Non-stoichiometric Relationship between Clathrin Heavy and Light Chains Revealed by Quantitative Comparative Proteomics of Clathrin-coated Vesicles from Brain and Liver

We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Finally we identified 21 CCV-associated proteins in liver not yet characterized in mammals. Our results further validate the peptide accounting approach, reveal new information on the properties of CCVs, and allow for the use of quantitative proteomics to compare abundant components of organelles under different experimental and pathological conditions. We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Finally we identified 21 CCV-associated proteins in liver not yet characterized in mammals. Our results further validate the peptide accounting approach, reveal new information on the properties of CCVs, and allow for the use of quantitative proteomics to compare abundant components of organelles under different experimental and pathological conditions. Vesicle budding and trafficking via clathrin-coated pits (CCPs) 1The abbreviations used are: CCP, clathrin-coated pit; AP, adaptor protein; CHC, clathrin heavy chain; CLC, clathrin light chain; CCV, clathrin-coated vesicle; EM, electron microscopy; FENS-1, FYVE domain-containing protein localized to endosomes 1; GRASP, Golgi peripheral membrane protein p65; MPR, mannose 6-phosphate receptor; NECAP 1, adaptin-ear-binding coat-associated protein 1; RME-8, receptor-mediated endocytosis 8; TGN, trans-Golgi network. and vesicles (CCVs) provides a major route by which proteins are transported out of the trans-Golgi network (TGN) and by which receptors, transporters, and nutrients are endocytosed at the plasma membrane (1McPherson P.S. Kay B.K. Hussain N.K. Signaling on the endocytic pathway.Traffic. 2001; 2: 375-384Google Scholar, 2Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Biological basket weaving: formation and function of clathrin-coated vesicles.Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Google Scholar, 3Conner S.D. Schmid S.L. Regulated portals of entry into the cell.Nature. 2003; 422: 37-44Google Scholar). Many clathrin-dependent trafficking events mediate cargo transport that is needed in all cell types. These “housekeeping” forms of clathrin trafficking include the turnover of plasma membrane proteins and lipids, endocytic uptake of nutrients such as iron-saturated transferrin and low density lipoproteins, and endocytosis of a diverse range of activated growth factor receptors (1McPherson P.S. Kay B.K. Hussain N.K. Signaling on the endocytic pathway.Traffic. 2001; 2: 375-384Google Scholar, 2Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Biological basket weaving: formation and function of clathrin-coated vesicles.Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Google Scholar, 3Conner S.D. Schmid S.L. Regulated portals of entry into the cell.Nature. 2003; 422: 37-44Google Scholar). Moreover all cells have housekeeping trafficking at the TGN. An important example is the delivery of mannose 6-phosphate-tagged lysosomal hydrolases from the TGN to endosomes/lysosomes via the mannose 6-phosphate receptor (MPR) (4Le Borgne R. Hoflack B. Protein transport from the secretory to the endocytic pathway in mammalian cells.Biochim. Biophys. Acta. 1998; 1404: 195-209Google Scholar). In addition to these housekeeping activities of CCVs, some tissues have specialized trafficking needs. For example, in secretory cells, clathrin coats are involved in the formation of secretory granules at the TGN (5Austin C. Hinners I. Tooze S.A. Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules.J. Biol. Chem. 2000; 275: 21862-21869Google Scholar), and polarized cells utilize CCVs for the trafficking of certain receptors from the TGN to the basolateral membrane necessary for the maintenance of polarity (2Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Biological basket weaving: formation and function of clathrin-coated vesicles.Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Google Scholar). At the plasma membrane, intestinal epithelial cells in rat or placental cells in humans use CCVs for the uptake of maternal immunoglobulins, a necessary aspect of maternal derived immunity (6Pearse B.M. Bretscher M.S. Membrane recycling by coated vesicles.Annu. Rev. Biochem. 1981; 50: 85-101Google Scholar). A striking example of specialized CCV function is seen in neurons, which communicate by releasing neurotransmitters through fusion of synaptic vesicles with the plasma membrane following transient increases in Ca2+ concentration (7Sudhof T.C. The synaptic vesicle cycle.Annu. Rev. Neurosci. 2004; 27: 509-547Google Scholar). These vesicles are then retrieved through CCVs (8Murthy V.N. De Camilli P. Cell biology of the presynaptic terminal.Annu. Rev. Neurosci. 2003; 26: 701-728Google Scholar, 9Bauerfeind R. David C. Grabs D. McPherson P.S. Nemoto Y. Slepnev V.I. Takei K. De Camilli P. Recycling of synaptic vesicles.Adv. Pharmacol. 1998; 42: 253-257Google Scholar, 10Bauerfeind R. David C. Galli T. McPherson P.S. Takei K. De Camilli P. Molecular mechanisms in synaptic vesicle endocytosis.Cold Spring Harb. Symp. Quant. Biol. 1995; 60: 397-404Google Scholar). Thus, neurons need CCVs not only for housekeeping forms of clathrin-mediated endocytosis but also to retrieve synaptic vesicle membranes. It has been unclear whether or not the mechanisms mediating these two related but distinct events taking place at the plasma membrane could be distinguished. Moreover the relative amount of brain CCVs specialized for synaptic function has never been assessed. The presence of clathrin adaptor proteins (APs) can provide one level of discrimination of vesicle type as CCVs arising from the TGN contain AP-1 and CCVs derived from the plasma membrane contain AP-2. AP-1 and AP-2 are heterotetramers composed of four subunits each, namely γ-, β1-, μ1-, and ς1-adaptin for AP-1 and α-, β2-, μ2-, and ς2-adaptin for AP-2 (11Boehm M. Bonifacino J.S. Adaptins: the final recount.Mol. Biol. Cell. 2001; 12: 2907-2920Google Scholar). Two genes code for α-adaptin giving rise to αA and αC variants with an alternative brain-specific splice form for αΑ (12Ball C.L. Hunt S.P. Robinson M.S. Expression and localization of α-adaptin isoforms.J. Cell Sci. 1995; 108: 2865-2875Google Scholar). AP-1 and AP-2 provide a link between membranes and clathrin, the major component of CCVs (13Ritter B. McPherson P.S. Molecular mechanisms in clathrin-mediated membrane budding.in: Keranen in of Membrane Scholar). In AP-2 clathrin is for AP-1 R. proteins of brain coated vesicles. An of and Biol. Chem. Scholar). The clathrin of AP-1 at the TGN be by proteins such as R. M. D. Bonifacino J.S. McPherson P.S. a protein identified through Cell Biol. Scholar). is composed of of clathrin heavy chain and one of two clathrin light and dimers form a to as a that has been in CCVs derived from brain to be composed of and D. of clathrin 1981; Scholar, T. Protein in clathrin 1981; Scholar). The stoichiometry of to in brain CCVs has been by quantitative proteomics B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). the of clathrin has been to all tissues further and has the and function of CCVs, we have of an that we to determine the relative levels of proteins tandem B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). The on the that the more abundant a protein the more These be more in the mass giving more on the of the protein as proteins more and also on the of the protein as certain be more readily and detected than in peptide between proteins a provide a of relative we have been to the of abundant components of CCVs from rat brain B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). The was by A for and of relative protein in Chem. 2004; protein with of that in the of identified for a protein to in concentration of A for and of relative protein in Chem. 2004; Scholar). In we a of highly CCVs from rat liver. of the peptide accounting approach, compared with a on brain CCVs, to reveal new information the for CCV for CHC, AP-1 and AP-2 were from which and was from F.M. characterized with of in forms of Cell Biol. was a of CCVs were vesicles in the receptor-mediated endocytosis of Cell Biol. Scholar, M. McPherson P.S. of clathrin-coated vesicles by and density Bonifacino M. K. in Cell and from that been of liver CCVs were on to P. P. of I. The of from of Cell Biol. and were then for electron an and M. of low as in electron The and involved in the Cell Biol. Scholar). The of liver CCVs was by the of coated vesicles and in from from CCV proteins were by and was into was for tandem as B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). were by to identify peptide to the for protein with a level of or J.S. protein by mass spectrometry Scholar). and with an or than the were and for from the were then on and as specific for level of only proteins in two of and with or more were C. proteomics Golgi Biol. Cell. 2004; Scholar). CCVs were from rat as R. M. D. Bonifacino J.S. McPherson P.S. a protein identified through Cell Biol. Scholar, M. McPherson P.S. of clathrin-coated vesicles by and density Bonifacino M. K. in Cell and Scholar). For specific CCVs were from the of the For on of liver or brain CCVs was on in A 1 and in a at for The were from the and proteins from of were by and tissues and cell were in A as M. McPherson P.S. of clathrin-coated vesicles by and density Bonifacino M. K. in Cell and Scholar). The were at for and the was further at for 1 of protein from the from were by and to an for the The was also by In were from 1 of two different tissues to or from of a For these were with of from a and of tissues to for the of the tissues CCVs were from the of a vesicles in the receptor-mediated endocytosis of Cell Biol. Scholar, M. McPherson P.S. of clathrin-coated vesicles by and density Bonifacino M. K. in Cell and Scholar). for protein to the of CHC, and the α-, and subunits of the AP-1 and AP-2 complexes was in the of the CCV The of CHC, and was by on the highly enriched CCV to a of the M. McPherson P.S. of clathrin-coated vesicles by and density Bonifacino M. K. in Cell and Scholar, P. P. of I. The of from of Cell Biol. the presence of CCVs on and coated vesicles in and were also of the of CCVs compared with in from that the CCVs were for a of a The proteins from CCV were on and into was for by the of only proteins in which with a or were in at two of the with at in were identified proteins were by to that all were into a of the high of between different all were into one of two and these we identified proteins in the liver CCV including 21 proteins and all CCV proteins were identified with the of for and and the of the of for the subunits of AP-1 and AP-2 from liver a distinct from that seen in brain for a of CHC, the level of was in liver compared with brain the ratio of AP-1 to AP-2 was distinct in the two tissues with a high AP-1 to AP-2 ratio in liver and a ratio in brain proteins in liver to protein to protein to protein to to protein to protein transport protein protein domain-containing to to A cell protein to in protein; protein; in a new for protein peptide counts provide a of the relative of proteins in B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). to and the amount of and the ratio that there are than in liver CCVs for and for is is that and form as stable to a ratio in all tissues, and peptide accounting on brain CCVs reveal a ratio for the proteins B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). with peptide with a that for a there is for in CCVs from liver than CCVs from brain from brain more on P. of clathrin light of light chain in mammalian Biol. Chem. Scholar, P. F.M. expression of clathrin light Neurosci. Scholar). the an that is between the different splice forms of and that are in different Moreover we the of to the of the in not the between and by peptide The used to liver CCVs for on that of vesicles in the receptor-mediated endocytosis of Cell Biol. Scholar), was different from that used for of CCVs from which was on the of S.A. 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R. vesicles in are involved in synaptic vesicle Cell Biol. for and not Thus, the in the ratio of to was not to different We the relative ratio of to in to compare tissues and to to of of For a of CHC, to that in the a deficit of compared with brain Moreover for a of CHC, were abundant in cell with the ratio in cells to that seen in the brain A deficit in relative to was also seen in from the cell compared with brain further out a of we with which is specific to but is on and is of on were seen in from tissues with the of from which of was also seen in liver and brain tissues were with to of but was not liver and brain with tissues Thus, the of in to from to for the of seen in non-brain Our results the that and are not in liver and non-brain It is that the deficit in relative to in liver CCVs on a of CCVs, that CCVs from or CCVs from the plasma the deficit be seen on CCV we liver and brain CCVs and to on In AP-2 CCVs one to the of the than AP-1 CCVs, suggesting in CCVs are and to the suggesting that the ratio of the two proteins is at the AP-1 and AP-2 vesicle Thus, there not to be a of relative to on a specific of the clathrin proteins with the CCVs, which the major of CCVs in liver In CCVs into the than CCVs that the AP-2 was to the of the protein on the and on the as that the ratio of the proteins is in brain AP-2 is a of plasma CCVs and in brain was in a to the adaptor AP-1 B. 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In and CCVs and of the of CCVs is to an ratio of In CCVs of the for by the ratio of is to one that by of CCVs in brain are involved in general endocytic housekeeping to all The is to have a specialized function the CCVs that are derived from the plasma membrane housekeeping and we can that of the CCVs are specialized for synaptic vesicle In proteins were identified in the liver CCV that were into Golgi proteins such as peripheral membrane protein or which are all with high in the Golgi C. proteomics Golgi Biol. Cell. 2004; Scholar), were not detected in the CCV We not abundant proteins such as or that C. proteomics Golgi Biol. Cell. 2004; in Golgi of the CCV include abundant liver and proteins the proteins 21 are and We NECAP 1, and in in as were to as were identified in the brain CCV B. M. Hussain N.K. D. McPherson P.S. of brain clathrin-coated vesicles in synaptic vesicle Sci. 2004; Scholar). two of the and have been to be involved in endocytosis and vesicle trafficking in M. I. 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Moreover is to to all non-brain tissues and used cell The to with the in that for all non-brain tissues and cell with the of the deficit in relative to be by Moreover the deficit in seen in cell and the to the proteins in of of the not that the deficit is to the levels of and and is not to a of into J.S. of coated vesicles from liver and by electron Cell Biol. a stoichiometry between and in liver ratio was by of that were to to and in CCV We detected from to to but we also detected from proteins identified in the In of the in Thus, is not to proteins to a specific on can provide a to determine protein in the of are and with at at the plasma membrane and the TGN I. of in Cell Biol. Scholar, M. R. K. T. by and of clathrin-coated 2004; Scholar), and are to function at function in a function for is to clathrin brain clathrin light heavy chain in Biol. Chem. Scholar), and proteins are to M. McPherson P.S. protein clathrin through to the of the clathrin light Biol. Chem. 2004; Scholar, C.Y. 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Scholar). The CCVs that to to the to as from the to at Ca2+ Robinson Ca2+ and synaptic vesicle endocytosis at the Neurosci. 2000; Scholar). In synaptic vesicle endocytosis proteins in with high Ca2+ with and endocytosis of at Neurosci. 2001; have that increases in Ca2+ concentration an of endocytosis of synaptic vesicles. In non-brain where the Ca2+ concentration is low and yet the ratio of to is a in the amount of on could the for clathrin Thus, through quantitative we are to provide a of the specialized for in the regulation of synaptic vesicle new into the of coats and of CCVs for We are to M. for and for the of the We and for with a We also all of the of the and for with

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