Cell Bio - Vesicle transport Flashcards
what is the secretory pathway
flow of membrane bound and soluble proteins destined for certain organelles or extracellular space
what is the endocytic pathway
plasma membrane capture of extracellular components and internalisation of membrane proteins into vesicles for recycling or degradation
purpose of coat proteins
Provides shape to membranes to “curve” and bud
Determine the size and shape of the vesicle
Concentrate the protein in the vesicle
Provide selectivity for the “cargo”
Determine the vesicle’s destination
where do clathrin coated vesicles go from and to
trans-Golgi network (TGN) to endosome and plasma membrane (via endocytosis)
where do COPI and COPII coated vesicles go from and to
COPI - Golgi complex to the ER (retrieval)
COPII - ER to Golgi
how does a protein associate with the plasma membrane via a helix
protein forms an amphipathic α-helix within the cytosolic face that anchors it
how does a protein covalently and and non-covalently associate with the plasma membrane
covalent - Covalent attachment of lipid group – fatty acid or prenyl group
non-covalent - Non-covalent interactions with other membrane bound proteins
what forms non-covalent bonds with proteins in the plasma membrane
peripheral proteins
structure of clathrin and where is it formed
subunits made of 3 heavy and 3 light chains - assemble to form triskelions
formed at the trans-Golgi network/plasma membrane
clathrin forms an outer protein lattice
what are the various types of endocytosis
receptor-mediated endocytosis
phagocytosis
pinocytosis
during endocytosis what is required for clathrin recruitment and coat formation
recruitment of AP2 adaptor protein
what is the purpose of AP2 adapter protein
binds to specific phospholipids results in conformational change that allows binding to cargo receptors on cell surface, triggers membrane curvature
structure of AP2 adapter protein
heterotetrameric, multi sub-unit:
α-adaptin
β2-adaptin
σ2-chain
µ2-chain
what is required for AP2 adaptor protein to enter its open conformation and exposes the clathrin binding site
clathrin binding site is buried in AP2 in locked, soluble state
binding to PIP2 on membrane exposes the clathrin binding motif in β2-adaptin
leads to AP2 open conformation
how does μ2-adaptin facilitate clathrin coat assembly
μ2-adaptin interacts with cargo which stabilises AP2 complex open conformation
thus aids in cathrin binding
what is the function of dynamin and what does it require
assists in vesicle and budding formation
requires GTP
what happens to the clathrin coated vesicle once it separates from the membrane
the clathrin coat dissociates immediately and the components are recycled
leaves behind a naked vesicle that is transported to its destination
structure of dynamin and what does it do in the presence of GTP
dynamin oligomerises to form a helical ring around the neck of the bud, recruits other proteins, and tethers itself to the membrane through lipid binding domains
dynamin constricts in the presence of GTP
what does GTP hydrolysis of dynamin result in
GTP hydrolysis of dynamin results in the lengthwise extension of helix, and fission of membrane
how are acid hydrolase enzymes modified to bind to what
N-glycosylated and phosphorylated by mannose-6 in Golgi
allows binding to M6P-receptor and trafficking to lysosome
structure of COPII protein
has 5 subunits
associated GTPase (SAR 1)
location and function of Sar-1 GEF
embedded in the donor membrane
recruits and activates Sar1 - loading with GTP
function of Sar1-GTPand what does it lead to
recruits Sec23/24 which interacts with cargo forming an inner coat
function of Sec13/31
forms the outer coat
what do ER proteins have at their C-terminus and what is it recognised by
KDEL
recognised by KDEL receptors in cis-golgi
what is required for coatemer recruitment and what activates it
ARF1 GTPase is required for coatomer recruitment,
activated by Golgi-localised GEF proteins
what do vesicles originating from plasma membrane require
Rab5
what is acquired from vesicle transport and maturation
Rab7
function of GDI
keeps Rab inactive in cytosol
outline Rab-GTPase activation
GDF – GDI displacement factor → displaces GDI from GDP bound form of Rab, thus allowing membrane anchor with its hydrophobic prenyl group
GEF mediated GDP to GTP exchange triggers a conformational change in the Switch 1 and 2 regions of Rab allowing interactions with effector proteins
what does activation of RabA-GEF to membrane lead to
locally activates RabA
function of RabA
activates effector proteins
RabB-GEF
what does activation of RabB by RabB-GEF lead to
activation of RabB effector proteins
RabA-GAP
function of RabA-GAP
inactivates RabA
each V-SNARE protein on the surface of the vesicle has a corresponding what
corresponding T-SNARE protein on the surface of target membrane
what is v/t-SNARE protein docking initiated by
Rab-GTPase
what is step 1 of vesicle docking
Rab-GTP protein on vesicle surface binds to specific Rab-effector in target membrane
This brings v-SNAREs and t-SNAREs into close proximity -allowing docking
what is step 2 of vesicle docking
a-helices of v-SNARE and t- SNARE form coiled-coils (trans-SNARE complex)
exerts inward force that brings the two membranes close together
step 1 of membrane fusion
Lipid bilayers fuse by flowing into each other after being forced into close proximity
step 2 of membrane fusion
A complex of two proteins (NSF and a-SNAP) binds to the “empty” SNARE complexes (cis-SNARE complex)
step 3 of membrane fusion
ATP hydrolysis (catalysed by NSF) causes disassembly of the SNARE complexes and recycling
outline the mechanism of coordinated synaptic membrane fusion and neurotransmitter release
Synaptic vesicles dock at presynaptic plasma membrane, with complexin keeping the trans-SNARE complex in a primed position
Calcium induces a conformational change in the complex allowing coordinated vesicle fusion with plasma membrane leading to neurotransmitter release
what do multivesicular body’s (MVB’s) contain
intraluminal vesicles
lower pH
what is the purpose of multivesicular body’s (MVB’s)
shield receptors from the cytosol - turns off potential transduction signals
is a method of control of signals via internalisation
what happens as a consequence of ligand-receptor internalisation
results in the degradation of the receptor
inactivation of signalling cascade
what is required for intraluminal vesicle formation
ESCRT (endosomal sorting complexes required for transport)
what does ECRT-0 contain
ESCRT-0 contains ubiquitin binding domain which interacts with ubiquitylated receptor cargo
ESCRT-0 also contains binding domain for interaction with PI3P rich phospholipid on endosomal membrane
what does the IP3 in early endosome act as
acts as a binding site for effector proteins
what do ESCRT-0/I/II bind to
complexes bind to ubiquitylated cargo and the membrane phospholipid PI3P
what is necessary for budding and scission
ESCRT’s-III and Vps4’s
what is Hrs
Hrs is an ESCRT-0 protein that interacts with ubiquitin on cargo
what is VPS4 (vacuolar protein sorting-associated protein 4)
is an ATPase that hydrolyses ATP to disassemble ESCRT complex allowing intraluminal vesicle to form
what is the machinery required that can select cargo to be captured by autophagy and non-selective capture of cytosol during starvation
Atg8
Atg5-Atg12-Atg16 complex
what is Atg8
Atg8 (also known as LC3) is a membrane protein that decorates inner and outer leaflets of autophagosome
how is an amphisome formed
Following closure of the autophagosome, there are fusion events with endosomes and MVBs to form an amphisome
what does formation of an amphisome lead to
results in a gradual reduction in internal pH and acquisition of machinery to facilitate fusion with the lysosome (e.g. SNARE components)
to form an autolysosome, resulting in proteolytic degradation of components
what is non-selective autophagy used for
During nutrient starvation leading to low ATP levels or low amino acids, autophagy is activated resulting in the removal of bulk cytosol for harvesting of amino acids required for protein synthesis and energy production
how are damaged cargo recognised by autpphagy receptors
they are decorated with polyubiquitin
what do endocytic vesicles and clathrin coats also recruit
actin-nucleation promoting factor
what is WASP
nucleation promoting factor that activated Arp2/3 complexes
in terms of actin what does Arp2/3 promote and what does this lead to
Arp2/3 promotes actin polymerisation which drives internalised vesicles away from the plasma membrane
how is WASP stored
WASP is held inactive in cytosol through intramolecular interaction that masks WCA domain
how is WASP activated
interaction via GTPase through RBD
what happens when WASP is activated
intramolecular interaction is relieved and W domain is exposed to bind actin and the A domain activates Arp2/3
what is the angle between new and old fialment
70 degrees
what does listeria possess instead of WASP
ActA
what is required for a cell to migrate in the forward direction
Arp 2/3 activation and formation of branched actin at leading edge promotes membrane protrusion
how much does the myosin head swing when moving an actin filament
30-40nm
structure of golgins
Golgins are large proteins (over 30 genes), with coiled-coil domains adopting a rod-like shape
features of golgins
Golgins involved in transport and vesicle tethering around regions of the Golgi
Act as Rab effector proteins
Golgins interact directly with microtubules, with microtubule associated proteins or microtubule motors, such as dynein
Contribute to Golgi positioning and morphology
what does a loss of dynein lead to in terms of lysosomes
Lysosomes are positioned in perinuclear regions - loss of dynein leads to a dispersal of lysosomes throughout the cytoplasm
structure of dynein
Cytoplasmic dynein complex contains a pair of identical heavy chains (homodimer)
Dynein heavy chain has an ATP-dependent motor (head), Microtubule binding stalk region, and N-terminal stem that binds cargo or adaptors
N-terminal stem interacts with intermediate and light chain proteins
what does each dynein motor head contain
Each motor head domain contains a hexameric AAA ring – that has stalk, buttress, and linker regions protruding from AAA ring
what is the size of each dynein step
8nm
function of dynactin
a large complex linking dynein to cargo and regulating dynein activity
This complex can interact with a range of adaptor proteins, thus providing specificity for different cargo
what are melanophores
cells in the skin that contain melanin-filled pigment granules called melanosomes
what are melanosomes transported by
transported by kinesin-2 during dispersal, also tethered in the periphery by myosin actin motors (myosin V)
what is responsible for melanosome aggregation
dynein-dynactin motors
what regulates dispersion and aggregation of melanosomes
intracellular cAMP levels
