Membrane and Protein Trafficking

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Membrane and proteintrafficking

Membranes and their protein components areconstantly being turned over through a mechanismthat has multiple components and pathways. Mostemphasis will be focused on the proteins that aresynthesized on the endoplasmic reticulum and thenbegin their journey through the cell through a number

of pathways some of which are illustrated inModule4: Figure membrane and protein trafficking:

1. Endoplasmic reticulum/Golgi transport mechanismsdescribe the two-wayprotein transport pathways that operate between the ER and the Golgi. Thecoat protein complex II (COPII) vesicles carry cargo from the ER to the Golgi,whereas the COPI vesicles return certain ER proteins from the Golgi back tothe ER.

2. Golgi protein sorting and packaging.3. Golgi-to-plasma membrane transfer

The trans-Golgi network (TGN) is a major proteinsorting organelle functioning to direct newlysynthesized proteins either to the plasma membraneor to various endosomal compartments. Control ofvesicle formation at the TGN depends onCa2+regulation of the PtdIns 4-KIII that forms the

PtdIns4P necessary for this trafficking process. Inaddition, it can also receive cargo back from theendosomes. This bidirectional transfer between theTGN and endosomes is particularly important forsorting and directing lysosomal hydrolases towardsthe lysosome. These hydrolases are carried on thecation-independent mannose 6-phosphate receptor

(CI-MPR), which is then returned to the TGN by
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theearly endosome to trans-Golgi network (TGN)traffickingpathway (Module 4: Figure endosomebudding TGN).

4. Exocytosis5. Endocytosisand the transport of vesicles to the early endosome by myosin

motors such asmyosin VI.6. Endosome vesicle fusion to early endosomes7. Early endosome protein sorting and intraluminal vesicle formation.

The endocytic vesicles that bud off from the plasmamembrane fuse with the early endosome to deliver a

number of different proteins. These proteins have tobe sorted and then packaged for transport to anumber of other cellular locations.

8. Early endosome to plasma membrane trafficking9. Early endosome to trans-Golgi network (TGN) trafficking10.Early endosome maturation to lysosomes

As the early endosome begins to accumulate

intralumenal vesicles it matures into a multivesicularendosome (MVE). At this stage, there is a largeaccumulation of PtdIns3,4,5P2, which isaphosphoinositide lipid signalling molecule(Module2: Figure localized inositol lipid signalling) thatactivates theTRPML1channels to create the localdomains of Ca2+that triggers the fusion events to

form the lysosomes.

Endoplasmic reticulum/Golgitransport mechanisms

The first step inmembrane and protein traffickingis

the transfer of proteins from the ER to the Golgi (see
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step 1 inModule 4: Figure membrane and proteintrafficking). The Golgi is a highly dynamic organellethat processes large amounts of protein that not only

is being exported to the plasma membrane, but isalso constantly being exchanged with the ER and theendosomal system. To carry out these dynamic Golgifunctions, it is essential that this organelle maintainsits characteristic morphology. ThePtdIns4 signallingcassette(Module 2: Figure localized inositol lipidsignalling) seems to play an important role in

orchestrating both the morphology and function of theGolgi. The PtdIns4P binds to proteins such asoxysterol-binding protein (OSBP),phosphatidylinositol-Four-P AdaPtor Protein (FAPP)and the ceramide transfer protein (CERT). CERTfunctions in thegeneration and function of ceramideand sphingosine 1-phosphate (S1P)(see Step 2inModule 2: Figure sphingomyelin signalling). Inaddition, GOLPH3 binds to PtdIns4P to provide ananchor, which is linked to myosin 18A and then toactin to provide a tensile force that stretches out themembrane stacks to maintain the characteristicshape of the Golgi.

In this section, we will consider the two-way transportbetween the ER and the Golgi that is orchestrated bycoat protein complex I and II (COPI andCOPII). COPII-mediated transport from ER to Golgiisresponsible for the anterograde transport system(Module 4: Figure COPII-coated vesicles),whereasCOPI-mediated transport from Golgi toERtakes care of the retrograde transport of certain
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proteins that are returned to the ER (Module 4:Figure COPI-coated vesicle).

COPII-mediated transport from ER toGolgi

The anterograde transport of newly synthesizedproteins from the ER to the ERGolgi intermediatecompartment (ERGIC) is carried out by coat proteincomplex II (COPII) through the following sequence of

events (Module 4: Figure COPII-coated vesicles):1. The ER exit sites (ERES), which are specialized to communicate with the

Golgi, have the secretory 12 (Sec12) type II transmembrane protein thatfunctions as a guanine nucleotide-exchange factor (GEF) for the smallGTPase Sar-1. When cytoplasmic Sar-1 interacts with Sec12, it exchanges itsGDP for GTP, which then induces a conformational change causing theprotrusion of an N-terminal amphipathic -helix that attaches Sar-1 to themembrane.

2. The membrane-bound Sar-1.GTP complex then recruits components of theCOPII complex beginning with the Sec23/Sec24 dimer. The Sec23 has two

functions. First, it is a GTPase-activating protein (GAP) that will come intoplay later when the COPII coat is removed (see step 6 below). Secondly, itfunctions to attach the vesicle to the Golgi surface by binding to the tetheringcomplex trafficking protein particle 1 (TRAPP1). The Sec24 is responsible forcapturing the cargo that is to be transported to the Golgi.

3. The Sec13/Sec31 heteromeric complex then attaches to the Sec23/Sec24dimer to complete the formation of the COPII complex. As these COPIIcomplexes accumulate, they induce a localized curvature of the membranethat then matures into a bud. SNARE proteins, which will be used for vesiclefusion to the Golgi, are also incorporated into the maturing vesicle.

4. The mechanism of bud scission is still not fully understood. There areindications that the hydrolysis of phosphatidylcholine (PC) byphospholipaseD (PLD)to formphosphatidic acid (PA)may play a role in deforming themembrane during bud scission. PA may also be formed from diacylglycerol(DAG) through the activity ofDAG kinase (DAGK).Both DAG and PA arecone-shaped lipids that can bring about negative curvature of the membranethat can facilitate scission of the vesicle. The BFA-induced ADP-ribosylationsubstrate (BARS), whose activity seems to depend on PA, has also beenimplicated in the process of cutting off the vesicle.

5. Once the vesicle has been released from the ER, it is carried alongmicrotubules towards the Golgi by thedyneinmotor. It is the p50 component

of dynactin, which is adynein adaptor(Module 4: Figure dynein), that usesthegolginbicaudal D andRab6to attach the motor to the COPII vesicle.
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6. As the COPII-coated vesicle approaches the ERGolgi intermediatecompartment (ERGIC) membrane system, it begins to shed its COPII coat.Part of this shedding process seems to be driven by the inactivation of theSar-1.GTP complex when the GTP is hydrolysed to GDP through a processfacilitated by the GAP activity of Sec23 (see step 2 above) (Module 4: Figure

COPII-coated vesicles).7. The Sec23 has an additional function of attaching the vesicle to the TRAPP1

tethering complex, which contains six main subunits. It is the Bet3 subunit thatis responsible for binding to the Sec23 subunit on the vesicle. The TRAPP1 isalso a guanine nucleotide-exchange factor (GEF) forRab1and thus convertsinactive Rab1.GDP into active Rab1.GTP that functions in vesicle tethering.Sec31 can bind the penta-EF-hand protein apoptosis-linked gene 2 (ALG-2),which also interacts withAnnexin-11 (ANX11). Alg-2 and ANX11 are Ca2+-binding proteins that may carry out a Ca2+-dependent regulatory step tomodify the membrane events related to either vesicle formation or the laterfunction of COPII vesicles to the Golgi. To what extent Ca2+plays a role in the

regulation of this ER-to-Golgi transport system remains to be determined.8. The next step is for the SNARE complexes on the two membranes to begin to

interact with each other. This final approach may be facilitated bythegolginprotein p115, which attaches to Rab1 on the Golgi membrane.

9. Once the SNAREs begin to interact with each other, they drive vesicle fusionthus enabling the ER cargo proteins to reach the Golgi

COPI-mediated transport from Golgi to ER

The COPI retrieval pathway functions to return thoseER-resident proteins that escaped to the Golgithrough the COPII-mediated transport from ER toGolgi(Module 4: Figure COPII-coated vesicles). Thisretrieval pathway (see step 2 in Module 4: Figuremembrane and protein trafficking) is somewhat morecomplex than the anterograde pathway because it

can originate from multiple locations within the Golgi.ER-resident proteins that move along the Golgi as itmatures can be removed at all levels and movedbackwards to be returned to the Golgi. Thisretrograde transport to the ER, which depends on thecoat protein complex I (COPI), occurs through thefollowing sequence of events (Module 4: Figure

COPI-coated vesicles):
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1. The initial step in this retrograde pathway depends on the activation of thesmall GTPaseRab1b,which then recruits thegolginprotein p115.

2. The Rab1b/p115 complex helps to recruit the Golgi-specific brefeldin A-resistant factor 1 (GBF1), which is a guanine nucleotide-exchange factor(GEF) for the small monomeric G proteinADP-ribosylation factor 1

(Arf1)(Module 2: Table monomeric G protein toolkit). TheArf signallingpathwayhas an important role in controlling key events that occur during coatformation, actin polymerization and Golgi vesicle budding (Module 2: FigureArf signalling).

3. When cytoplasmic Arf1.GDP interacts with GBF1, it exchanges its GDP forGTP, which then induces a conformational change causing the protrusion ofan N-terminal amphipathic -helix capped by a myristoyl group that attachesArf1.GTP to the membrane that sets the stage for assembling the COPI coat.

4. The membrane-bound Arf1.GTP complex then begins to assemblecomponents of the COPI complex beginning with the p23/p24 heterodimerand this is then followed by the large COPI complex that consists of multiple

subunits organized into two groups: F-COP (, , , and ) and B-COP (,'and ). It is the -COP and the -COP that bind to the Arf1.GTP. At thisstage, the cargo marked out for transport also associates with this large COPIcoat.

5. As these COPI complexes begin to accumulate, they induce a localizedcurvature of the membrane that then matures into a bud. SNARE proteins,which will be used for vesicle fusion to the Golgi, are also incorporated intothe maturing vesicle. It is during this budding stage that the ArfGAP1 and thetwo closely related ArfGAPs 2 and 3 (ArfGAP2/3) associate with thedeveloping bud through separate mechanisms. ArfGAP1 recognizes thecurvature of the bud using an ArfGAP1 lipid sensory (ALPS) motif, which isnormally unstructured, but when it detects a curved membrane it forms anamphipathic -helix enabling the molecule to bind to the developing vesicle.

The mechanism of bud scission is still not fullyunderstood. There are indications that the hydrolysisof phosphatidylcholine (PC) byphospholipase D(PLD)to formphosphatidic acid (PA)may play a role.

PA may also be formed from diacylglycerol (DAG)through the activity ofDAG kinase (DAGK).BothDAG and PA are cone-shaped lipids that can bringabout negative curvature of the membrane that canfacilitate scission of the vesicle. The BFA-induced

ADP-ribosylation substrate (BARS), whose activityseems to depend on PA, has also been implicated inthe process of cutting off the vesicle.
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6. Once the vesicle has been released from the Golgi, it is carried along actinfilaments towards the ER by akinesin-2motor.

7. As the COPI-coated vesicle approaches the ER, it begins to shed it's COPIcoat. Part of this shedding process seems to be driven by the inactivation ofthe Arf1.GTP complex when the GTP is hydrolysed to GDP through a process

facilitated by the GAP activities of both ArfGAP1 and ArfGAP2/3.8. The initial contact between the vesicle and the ER depends on a tethering

complex called syntaxin 18, which may also act to draw in the SNAREproteins necessary for subsequent membrane fusion.

9. The next step is for the SNARE complexes on the two membranes to begin tointeract with each other.

10. This SNARE interaction finally drives vesicle fusion thus enabling theGolgi proteins to reach the ER.

Golgi sorting and proteinpackaging

The Golgi consists of a series of flattened cisternalmembranes, which are stacked on top of each other(Module 4: Figure membrane and protein trafficking).

The Golgi is polarized with the cis-face exchangingproteins and lipids with the endoplasmic reticulumwhile the trans-face sends secretory proteins to theplasma membrane and also communicates with theendosomal system.

This stack like organization appears to be heldtogether by interactions with the cytoskeleton andalso through the action of the coiled-coil proteins ofthegolginfamily.

Golgins

The Golgins are a family of coiled-coil proteins thatoperate within the Golgi during the process ofGolgi

sorting and protein packaging. The golgins help to
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maintain the structural organization of the Golgi andmay also function as membrane tethers duringvesicle transfer between different vesicle

compartments. The golgins function as dimers heldtogether by their coiled-coil regions, and some of thefamily members have interaction domains that enablethem to interact with various small GTPases suchasRab1,ADP-ribosylation factor (Arf)and Arf-like(ARL) (Module 2: Table monomeric G protein toolkit)that appear to control their interaction with the Golgi

membranes. The following are some of the maingolgin family members:

1. Transmembrane golgins such as Giantin, Golgin-84 and CCAAT-displacement protein (CDP) alternatively spliced product (CASP) are attachedto membranes through a C-terminal transmembrane domain.

2. Adaptor-associated golgins are attached to membranes through the Golgireassembly stacking protein (GRASP) adaptors, which associate withmembranes through an N-terminal myristoyl group. For example, GM130 isattached to GRASP65, whereas Golgin-45 is attached to GRASP55.

3. Rab-associated golgins are recruited to membranes through the small RabGTPases.Rab1recruits p115 duringCOPI-mediated transport from Golgi toER(Module 4: Figure COPI-coated vesicle). The Rab1/p115 complex alsofunctions as a tether on the Golgi surface duringCOPII-mediated transportfrom ER to Golgi(Module 4: Figure COPII-coated vesicles).Rab6binds tothe golgin Bicaudal D, which functions in vesicle transport between the Golgiand the ER, to attach thedyneinmotor complex to the vesicle (Module 4:Figure dynein) for its transfer from the ER to the Golgi membranes.

4. ARL-associated golgins are recruited to membranes through the Arf-like(ARL) small GTPase. The golgin-97 and golgin-245 attach to ARL1 through

their GRIP domains.5. Arf-associated golgins, such as GMAP-210, are recruited to membranesthrough the small GTPaseADP-ribosylation factor (Arf).

Golgi to plasma membranetransfer

The trans-Golgi network (TGN) is a major protein-sorting organelle functioning to direct newly
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synthesized proteins either to the plasma membraneor to various endosomal compartments (see step 3inModule 4: Figure membrane and protein

trafficking). The formation of vesicles at the TGNappears to be regulated by a local Ca2+signal thatstimulates the PtdIns4 signalling cassette(Module 2:Figure localized inositol lipid signalling). A keycomponent of this Golgi lipid signalling system isPtdIns 4-KIII. At resting levels of Ca2+, this lipidkinase is kept inactive when bound to the Ca2+-

sensing proteins calneuron-1 or calneuron-2. Inresponse to a local pulse of Ca2+, the inhibitorycalneurons are replaced by neuronal Ca2+sensor 1(NCS-1)that stimulates PtdIns 4-KIII to begin toproduce the PtdIns4P necessary for vesicleformation.

ExocytosisMany aspects of cell communication depend uponthe process of exocytosis to release signallingmolecules such as hormones and neurotransmitters.These signalling molecules are stored in membranevesicles that are released during cellcellcommunication. This regulated release of storedvesicles occurs through a process of Ca2+-dependentexocytosis. There are twoexocytotic mechanisms:the classicalexocytotic/endocytotic cycleand abrieferkiss-and-run vesicle fusionmechanism. Inboth cases, the problem is to understand how an

elevation in Ca

2+

can trigger the initial event ofmembrane fusion. Since there is a natural reluctance
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for membranes to fuse with each other,special exocytotic machineryis used to force the twomembranes together so that fusion occurs.

There are two types of Ca2+-dependent exocytosis(Module 4: Figure Ca2+-dependent exocytosis):

Exocytosis triggered by Ca2+entry through voltage-operated channels (VOCs) Exocytosis triggered by Ca2+release from internal stores

Exocytotic mechanisms

Membrane vesicles lying close to the plasmamembrane are primed to fuse with the plasmamembrane in response to a pulse of Ca2+.Theneuronal Ca2+sensor-1 (NCS-1)can enhanceexocytosis and this may depend upon its ability tofacilitate the priming step. NCS-1 activates thePtdIns4-kinase (PtdIns 4-K), which contributes tothePtdIns4,5P2regulation of exocytosis.Once thevesicles are primed, membrane fusion is triggered bya brief pulse of Ca2+. When fusion occurs, thecontents of the vesicles are free to diffuse outthrough the fusion pore. Just how much of thecontent is released depends upon the subsequent

events. In the case of theclassicalexocytotic/endocytotic cycle, all of thecontents are released. On the other hand, thekiss-and-run vesicle fusionmechanism is much brieferand can be repeated a number of times, thusenabling the same vesicle to function repeatedly.

Exocytotic/endocytotic cycle
http://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytosis_triggered_by_Ca_entryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytosis_triggered_by_Ca_entryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytosis_triggered_by_Ca_entryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytosis_triggered_by_Ca_entryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exo_triggered_Ca_release_internal_storeshttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exo_triggered_Ca_release_internal_storeshttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exo_triggered_Ca_release_internal_storeshttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exo_triggered_Ca_release_internal_storeshttp://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns_4_kinasehttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns_4_kinasehttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns_4_kinasehttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns45P2_regulation_of_exocytosishttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns45P2_regulation_of_exocytosishttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns45P2_regulation_of_exocytosishttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns45P2_regulation_of_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_endocytotic_cyclehttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_endocytotic_cyclehttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_endocytotic_cyclehttp://www.biochemj.org/csb/004/csb004_sec005.htm#Kiss_and_run_fusionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Kiss_and_run_fusionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Kiss_and_run_fusionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Kiss_and_run_fusionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Kiss_and_run_fusionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_endocytotic_cyclehttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns45P2_regulation_of_exocytosishttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns_4_kinasehttp://www.biochemj.org/csb/002/csb002_sec008.htm#PtdIns_4_kinasehttp://www.biochemj.org/csb/004/csb004_sec003.htm#Neuronal_calcium_sensor_1http://www.biochemj.org/csb/004/csb004_sec005.htm#Exo_triggered_Ca_release_internal_storeshttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytosis_triggered_by_Ca_entryhttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machinery


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The classical exocytotic/endocytotic cycle (Module 4:Figure vesicle cycle) depends upon sequentialprocesses of docking, priming, exocytosis and

endocytosis. During this process, the vesicle fuseswith the plasma membrane to release all of itscontents, and this is then followed by the membraneof the empty vesicle being taken up again through theprocess of endocytosis.

Kiss-and-run vesicle fusion

As its name implies, the kiss-and-run vesicle fusionprocess depends upon individual vesicles fusingrepeatedly with the membrane, during which processthey release a small proportion of their contents. Thismechanism is particularly evident in the smallsynaptic endings found in the brain, which have 20

30 synaptic vesicles that can be re-used repeatedlyto give transient pulses of neurotransmitter duringsynaptic transmission. Another example of kiss-and-run fusion has been described inchromaffincells(Module 7: Figure chromaffin cell exocytosis).

Exocytosis triggered by Ca2+entry

through voltage-operated channels(VOCs)

The most extensively studied form of exocytosis isthe release of synaptic vesiclesat neuronalpresynaptic endings, which depends uponCa2+-dependent exocytosistriggered by the entry of

Ca2+through theCaV2 family of N-type, P/Q-type and
http://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/007/csb007_sec020.htm#Chromaffin_cellhttp://www.biochemj.org/csb/007/csb007_sec020.htm#Chromaffin_cellhttp://www.biochemj.org/csb/007/csb007_sec020.htm#Chromaffin_cellhttp://www.biochemj.org/csb/007/csb007_Fig128.htm#Fig7_chromaffin_cell_exocytosishttp://www.biochemj.org/csb/010/csb010_sec006.htm#Synaptic_vesiclehttp://www.biochemj.org/csb/010/csb010_sec006.htm#Synaptic_vesiclehttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_sec005.htm#Ca_dependent_exocytosishttp://www.biochemj.org/csb/010/csb010_sec006.htm#Synaptic_vesiclehttp://www.biochemj.org/csb/007/csb007_Fig128.htm#Fig7_chromaffin_cell_exocytosishttp://www.biochemj.org/csb/007/csb007_sec020.htm#Chromaffin_cellhttp://www.biochemj.org/csb/007/csb007_sec020.htm#Chromaffin_cellhttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cycle


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R-type channels.A remarkable aspect of this processis its rapid kinetics. The phenomenal computationalability of the brain depends upon neurons being able

to communicate with each other in less than 2 ms(Module 10: Figure kinetics of neurotransmission).During this process of synaptic transmission, thearrival of an action potential at the synaptic endingcan trigger the release of neurotransmitter in lessthan 200 s. The organization of theexocytoticmachineryappears to be specially designed to

achieve these high reaction rates.

Exocytosis is part of an orderly vesicle cycle (Module4: Figure vesicle cycle). Vesicles move from areserve pool to dock with the membrane, duringwhich the exocytotic machinery is assembled andprimed to respond to the final event of Ca2+-induced

exocytosis. The key to achieving such rapidresponses is therefore to have the exocytoticmachinery assembled and primed prior to the arrivalof the Ca2+signal, which then functions just to triggerthe final step of membrane fusion.

Exocytosis triggered by Ca2+release from

internal stores

Some cells seem to be capable of stimulatingexocytosis by releasing Ca2+from internal stores(Module 4: Figure Ca2+-dependent exocytosis). Theconcentration of Ca2+within the microdomains thatform around the opening of internal release channels,

such as the inositol 1,4,5-trisphosphate receptors
http://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channelshttp://www.biochemj.org/csb/010/csb010_Fig020.htm#Fig10kinetics_of_neurotransmissionhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/003/csb003_sec016.htm#InsP3_receptors_InsP3Rshttp://www.biochemj.org/csb/003/csb003_sec016.htm#InsP3_receptors_InsP3Rshttp://www.biochemj.org/csb/004/csb004_Fig015.htm#Fig4_Ca_dependent_exocytosishttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_Fig016.htm#Fig4_vesicle_cyclehttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/004/csb004_sec005.htm#Exocytotic_machineryhttp://www.biochemj.org/csb/010/csb010_Fig020.htm#Fig10kinetics_of_neurotransmissionhttp://www.biochemj.org/csb/003/csb003_sec004.htm#CaV2_family_N_PQ_R_type_channels


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(InsP3Rs)andryanodine receptors (RYRs), is veryhigh and thus will be capable of triggering theexocytotic process. There are a number of examples

of vesicle release being triggered by release ofCa2+from internal stores:

Astrocytes use InsP3-induced release of Ca2+to trigger the exocytosis of

vesicles containing glutamate as part of theastrocyteneuronalcommunication system(Module 7: Figure astrocyte tripartite synapse).

Mossy fibre presynaptic Ca2+release. Cerebellar basket cell presynaptic Ca2+release. Hypothalamic neuronal presynaptic Ca2+release. Neocortical glutamatergic presynaptic Ca2+release. Release of luteinising hormone (LH) and follicle-stimulating hormone (FSH)

fromgonadotrophs(see step 5 inModule 10: Figure gonadotroph regulation). Release ofthyroid-stimulating hormone (TSH)from thethyrotrophsis

triggered by Ca2+released from the internal store (Module 10: Figurethyrotroph regulation).

Release of 5-HT fromtype III presynaptic cellsduring taste reception (Module10: Figure taste receptors).

Exocytotic machinery

The exocytotic machinery is made up of manydifferent components that are distributed between theplasma membrane and the synaptic vesicle. Thelatter are encrusted by a large number of proteinsthat include those that function in exocytosis such assynaptobrevin, and synaptotagmin (Module 10:

Figure synaptic vesicle). Membrane fusion is drivenby an interaction between these vesicle proteins andthe plasma membrane proteins (Module 4: FigureCa2+-induced membrane fusion). Some of the keyplayers in this membrane fusion mechanism are thesoluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNAREs), composed

of the vesicle SNAREs (v-SNAREs) and the target
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/010/csb010_Fig019.htm#Fig10synaptic_vesiclehttp://www.biochemj.org/csb/010/csb010_sec006.htm#Synaptic_vesiclehttp://www.biochemj.org/csb/010/csb010_Fig088.htm#Fig10taste_receptor_cellshttp://www.biochemj.org/csb/010/csb010_Fig088.htm#Fig10taste_receptor_cellshttp://www.biochemj.org/csb/010/csb010_sec015.htm#Type_III_presynaptic_cellshttp://www.biochemj.org/csb/010/csb010_Fig067.htm#Fig10thyrotroph_regulationhttp://www.biochemj.org/csb/010/csb010_Fig067.htm#Fig10thyrotroph_regulationhttp://www.biochemj.org/csb/010/csb010_sec014.htm#Thyrotrophshttp://www.biochemj.org/csb/010/csb010_sec014.htm#Thyroid_stimulating_hormone_TSHhttp://www.biochemj.org/csb/010/csb010_Fig062.htm#Fig10gonadotroph_regulationhttp://www.biochemj.org/csb/010/csb010_sec014.htm#Gonadotrophshttp://www.biochemj.org/csb/010/csb010_sec008.htm#Neocortical_glutam_presyn_Ca_releasehttp://www.biochemj.org/csb/010/csb010_sec008.htm#Hypothalamic_neuronal_presyn_Ca_releasehttp://www.biochemj.org/csb/010/csb010_sec008.htm#Cerbellar_basket_cell_presyn_Ca_releasehttp://www.biochemj.org/csb/010/csb010_sec008.htm#Mossy_fibrel_presyn_Ca_releasehttp://www.biochemj.org/csb/007/csb007_Fig006.htm#Fig7_astrocyte_tripartite_synapsehttp://www.biochemj.org/csb/007/csb007_sec005.htm#Astrocyte_neuronal_communicationhttp://www.biochemj.org/csb/007/csb007_sec005.htm#Astrocyte_neuronal_communicationhttp://www.biochemj.org/csb/003/csb003_sec016.htm#Ryanodine_receptors_RYRshttp://www.biochemj.org/csb/003/csb003_sec016.htm#InsP3_receptors_InsP3Rs


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SNAREs (t-SNAREs) located on the plasmamembrane (Module 4: Figure Ca2+-inducedmembrane fusion):

Synaptobrevin [also known as vesicle-associated membrane protein 2(VAMP2)] is a v-SNARE.

Syntaxin 1a and 25 kDa synaptosome-associated protein (SNAP-25) areexamples of t-SNAREs.

These three SNAREs are weakly homologousproteins, especially with regard to a SNARE motifconsisting of a coiled-coil domain, that bind to each

other with high affinity through hydrophobicinteractions to form parallel arrays (Module 4: FigureCa2+-induced membrane fusion). The current modelsof exocytosis consider that the SNARE proteinszipper up with each other, thereby inducing fusion bydriving the two membranes together.

The rapidity of the fusion process described in thesection onExocytosis triggered by Ca2+entry throughvoltage-operated channels (VOCs)can be accountedfor by the fact that the fusion proteins may begin thezippering processes during the docking/primingevents, and are thus poised for completion whengiven the appropriate signal through the process

ofCa2+-dependent exocytosis.

Ca2+-dependent exocytosis

The process of membrane fusion during exocytosis inneurons is driven by an influx of Ca2+throughtheCaV2 family of N-type, P/Q-type and R-type

channels.A characteristic feature of these voltage-operated channels (VOCs) is that they possess a
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binding site that tightly anchors them to the exocytoticmachinery (Module 3: Figure CaV2 channel family),and they are thus positioned to provide the rapid,

high-intensity pulse of Ca2+necessary to triggermembrane fusion (Module 4: Figure Ca2+-inducedmembrane fusion). In many endocrine cells,activation of L-type Ca2+channels produces theglobal elevation of Ca2+necessary to triggerexocytosis. Just how this Ca2+triggers fusion is stillsomewhat of a mystery. The synapse has a number

of Ca2+sensors that may play different functions, asthey seem to be sensitive to differentCa2+concentrations. The zipper model implies that,once theexocytotic machineryhas been primed, it isprevented from going to completion by inhibitorymechanisms that are removed by the pulse of Ca2+. Ittherefore seems reasonable to imagine that the Ca2+-sensitive synaptotagmin family of proteins mightmediate this inhibition. Different synaptotagminsseem to play a role in exocytosis. Synaptotagmins Iand II are integral membrane proteins anchored inthe vesicle through membrane-spanning regions. Onthe other hand, synaptotagmin VII is located in the

plasma membrane. These synaptotagmins are ideallysuited to regulate fusion in that they can bind tosyntaxin and theirC2 domainscan bind tophospholipids in a Ca2+-dependent manner. TheseCa2+-sensitive C2 domains, which are separated fromeach other by a flexible linker, contain a -barrel andbind multiple Ca2+ions during which there is a

conformational switch that might activate the
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exocytotic machinery. One possibility is that theconformational change in synaptotagmin relieves theinhibition on the exocytotic machinery, thus enabling

fusion to occur (Module 4: Figure Ca2+-inducedmembrane fusion). The plasma membrane andvesicular synaptotagmins appear to have differentaffinities for Ca2+: those on the plasma membraneare high-affinity sensors involved in slow exocytosis,whereas those on the vesicle have low-affinitysensors capable of fast Ca2+-dependent exocytosis.

Another protein called piccolo/aczonin, which has C-terminal C2A and C2B domains, may be a low-affinityCa2+sensor that functions as a regulator whenCa2+accumulates during repetitive activity.
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Endocytosis

Cells take up a wide range of molecules through anumber of mechanisms such as clathrin-mediatedendocytosis (CME), caveolin-mediated endocytosis,clathrin/caveolin-independent endocytosis andmacropinocytosis (Module 4: Figure membrane andprotein trafficking). The endocytic vesicles, which

carry molecules away from the plasma membrane,are directed towards the early endosome and thesubsequent process of endosome vesicle fusion toearly endosomes, enabling the molecules taken upfrom the plasma membrane to enter the intracellularprotein trafficking system (Module 4: Figuremembrane and protein trafficking). A number of

signalling mechanisms function in thecontrol ofendocytosis.

Clathrin-mediated endocytosis(CME)

Most attention has focused on clathrin-mediatedendocytosis (CME), which has multiple functions,such as down-regulation of surface receptors,nutrient uptake and synaptic vesicle recycling. Theuptake of integral membrane proteins, such as thetransferrin receptor (TFR), occurs through variousstages (Module 4: Figure endocytosis):

Cargo selection by sorting proteins Membrane invagination and scission
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Vesicle transport Coat removal

Cargo selection by sorting protein

The initial step of cargo sorting depends on theassembly of various sorting proteins that recognizethe cargo. The sorting proteins function as adaptorsto connect cargo proteins to the clathrin coat duringthe process of endocytosis (Module 4: Figureendocytosis). In order to carry out this adaptor

function, the sorting proteins such as theadaptorproteins (APs)and the clathrin-associated sortingproteins (CLASPs)have to bind multiple partners. Forexample adaptor protein 2 (AP2) associates with themembrane by binding to PtdIns4,5P2, formed bythePtsIns4P 5-kinase I (PIPKI),and to the sortingsignals located on the cytoplasmic domain of the

cargo proteins (Module 4: Figure cargo sortingsignals). These sorting proteins then form amolecular platform that binds clathrin, which is anessential feature of the coated vesicles. Thecytoplasmic domain of the cargo proteins has thesorting signals that enable them to be recognized bythe sorting proteins. Many of these sorting signals areshort amino acid sequences that are found on cargoproteins that are taken up constitutively. However,the endocytosis of some proteins is regulated througha post-translational modification such as theubiquitination that occurs during the Cbl down-regulation of cell signalling components(Module 1:

Figure receptor down-regulation).
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Theadaptor protein (AP)family are particularlyimportant sorting proteins, with AP2 playing a majorrole in endocytosis. In addition to AP2, there are a

number ofclathrin-associated sorting proteins(CLASPs)that function in cargo recognition duringendocytosis.

Adaptor protein (AP)

The adaptor protein family has three members: AP1,

AP2 and AP3. Most attention has focussed onadaptor protein 2 (AP2), which has a primary role toplay in clathrin-mediated endocytosis (CME) (Module4: Figure endocytosis). AP2 consists of four subunits(, 2, 2 and 2) that form a heterotetramericcomplex that has a large trunk and two appendagedomains located on flexible linkers that come from

the - and 2-subunits (Module 4: Figure cargosorting signals). The trunk region attaches AP2 tothe cargo and the membrane, whereas theappendages bind to various accessory proteins thatcontribute to forming the molecular layer that coatsthe vesicle.

The YXX sorting signal, which is located onproteins such as the transferrin receptor (TFR), CD-M6PR, LAMP1, LRP1, PAR1, P2X4receptor and the2-subunit of the GABAAreceptor, is recognized by aregion on the 2-subunit of AP2. The latter also bindsto phosphatidyl 4,5-bisphosphate (PtdIns4,5P2),which also functions as an adaptor to glue AP2 to

the membrane. A separate group of cargo proteins
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such as CD4, CD3, LIMP2 and Nef have the sortingsignal [DE]XXXL[LI], which recognizes the 2-subunit.

Clathrin-associated sorting proteins(CLASPs)

The clathrin-associated sorting proteins (CLASPs)function as adaptors to select cargoduringendocytosis(Module 4: Figure cargo sorting

signals). Some of these CLASPs, such as disabled 2(DAB2), autosomal recessive hypercholesterolemia(ARH) andNumb, have a PTB domain,whichrecognizes the [FY]XNPX[YF] sorting signal found oncargo proteins such as the LDL receptor, LRP1,LRP2 (megalin), P-selectin and 1A integrin1 and 2.These CLASPs contribute to the coat by binding to

clathrin and AP2.

The epsins and the epidermal growth factor receptorsubstrate 15 (EPS15) contribute to endocytosis byfunctioning as sorting proteins to detect ubiquitinatedcargo. They have ubiquitin-interacting motifs (UIMs)that bind to the ubiquitinated cargo such as the

epidermal growth factor receptor (EGFR) as occursduring theCbl down-regulation of cell signallingcomponents(Module 1: Figure receptor down-regulation). Epsin 1 and EPS15 can recognize thepolyubiquitination of Lys-63 on the EGFR.

Thereceptor down-regulationofG protein-coupled

receptors (GPCRs)is carried out by-arrestinsthatbehave like CLASPs (Module 1: Figure homologous
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desensitization). When the arrestins bind to thehyperphosphorylated GPCRs, they reveal calthrin-and AP2-binding motifs that guide the complex into

the coated pits ready for internalization (Module 4:Figure cargo sorting signals).

Membrane invagination and scission

One sorting protein, such as the adaptor protein 2(AP2), have trapped and concentrated cargo

proteins, clathrin begins to coat the macromolecularcomplexes and the membrane invaginates to form aconcentric coated bud (Module 4: Figure scission ofendocytic vesicles). The clathrin, which consists oftwo subunits, an elongated heavy chain and a lightchain, polymerize to form triskelia. These clathrintriskelia, which have three legs radiating out from a

hub, interact with each other to form a web that coatsthe vesicular bulb. This bulb is then cut off through ascission process that is not fully understood, butsome of the main players have been identified. A keyevent appears to be the formation of amacromolecular spiral that wraps around the neck ofthe vesicular bud. The main components of this spiral

are SNX9 and the large GTPasedynamin. The PXdomainon SNX9 binds to PtdIns4,5P2, which ispresent at high levels, to induce a conformationchange resulting in its oligomerization and this thenprovides a platform to draw in other proteins. Forexample, dynamin binds to both SNX9 and toPtdIns4,5P2to form part of the spiral.
http://www.biochemj.org/csb/001/csb001_Fig035.htm#Fig1_homologous_desensitizationhttp://www.biochemj.org/csb/004/csb004_Fig019.htm#Fig4_cargo_sorting_signalshttp://www.biochemj.org/csb/004/csb004_Fig019.htm#Fig4_cargo_sorting_signalshttp://www.biochemj.org/csb/004/csb004_Fig019.htm#Fig4_cargo_sorting_signalshttp://www.biochemj.org/csb/004/csb004_sec006.htm#Adaptor_protein_APhttp://www.biochemj.org/csb/004/csb004_sec006.htm#Adaptor_protein_APhttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_sec006.htm#Dynaminhttp://www.biochemj.org/csb/004/csb004_sec006.htm#Dynaminhttp://www.biochemj.org/csb/006/csb006_sec003.htm#PX_domainhttp://www.biochemj.org/csb/006/csb006_sec003.htm#PX_domainhttp://www.biochemj.org/csb/006/csb006_sec003.htm#PX_domainhttp://www.biochemj.org/csb/006/csb006_sec003.htm#PX_domainhttp://www.biochemj.org/csb/006/csb006_sec003.htm#PX_domainhttp://www.biochemj.org/csb/004/csb004_sec006.htm#Dynaminhttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_sec006.htm#Adaptor_protein_APhttp://www.biochemj.org/csb/004/csb004_sec006.htm#Adaptor_protein_APhttp://www.biochemj.org/csb/004/csb004_Fig019.htm#Fig4_cargo_sorting_signalshttp://www.biochemj.org/csb/004/csb004_Fig019.htm#Fig4_cargo_sorting_signalshttp://www.biochemj.org/csb/001/csb001_Fig035.htm#Fig1_homologous_desensitization


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The SNX9/dynamin spiral provides a platform toassemble actin remodellingproteins suchas cortactin,N-WASPandArp2/3responsible for the

nucleation of actin filaments (Module 4: Figurescission of endocytic vesicles). In addition, the SNX9also provides another connection to actin by bindingtomyosin 1E. Another molecular motor (myosin VI)is connected to the vesicular region of the bulb bybinding to both the sorting protein DAB2 and toPtdIns4,5P2.

As these various molecules bind, the neck begins tothin and is then severed (scission) through a processthat seems to depend on the GTP-dependent actionof dynamin. In addition, the two motor proteins mighthelp to pull the vesicle into the cytoplasm throughtheir interaction with actin (Module 4: Figure scission

of endocytic vesicles). Myosin 1E is a plus-endmotor that will pull the dynamin ring towards theplasma membrane, whereas the minus-end motormyosin VI will pull the vesicle in the opposite directiontowards the cytoplasm.

Dynamin

Dynamin is a large GTPase that functions in clathrin-mediated endocytosis (CME) (Module 4: Figurescission of endocytic vesicles). At the time ofscission, dynamin is part of a macromolecularcomplex containingamphiphysin, endophilin, epsin,Eps15, synaptojanin, syndapin, N-WASP, cortactin,

mammalian actin-binding 1 (mAbp1), intersectin and
http://www.biochemj.org/csb/004/csb004_sec018.htm#Actin_remodellinghttp://www.biochemj.org/csb/004/csb004_sec018.htm#Actin_remodellinghttp://www.biochemj.org/csb/004/csb004_sec018.htm#Cortactinhttp://www.biochemj.org/csb/004/csb004_sec018.htm#Cortactinhttp://www.biochemj.org/csb/004/csb004_sec018.htm#WASPhttp://www.biochemj.org/csb/004/csb004_sec018.htm#WASPhttp://www.biochemj.org/csb/004/csb004_sec018.htm#WASPhttp://www.biochemj.org/csb/004/csb004_sec018.htm#Arp2_3_complexhttp://www.biochemj.org/csb/004/csb004_sec018.htm#Arp2_3_complexhttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_sec012.htm#Myosin_Ihttp://www.biochemj.org/csb/004/csb004_sec012.htm#Myosin_Ihttp://www.biochemj.org/csb/004/csb004_sec012.htm#Myosin_Ihttp://www.biochemj.org/csb/004/csb004_sec012.htm#Myosin_VIhttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_Fig020.htm#Fig4_scission_of_endocytic_vesicleshttp://www.biochemj.org/csb/004/csb004_sec006.

Membrane and Protein Trafficking

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