BIOL 2056 - Vesicle transport, cargo delivery and cytoskeletal components

vesicle transport pathways

• helps to compartmentalise the cell —> regulates transcription, translation, modification, trafficking of proteins

• mechanism or turning on/off signal transduction

• pH important in compartmentalisation as it alters the affinity of molecules for their receptors

SECRETORY PATHWAY

• flow of membrane bound or soluble proteins destined for certain organelles

ENDOCYTIC PATHWAY

• plasma membrane capture of EC components and internalisation of membrane proteins into vesicles

• recycling of receptors or degradation of lysosome contents

requirements of vesicle transport

BIDIRECTIONAL FLOW

• cells dont change size, there’s a constant flow

• lipid and protein components maintained

ID OF SPECIFIC CARGO

SORTING OF VESICLES AND ASSOCIATED CARGO

TRANSPORT

TRANSFER OF MATERIAL

• fission

• tethering

• fusion

vesicle coat protiens

CLATHRIN

• to the golgi from plasma memb

• found on the early endosome, matures into the lysosome where a protein goes to a lysosome or to the trans golgi face is an adaptor p protein

• ARF GTPase

COP 1

• golgi complex to the ER (retrieval)

• heptameric coatamers and ARF GTPase

COP 2

• goes from the Er to the golgi

• sec 13/14 and sec 23/24 w associated GTPase

protein interactions with membranes

• may be anchored with amphipathic alpha helix

• may be post translational modification of lipid anchor

• may be non covalent interactions w other memb

types of endocytosis

1. receptor mediated —> triggered by a receptor embedded into the membrane

   • clathrin dependent

   • caveolin dependent

   • clathrin caveolin independent

2. phagocytosis

3. pinocytosis

clathrin coat

CLATHRIN COAT

• made of 3 large chain and 3 light chain polypeptides that assemble in triskeletons of the trans golgi network or at the plasma membrane

• these clathrins then form an outer protein lattice

FORMATION

• mediated by adaptor protein complex —> required for clathrin recruitment, coat assembly and eventual budding

• lipid modification sites of phosphoinositides on the cell membrane allow for attachment of APs.

• AP2 adaptor protiens bind to specific phospholipids and cause a conformational change which allows:

  • binding to cargo receptors on cells surface

  • triggers membrane curvature

  • recruitment of clathrin

AP2 adaptor protein complex

AP2 ADAPTOR PROETIN COMPLEX

• heterotrimeric subunit

  • a adaptin

  • b2 adaptin

  • sigma 2 adaptin

  • mu 2 adaptin

• AP1 adaptor complex found on the vesciles coming from teh golgi

• AP2 on clathrin coated vesicles originating from plasma membrane

• can recognise specific peptide motifs on the cargo receptor (endocytosis signal)

• interacts w plasma membrane lipids, cargo and clathrin —> link between clathrin and cargo

ACTIVATION

• always present but kept in closed state in the cytoplasm —> internal interactions so binding domain buried

• activation of CSM receptors causes clustering of receptors and PIP2 formation

• PIP2 binds to AP2 and holds it at the membrane so that it can bind to a receptor —> only occurs when there’s enough PIP2 at the membrane

• activation of AP2 with PIP2 at the membrane allows the clathrin binding site to be exposed

• mu 2 subunit is displaced which stabilises the AP2 complex in the open formation

summary of assembly of clathrin

1. binding of cargo to receptor

2. recruitment of adaptr complex

3. recruitment of clathrin

4. clathrin causes memb curvature

5. vesicle now needs to be excised from memb

dynamin

• cuts off the bud from the membrane

• is a GTPase

• coat is then lost after the vesicle buds off —> components recycled and the proteins needed for a fusion event are exposed

• hydrolysis of GTP causes a conf change in dynamin which helps to break the 2 membranes

• oligomerises to form a helical ring around teh neck of the band

• then will recruit other proteins and will thether itself via a lipid binding motif

• GTP hydrolysis results in the lengthwise extension of helix and fission of the memb

formation of cop 2 vesicles

• cop 2 has 5 protein subunits which has an associated sar 1 GTPase

• sec 12 is a receptor in the ER membrane which can switch on sar 1 allowing for the conversion of GTP

• cop 2 vesicles carry bulk proteins but also

  • enzymes for golgi processing

  • docking and fusion proteins

  • integral proteins that bind to specific targets

• the GTP bound GTPase embeds in the membrane and acts as a marker to recruit the coat complex

• sec23 & sec24 act as the inner coat whilst sec 13/14 act as the outer coat

• hydrolysis of GTP disassembles the coat and allows the vesicle to move onwards

• cargo receptors ensure the correct packaging of protein by interaction with the coat complex.

cop 1 vesicles

STRUCTURE

• function as a retrieval pathway

• ER resident enzymes have a specific KDEL motif at the C terminus which allows resident Er proteins to be retrieved

• ER has higher pH than golgi so in golgi the KDEL has high affinity for receptor —> low affinity in Er so is deposited

• is a heptamer in the cytoplasm

• it is not formed at the vesicle

• driven by an ARF1 GTPase for coatomer recruit

• clathrin and cop 1 have the coat proteins bought to the membrane where the coat is formed unlike cop 1

the importance of GTPases

• switch for vesicle transport

• activation w GTP allows for interaction w effector proteins

• rab family of GTPases provide compartments with identity

• rab family part of teh ras family

RAS FAMILY

• lipidated - allows membrane association

• small

• conserved regions which allows for selectivity of effector proteins

• 5 major subfamilies, one of them being rab

• various PT modifications

RAB FAMILY

• 61 members

• intracellular transport

• localised lipid binding motif

• PT addition of prenyl lipid group

• effector proteins allow vesicle formation, budding, transport, vesicle tension

NOTE: GEF = guanine exchange factor, GAP = GTPase activating proteins

maturation of vesicles

• vesicle carrying cargo originating from plasma membrane may require rab 5

• this may transition into early endosome w rab 4/11

• maturation of vesicle is changing of location via turning off/on molecular switch

recruitment of Rab GTPases

• Rabs kept inactive in the cytoplasm

• interaction of GDI masks the prenyl lipid group which inactivates it

• GDI = Guanine dissociation inhibitor

• when lipid group exposed can embed in the membrane and recruit proteins

• Resident GEFs in the membrane can activate Rab so it can hydrolyse GTP

direction of vesicles and vesicle identity

IDENTITY

• changing/maturation of vesicles determined by switching on of GEFs to actuvate next rab whist the previous GAP turns off the previous rab

DIRECTION

• budding off of vesicles causes uncoating to reveal proteins on the surface

• uncoating driven by GTPases

• vesicle now exposed to snare proteins/other Rabs

• SNARE proteins help with the fusion of membranes and delivery of cargo

• Rab GTpases can bring the vesicles close to target membranes so snares can interact

id of target membrane and vesicle docking

ID OF TARGET MEMBRANE

• different snare proteins exist in pairs

  • vsnare on surface of vesicles

  • tsnare on membrane of target

• v and t snares have helices which interact with one another and dock the vesicle to the target membrane

• initiated by a sepcific Rab GTPase

• can also be used for binding of vesicles which are intended for extracellular space

DOCKING

• Rab GTP protein on surface binds to specific Rab effector in target memb (VAMPS)

• this brings v and t snares in close proximity so they can form coiled coils called a trans snare complex

• this exerts an inwards force that bringd the two membranes close together

membrane fusion

• the two membranes are slightly repulsive so need to be forced together

• the alpha helices bring these together to release cargo

• a cis snare complex is formed on one membrane —> this will drive selectivity

• cis snare complex formation driven by alpha snap and NSF —> also disaasembles cis snare complex and allows for components to be recycled

vesicles in neurotransmission

• synaptic membrane is recycled to form vesicles

• in neurotransmission vesciles must be primed so their release is not random

• v snare - synaptobrevin

• t snare - syntaxin and snap 25

• complexin helps with docking - keeps it in the locked position where its held close to the membrane

• ca2+ causes synaptotagmin to undergo conf change and kick out complexin so vesicles can fuse at the same time

• removal of one complexin causes the removal of all the complexin

endocytosis

• receptors on the plasma membrane get taken up by vesicles which go to an early endosome

• receptors sometimes recycled back to the membrane

• also a method of uptake for ligand bound receptor

• eventually goes to lysosome via a multivesicular body (intermediate organelle)

• maturation of the lysosome is required so must acquire proteins which facilitate sorting, transfer, transport and vesicle fusion

late endosome

• receives both the early endosome from the plasma membrane and receptors from the golgi which need to fuse with the plasma membrane

• many different sub domains which have diff target locations

• some of this trafficking done by Rab GTPases

multivesicular body

• low pH driven by proton gradient caused by ATPases

• intralumenal vesicles which shield the cytoplasmic tails of receptors so they are unable to drive signalling

• requires a specialised set of machinery to form intralumenal vesicles

receptor signalling

• activation of receptors drives vesicle formation

• controlled by receptor clustering —> this means that when receptors are clustered on the surface of vesicles they can drive signalling

• receptor internalisation can turn off signalling pathways

UBIQUITYLATION

• activated receptors at the plasma membrane are internalised and trafficked via an endosome

• activated receptors are ubiquitylated which adds a signal for receptor sequestration within an intralumenal vesicle

• receptors will then be degraded when they fuse with the lysosome

ESCRT complex

• ESCRT complex required for intralumenal vesicle transport

• identifies cargo for intralumenal vesicles

• ESCRT has binding domains for:

  • ubiquitin which interacts with ubiquitinated receptor cargo

  • also contains interaction for PIP3

• multiple ESCRT proteins shape the membrane to form an invagination for the budding of the intralumenal vesicle

• note this forms a negative membrane curvature which requires this different set of machinery as opposed to the adaptor proteins

• selectivity of vesicle formation is driven by different phospholipids which are present in the membrane

ESCRT proteins

• form tetramers or dimers depending on the proteins

• ESCRT 1 and 2 help cluster the cargo once its been recognised

• ESCRT 3 and Vsp 4 help with budding and excision

• ESCRT 3 is a cytosolic monomer but forms a filamentus structure

• VSP4 is a cytosolic monomer but is a transient hexamer

• Vsp4 is an ATPase and wraps around ESCRT 3 to form the inward budding

• ATP hydrolysis of Vsp 4 will then cause disassembly of the complex

• this results in a uniform vesicle in which receptors are unbound to their cargo

viral shedding using ESCRT

• virions use ESCRT/ Vsp4 machinery

• also useful during cytokinesis

• this is useful for when the membrane must be cut from the outside

autophagy

• cytoplasmic degradation pathway

• housekeeping function that sends cytoplasmic cargo to the lysosome for turnover

• cytoplasmic contents are soluble and not memb bound

• always ends up in a lysosome

• requires an autophagosome

2 FORMS:

nutrient starvation

• Active pathway during starvation or stress areas of the cytoplasm turned over to harvest AAs

• mtorc acts as an energy sensor

• is AAs low or ATp low mtorc inactivated which causes activation of autophagy

• captures random peices of cytoplasm to stay alive

selective pathway

• take up organelles when they produce damage signals

• depends on the cargo selected how the signals put on

• multiple ubiquitins in specific conformation is recognised by autophagy receptors

• autophagy receptors bind to the ubiquitin or LC3 protein and select for damaged cargo like this

• can take muliple forms such as damaged cell components, tau proteins, damaged organelles

LC3

• plays a role in the selective pathway

• present in the cytoplasm but becomes lipidated and can insert in memb

• acts as a platform for other proteins to bind which may be involved in cargo recognition or transport or curving the membrane

autophagosome

• double membrane bound

• turnover bulk portions of cytoplasm or target specifc structures

• derived from other intracellular membranes

• eventually gets sealed, pH lowered and transported to the lysosome

MATURATION

• must fuse with other organelles, some of which are in the endocytic pathway

• reduction in internal pH is required for fusion

• after closure and fusion w endosomes and MVBs —> forms an amphisome