Protein trafficking Flashcards
anterograde transport
forwards
ER to golgi to plasma membrane
retrograde pathway
eg golgi to ER
endocytic pathways
cell exterior to early to late endosome
how molecules move from one organelle in the endomembrane system to another
vesicle buds from donor compartment, pinches off and translocates to acceptor compartment. this is a scission event. vesicle docks with acceptor membrane; targeting event. vesicle fuses with acceptor compartment and releases its contents into the lumen
how are buds formed/what triggers their formation?
coat proteins bind to specific sequences on cytoplasmic tail of transmembrane proteins acting as receptors. a cluster forms due to the affinity of coat proteins to eachother and the receptors. cargo is selectively recruited into buds as they form.
clathrin coat budding event
stalk-like structure
scission occurs by dynamin (GTPase) that constricts its neck
clathrin coated vesicle detaches from membrane then uncoats and the naked vesicle fuses with target
structure of clathrin coat
triskelion
trimer: 3 heavy chain subunits and 3 light chain subunits
assemble into cages via protein-protein interactions and deform membrane into a sphere
where does budding associated with clathrin occur
from the golgi apparatus and plasma membrane
differences in adaptor proteins involved
buds used for golgi retrograde trafficking
COPI
protein used for buds for anterograde transport from ER
COPII
clathrin coated vesicles (1): coat proteins, origin and destination
clathrin and adaptin 1
golgi body
lysosome, via endosomes
clathrin coated vesicles (2): coat proteins, origin and destination
clathrin and adaptin 2
plasma membrane
endosomes
COP coated vesicles: coat proteins, origin, destination
COP proteins
ER, golgi cisternae, golgi apparatus
golgi apparatus, golgi cisternae, ER
bulk sorting
some molecules are passively included in vesicles (not selected)
how is cargo selected
active recruitment
selective exclusion
passive inclusion
what controls coat formation
coat recruitment GTPases
activation of SAR-1
Sar1 is a small GTPase that is initially in its inactive, soluble form bound to GDP (Sar1-GDP) in the cytosol.
Sar1-GDP interacts with a guanine nucleotide exchange factor (Sar1-GEF) located on the ER membrane. This interaction facilitates the exchange of GDP for GTP, converting Sar1 to its active form (Sar1-GTP).
The active Sar1-GTP exposes an amphiphilic helix that allows it to insert into the ER membrane.
cargo selection and vesicle formation
Sar1-GTP recruits coat protein complex II (COPII) subunits to the ER membrane. COPII subunits include proteins such as Sec23/24.
Sec23 has an affinity for the Sar1 protein, which is anchored in the ER membrane in its active form (Sar1-GTP).
Sec24, another component, binds to specific tails on cargo receptors or integral membrane proteins that are destined to leave the ER. This ensures that the right cargo is selected for transport.
Recruitment of Outer Coat Proteins:
Outer coat proteins, such as Sec13/31, are recruited because of their affinity for the inner coat proteins (Sec23/24). This interaction begins to deform the ER membrane, forming a vesicle bud.
Selective Recruitment by Integral Membrane Proteins:
The integral membrane proteins that are bound to the coat help selectively recruit the coat to ensure specific cargo is included in the vesicle.
Cargo receptors have a specific affinity for cargo molecules, guiding them into the lumen of the budding vesicle
how is the coat removed so that the vesicle can fuse with the golgi membrane
hydrolysis of GTP on Sar 1
conformational change
retracts amphipathic helix
loses affinity for inner coat
inner coat drops off, followed by outer coat
DXE motif
on cargo receptor
binds to sec 24
D=aspartic
X=any
E=glutamic
diacidic motif
protein exclusion from budding vesicles
by chaperones, BIP
keeps proteins in ER lumen
eg antibodies until correctly assembled: BIP binds to heavy chain, retaining unassembled antibody in ER. it cannot bind to a fully assembled antibody
retrograde transport: returning molecules to the ER from the golgi.
ER resident proteins have a KDEL sequence at their C-termini (lysine, aspartic acid, glutamic acid, leucine).
these are retrieved in COPI vesicles
KDEL recognition by golgi
KDEL receptors (transmembrane protein)
selectively packaged into COPI coated vesicle
in ER, KDEL receptor released KDEL containing protein and is returned to golgi via COPII vesicles.
how are vesicles targeted to the correct membrane?
Rab proteins (small GTP binding proteins)
SNARE proteins
give vesicles and target membranes a molecular identity
tethering
Vesicle with Cargo: A vesicle carrying cargo approaches the target membrane. On its surface, there’s a protein called Rab-GTP.
Rab-GTP Interaction: The Rab-GTP protein on the vesicle interacts with a tethering protein (Rab effector) on the target membrane. This interaction pulls the vesicle closer to the target.
docking
Closer Positioning: Now that the vesicle is near the target membrane, it uses v-SNARE proteins on its surface.
SNARE Interaction: The v-SNARE on the vesicle interacts with t-SNARE proteins on the target membrane, positioning the vesicle for fusion.
SNARE PAIRING
SNARE driven fusion
water is forced out as the membranes are brought close together. stalk formed, then hemifusion then complete fusion
BoTox
produced by bacteria
cleaves SNARE proteins involved in neurotransmitter release
how are the SNARE complexes disassembled
NSF uses ATP hydrolysis to disassemble SNARE complexes
v-SNARES recycled
distribution of enzymes in the golgi
early acting enzymes in cis cisternae
late acting enzymes in trans cisternae
modifications to some proteins in the golgi that are trafficked into lysosomes
modified by mannose 6 phosphate
sorting to the lysosome in the cis golgi
N-acetylglucosamine phosphotransferase in vis golgi binds to the acid hydrolase being trafficked to the lysosome. the hydrolase has an N-linked oligosaccharide with terminal mannose residue. The phosphotransferase also binds UDP-GIcNAc. GIcNAc-phosphate is added to mannose residues.
sorting to the lysosome in the trans golgi
GIcNAc is cleaved off, leaving mannose 6 phosphate which binds to M6PR (receptor). clathrin coat forms etc
