2.3 Vesicular Transport Flashcards
vesicular transport
mediate exchange of components between compartments
clathrin
mediate endocytosis from plasma membrane to endosome to lysosome (bring nutrients in for cell)
COPI
mediate vesicular transport from Golgi to ER
COPII
mediate vesicular transport from ER to Golgi
anterograde pathway
ER to Golgi, mediated by COPII vesicles
retrograde pathway
Golgi to ER, mediated by COPI vesicles
LDL receptor
bring in LDL particles (contains nutrients such as cholesterol) into the cell
Stage 1: LDL Receptor Cycle (4 steps)
Vesicle formation
1) cargo (LDL) binds to cargo receptor (LDL receptor) on exterior of PM
2) adaptor proteins and Arf bind to cytosolic side of the cargo receptor (forms binding site)
3) coat assembly - clathrin binds to binding site of AP+Arf
4) vesicle forms
Arf
small monomeric GTPase that forms a binding site for clathrin with adaptor proteins (AP) during endocytosis
how is a clathrin coat formation possible (special characteristic of clathrin)
clathrin self polymerizing
- forms a triskelion that spontaneously self-assembles into polyhedral cage
Stage 2: LDL Receptor Cycle (5 steps)
Fission and Uncoating
1) dynamin assembles as ring around bund
2) GTPase domain of dynamin regulates pinching off of vesicle
3) non-cytosolic leaflets of membrane to fuse together
4) dynamic recruits other proteins to budding vesicle to bend patch of bilayer
5) Arf GTPase: vesicle rapidly loses clathrin coat
Rab
large and diverse subfamily of small monomeric GTPase
- recognizes naked vesicle
- important for vesicle trafficking
how is it ensured that the endocytosis vesicle is transported to the right location
depends on the type of Rab that is recruited (specific Rabs for different organelle targets)
Rab structure
small, has:
- GTPase (GDP in free state)
- amphipathic helix
Binding Mechanism for Rab
1) Free Rab (GDP form) recognized by Rab-GEF on donor membrane)
2) GDP phosphorylated to GTP
3) Rab becomes membrane bound (amphipathic helix inserts into the outer membrane)
how are Rabs selectively distributed at the membrane
PiP (inositol lipids)
where are the kinases and phosphatases for converting PiP located
on the cytosolic side of the organelle (some are integral and some are peripheral membrane proteins)
Stage 3: LDL Receptor Cycle
Recruitment of Rab GTPase to the vesicle
Stage 4: LDL Receptor Cycle (4 steps)
1) tethering - membrane Rab proteins on the vesicle binds to the Rab effectors on target membrane
2) docking - complementary SNAREs on the vesicle and target membrane pair together as vesicle approaches the membrane. A force is applied
3) fusion - as the SNAREs twist together, the two bilayers fuse together (separate layers>stalk>hemifusion>fusion pore)
4) synaptotagmin finishes process
synaptotagmin
activated by Ca2+, binds to SNARE complex and causes fusion clamp to tighten further and creates additional disturbance in lipid bilayer and finishes the vesicle fusion process
tetanus
neurotoxin that cleave SNARE proteins in nerve terminals (very, very toxic, LD50=1 ng/kg)
botulism
neurotoxin that cleave SNARE proteins in nerve terminals
Stage 5: LDL Receptor Cycle (4 steps)
Recycling of SNAREs
1) SNAREs are separated by NSF and a-SNAP
- NSF (wrench): ATPase that hydrolyzes ATP to catalyze dissociation of SNARE pairs
- a-SNAP (socket): soluble NSF attachment protein
NOTE: like a wrench system
what is needed to dissociate SNARE pairs
1) NSF
2) a-SNAP
3) ATP
what is defective 34
accumulation in cytosol
what is defective 34
accumulation in ER
what is defective 34
accumulation in ER to Golgi transport vesicles (COP1, COP2, VTC, KDEL)
what is defective 34
accumulation in Golgi (Golgi, Secretory Vesicles)
what is defective 34
accumulation in secretory vesicles
VTC
vesicular tubular cluster
- mediates transport from ER to to Golgi
homotypic fusion
fusion of membranes from same compartment, forms vesicular tubular clusters
VTC steps (4)
1) vesicles bud off from ER exit sites and shed COPII coat
2) homotypic fusion of vesicles to form vesicular tubular clusters
3) vesicular tubular clusters move along microtubules with help of motor proteins
4) bud off process of COPI coated transport vesicles that carry back resident ER proteins and cargo receptors
describe the mechanism for COPII vesicle formations
very similar process to clathrin coat/assembly and disassembly
homotypic fusion steps (3)
1) NSF with ATP unwinds the SNARE bundles on the vesicles
2) SNAREs bundle together and pull the vesicles together
3) vesicle membrane fusion
how are proteins packaged into vesicles (general)
selective process
1) proteins recognized by receptors
2) receptors recognized by adaptor proteins
3) adaptor proteins are recognized by COPII
4) coat assembly
why is the retrograde pathway important (from Golgi to ER)
- sometimes proteins with exit proteins (resident ER proteins) sometimes randomly enter the vesicle and needs to be returned
what are the ER retrieval signals and the proteins that are associated with them?
1) KKXX @ C-term: for resident ER membrane proteins
2) KDEL: soluble ER protein
KKXX
ER retrieval signal for ER membrane proteins that have been accidently taken away by COPII vesicles during anterograde transport
- binds directly to COPI coats
KDEL
ER retrieval signal for soluble ER proteins that have been accidently taken away by COPII vesicles during anterograde transport
what do ER resident proteins bind to?
KDEL receptor on cis-Golgi network with their KDEL ER signal sequence
how is the affinity of the KDEL receptor for KDEL sequence regulated
pH
- binds protein with KDEL sequence at low pH (Golgi)
- releases protein with KDEL sequence at high pH (ER)
how is the pH regulation of the KDEL sequence to KDEL receptor useful
prevents the KDEL sequence from interacting with the KDEL receptor in the ER where the binding is not necessary
describe the faces of the Golgi stack
cis face (entry) and trans face (exit)
what are the Golgi called and how are they different from each other?
cisterna, contain characteristic set of processing enzymes
how are the resident ER proteins associated with the Golgi apparatus and why is this desired?
Golgi resident proteins are all membrane bound. This makes retrieval easier because it can be regulated via the COPII mechanism
what are the characteristics of the constitutive secretory pathway
unregulated membrane fusion
- can release the newly synthesized soluble proteins right away to environment
what are the characteristics of the regulated secretory pathway
requires a signal (hormone/neurotransmitter)
- triggers secretion of the secretory proteins that are being stored in secretory vesicles
how are lysosome proteins regulated
by pH. synthesized as proenzyme and requires an acidic environment for activation (pH 4.5-5.0)
how is the environment of the lysosome acidic
vacuolar H+ ATPase uses ATP to pump H+ into lysosome
why does the membrane of the lysosome not just break down to it’s own enzymes?
the membrane is highly glycosylated to protect itself from its own proteases and lipases
M6P targeting steps
1) lysozyme proenzyme with a mannose is brought to the cis Golgi network
2) P-GLcNAc is added
3) M6P signal is uncovered
M6P targeting steps
1) lysozyme proenzyme with a mannose is brought to the cis Golgi network
2) P-GLcNAc is added onto mannose residue with GlcNAc phosphotransferase
3) M6P signal is uncovered (GlcNAc removed via GlcNAc hydrolase
4) M6P binds to M6P receptor at the trans Golgi network
5) receptor-dependent transport to early endosome
6) acidic pH dissociates the proenzyme from the M6P receptor
7) phosphate is removed from the lysosomal proenzyme
8) M6P receptor is recycled and brought back to the trans Golgi network
describe lysosomal storage disease
GlcNAc phosphotransferase in the cis Golgi network is defective
- lysosomal hydrolases are not tagged in the cis Golgi network and then not recognized by the M6P receptors in the trans Golgi network
- hydrolases are secreted at the cell surface instead of getting transported to lysosomes
- lysosome has no lysosomal hydrolases so the lysosomal substrates accumulate in the lysosome
P-GlcNAc definition
GlcNAc phosphotransferase
- adds GlcNAc-phosphate to mannose residues onto lysosomal proenzymes
I cells
inclusion cells, undigested substrates accumulate in lysosomes
what is the default secretory pathway
Golgi to extracellular space
what are the 3 pathways for bringing materials to the lysosome
1) intracellular traffic
2) autophagy
3) phagocytosis
describe intracellular traffic pathway of delivery of materials to lysosomes
1) macromolecules taken up from extracellular space via endocytosis
2) endosomes mature to lysosomes
describe autophagy pathway of delivery of materials to lysosomes
degradation pathway of parts of cells
1) formation of autophagosome (double membrane forms around whatever)
2) autophagosome fuses with lysosome/late endosome
3) thing digested and metabolites are derived
describe phagocytosis pathway of delivery of materials to lysosomes
digestion of large particles and microorganisms
1) macrophages and neutrophils engulf objects to form phagosome
2) phagosome fuses with lysosome
3) thing digested
how are late endosomes formed
endocytosis
how are endo-lysosomes formed
late endosomes fuse with pre-existing lysosomes
how are lysosomes formed
endo-lysosomes fuse with each other
how are components in early endosomes digested (4)
1) early endosome with stuff inside
2) sequestration (formation of invaginating buds) to form internal vesicles
3) multivesicular bodies fuse with late endosomal compartment
4) further acidification of late endosome activates the lysosomal enzymes
shape/charge of PC
cylindrical, neutral
shape/charge of PE
conical, neutral
shape/charge of SM
cylindrical, neutral
shape/charge of PS
cylindrical, negative
shape/charge of PI
cylindrical, negative
what are lipid-packing defects and where do they arise from
when the lipids aren’t stacked nicely with each other
- shape of lipids not cylindrical (PE and DAG are conical)
- unsaturated double bonds introduces kinks
shape/charge of DAG
conical, neutral
describe the packing of lipids in the ER and what can this be attributed to
loose packing
- unsaturated phospholipids
- low sterols
describe the packing of lipids in the PM and what can this be attributed to
tight packing
- saturated lipids
- high sterols
how are electrostatics related to packing defects
generally less packing defects = high electrostatics
how can the electrostatics of different membrane territories in the cell explained
by the degree of packing defects in the membrane (more defects = less electrostatics)
