter S. McPherson*‡ *Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, QC, Canada H3A 2B4; and †Montreal Proteomics Centre, McGill University, 740 Dr. Penfield Avenue, Montreal, QC, Canada H3A 1A4 Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT, and approved January 27, 2004 (received for review December 9, 2003) Tandem MS has identified 209 proteins of clathrin-coated vesicles Experimental Procedures (CCVs) isolated from rat brain. An overwhelming abundance of CCV Purification and Analysis. CCVs were isolated from adult rat peptides were assigned to the clathrin coat with a 1:1 stoichiom- brain as described (18). Suspensions of CCVs were deposited on etry observed for clathrin heavy and light chains and a 2:1 stoi- a 0.22-␮m nitrocellulose filter and processed for electron mi- chiometry of clathrin heavy chain with clathrin adaptor protein croscopy, using a double fixation protocol with osmium and heterotetramers. Thirty-two proteins representing many of the tannic acid (19). For quantification, all vesicles were circled by known components of synaptic vesicles (SVs) were identified, using PHOTOSHOP (Adobe Systems Canada, Ottawa), filled with supporting that a main function for brain CCVs is to recapture SVs white, and the regions outside were darkened artificially to after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive fac- remove all other white spots. The number of white objects was tor attachment protein receptors to target-N-ethylmaleimide-sen- automatically deduced by using the digital measurement system, sitive factor attachment protein receptors, similar to that previ- SIGMA SCAN PRO (SPSS, Inc., Chicago). The diameter of each ously detected on SVs, supports a single-step model for SV sorting vesicle was assessed as a function of its surface by SIGMA SCAN during CCV-mediated recycling of SVs. The uncovering of eight PRO. All antibodies used in this study are described in Table 1, previously undescribed proteins, four of which have to date been which is published as supporting information on the PNAS web site. linked to clathrin-mediated trafficking, further attests to the value After the separation of CCV proteins by 1D SDS͞PAGE, the of the current organelle-based proteomics strategy. gel lanes were divided horizontally into 60 or 61 slices that were individually processed to yield in-gel trypsin digestion products he sorting of receptors and other cell-surface proteins from (18). The resulting peptide mixtures were separated and ana- Tthe plasma membrane via clathrin-mediated endocytosis is lyzed in an automated system by nanoscale liquid chromatogra- the basis for a range of essential cellular processes, including the phy quadrupole time-of-flight MS͞MS, as described in detail in uptake of nutrient and signaling receptors, the control of cell and Supporting Experimental Procedures, which is published as sup- serum homeostasis through the internalization of plasma mem- porting information on the PNAS web site. After fragmentation brane pumps, and a contribution to learning and memory in the MS͞MS mode, spectra were analyzed by MASCOT software through the regulation of surface expression of neurotransmitter to identify tryptic peptide sequences matched to the National receptors (1). Until recently, it was thought that clathrin assem- Center for Biotechnology Information (NCBI) nonredundant bly into progressively curved lattices provided the driving force protein database with a confidence level of 95% or greater (20). for the formation of clathrin-coated pits (CCPs) and vesicles Each MS͞MS spectrum was then visually inspected. Spectra with (CCVs), and that the adaptor protein 2 (AP-2) complex was four clearly identified consecutive y ions were chosen as a solely responsible for recruiting clathrin to the membrane and validated subset of the previous MASCOT assignments. All other for binding to endocytic cargo, concentrating the cargo in CCPs MS͞MS spectra, amounting to 15% of those assigned by MAS- (1, 2). However, clathrin assembly may not be sufficient to drive COT, were rejected. For each protein with three or fewer peptide membrane curvature (3), and the previously accepted obligatory assignments, a further analysis was performed. Peptide sequence role for AP-2 in coat assembly and cargo recruitment has been tags were generated from the MS͞MS spectra as described (21). recently questioned (4–6). Only ions at least three times above background were taken into In neuronal tissues, CCVs are postulated to be responsible for the account. The sequence tags were searched against the NCBI recycling of synaptic vesicles (SVs) during neurotransmission (7). nonredundant protein database using the PepFrag search rou- As such, CCVs retrieve SV membranes from the plasma membrane tine (http:͞͞129.85.19.192͞prowl͞pepfragch.html), with param- after SV collapse, concomitant with neurotransmitter release. eters of trypsin digest with one missed cleavage, cysteines Many of the components of the endocytic machinery are concen- carboxyamidomethylated, and a tolerance of 1 mass unit for the trated in the presynaptic compartment (8), and disruption of these parent peptide or the fragment ion. PepFrag search results were proteins affects neurotransmission (9). Moreover, a number of SV rejected if different peptide sequences were retrieved for a given proteins have been identified as components of isolated CCVs (10, sequence tag. Greater than 95% of the MS͞MS spectra evalu- 11). Synaptic transmission involving intermittent fusion of SVs CELL BIOLOGY ated were found to be consistently assigned by MASCOT analysis. 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 This paper was submitted directly (Track II) to the PNAS office. retrieval model via CCVs, it remains unclear whether SVs are Abbreviations: CCV, clathrin-coated vesicle; cpCCV, cushion-pellet CCV; SV, synaptic vesicle; generated directly from CCVs (15, 16) or whether they require an MS͞MS, tandem MS; CHC, clathrin heavy chain; CLC, clathrin light chain; TGN, transGolgi additional sorting step through endosomal membranes localized in network; EM, electron microscopy; ER, endoplasmic reticulum. the presynaptic compartment (7, 17). Here, using a tandem MS ‡To whom correspondence may be addressed. E-mail: [email protected] or (MS͞MS) analysis of CCVs isolated from adult rat brain, several of [email protected]. these issues have been addressed. © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308186101 PNAS ͉ March 16, 2004 ͉ vol. 101 ͉ no. 11 ͉ 3833–3838 Downloaded by guest on October 1, 2021 Peptide assignments were based on the number of peptides found specifically in their cognate protein (defined as specific peptides) and those found in two or more different proteins (shared peptides) within a given band. The identifications pre- sented in Table 2, which is published as supporting information on the PNAS web site, are the minimal set of proteins to explain the peptides assigned by MASCOT on a band-to-band basis. Proteins are identified as a single entry with the band-to-band distribution shown (Table 2). For Figs. 2 and 4, the peptide numbers for each protein were a sum of all peptides counted as specific and a proportion of those peptides shared with other proteins in the gel slice with proportionality a consequence of the number of specific peptides. For example, ␤1- and ␤2-adaptin had 64 and 208 specific peptides, respectively. The 90 peptides shared between the two proteins were thus apportioned by the ratio of specific peptides (i.e., 21 to ␤1, 69 to ␤2). As a final criterion, only those proteins identified in at least two of the three CCV preparations analyzed were retained. Therefore, the protein assignments were a consequence of 9,239, 9,904, and 12,091 fragmented ions from the three CCV preparations. From these, 1,560, 2,225, and 2,348 MS͞MS profiles were assigned. Results Isolation of CCVs. Brain CCVs were isolated by differential Fig. 1. Isolation of CCVs. (A) Coomassie blue staining of proteins separated centrifugation, Ficoll density gradient centrifugation, and pel- by SDS͞PAGE from each step of the enrichment protocol for CCVs. Aliquots of ͞ 40 ␮g of protein were loaded on each lane. H, P1, S2, P2, SGp, SGs, cpCCV, and leting through a D2O sucrose cushion (10). These cushion-pellet CCVs (cpCCVs) were further enriched by centrifugation on CCV represent homogenate, first pellet, second supernatant, second pellet, sucrose͞Ficoll gradient pellet, sucrose͞Ficoll gradient supernatant, D2O cush- linear 20–50% sucrose gradients (18). A clear enrichment for ion pellet, and CCVs, respectively. The predicted migratory positions of CHC, polypeptides at the mobility of clathrin heavy chain (CHC), ␣ ␤ ␥ ␮ ␣ ␤ ␥ ␮ CLCs, and -, -, - and -adaptins are indicated. (B) Immunoblots detected the clathrin light chains (CLCs), and the -, -, -, and -adaptin presence CHC, CLCs, and ␣-adaptin as indicated.
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