Chapter 8 Flashcards
What are the two main transport routes?
exocytic pathway (secretory pathway) and endocytic pathway
New proteins destined for any locations within the endocytic/exocytic pathway must be first targeted to where?
ER
What are the two ways to exit the ER?
a. to fail to fold properly, retrograde transport out to be ubiquitinated and proteasomed
b. to exit via budding into a transport vesicle
What compartments form during endocytosis?
Endosomes
Transport has to be ___________ lest the donor compartments cease to exist
bi-directional
What mechanisms return some vesicle components to the donor compartments?
Recycling mechanisms
What mechanism returns resident proteins which escaped from their donor compartments?
Salvage mechanisms
When is pulse-chase experiment used?
To experimentally show the pathway that proteins take as they move through the secretory pathway
What is regulated selection?
when cells accumulate proteins to be secreted in vesicles near the plasma membrane releasing them upon stimulation
Regulated secretion is aka
inducible secretion
What are 3 examples of regulated secretion?
a. digestive enzymes (pepsinogen, trypsinogen)
b. hormones (insulin, ADH)
c. histamine
What is constitutive secretion?
when cells continuously secrete a protein
What 4 examples of constitutive secretion?
a. immunoglobulins
b. yolk protein
c. bacterial infection-promoting proteins
d. insulin
What compartment is the most abundant membrane in most eukaryotic cells?
ER
What is Golgi apparatus made of?
Golgi stack made of cis, medial, and trans cisternae
What part of Golgi is near the entry face and what part is nearest the exit face?
cis-Golgi near the entry face
trans-Golgi near the exit face
Within the Golgi stack, what are modified sequentially to highly sialylated structures?
high mannose oligosaccharides (added to proteins in the ER)
What is the function of cis-Golgi network (CGN)?
a. receive proteins from the ER export sites
b. in QC by allowing ER resident proteins that escaped to be returned
Trans-Golgi network sorts for distribution to what 3 places?
a. lysosomes
b. plasma membrane/constitutively secreted proteins
c. regulated secretion
What are the 4 endocytosis functions?
a. internalizing of nutrients
b. regulating the cell surface expression of receptors, transporters
c. uptake, recycling of EC debris
d. recovery of membrane from the plasma membrane
What is the range from least to most degradative?
early endosome, late endosome, lysosome
What are V-ATPases?
H+ ATPases that transport protons from cytosol into the organelle lumen
Why is the acidification of early endosomes important?
for the dissociation of internalized ligand receptor complexes and recycling of cell membrane receptors to the plasma membrane
Why is the acidification of late?
endosomes important?
for the delivery of lysosomal enzymes from the trans-Golgi network (TGN)
What 2 cells are professional phagocytes?
macrophages and dendritic cells
What do phagocytes do?
ingest pathogens and clear senescent or apoptotic cells via phagocytosis
RME
Receptor-mediated endocytosis
Where does transcytosis occur?
in epithelial cells lining the intestine and other body cavities
What does transcytosis allow? (2)
a. nutrient uptake via the formation of a clathrin-coated vesicle
b. allow infants to take Ig from mother’s milk by binding the Ig to gut apical receptors, transferring Ig through to the other side, and releasing the Ig into the blood plasma
What are 6 steps of vesicle-mediated transport?
- budding
- scission
- uncoating
- tethering
- tethering
- docking
- fusion
What does budding require?
requires coat and adaptor complexes, maybe PI lipids
What does scission require?
requires membrane fusion to generate a vesicle
What happens in uncoating?
coat proteins are moved and recycled
What happens in tethering?
vesicle is able to determine whether it is at the correct target
What happens in docking?
membranes are brought close enough together to fuse
Cargo selection involves binding of what to what?
protein to a cytoplasmic protein (coat protein)
Soluble proteins in the ER lumen bind or move to ?
a. bind to receptor protein in the ER membrane
b. move by bulk flow into a forming vesicles
What 2 coats are used in the exocytic pathway?
COPI and COPII
What coat is used in the endocytic pathway?
clathrin
What proteins on the vesicle form complexes with what proteins on the target organelle, thus docking the vesicle?
v-SNARE; t-SNARE
Signal-mediated movement through the endocytic or exocytic pathways requires what?
each cargo protein to contain 1 or more sorting signals for various steps along the way
Bulk flow movement through endocytic or exocytic pathways requires what?
cargo proteins to have signals to STOP in a compartment within the pathway (ER resident protein) or to divert it onto another pathway (lysosome)
Proteins to be exported (after completely folding, assembling into complexes) gather at where?
ER export sites
What is the only route of vesicular exit from the ER?
COPII-coated vesicles
What are the 3 soluble COPII components?
Sar1p, Sec23/24, and Sec13/31
What is Sar1p?
small GTPase, in cytosol is inactivate and bound to GDP
What is Sec12p?
an intrinsic membrane protein, indirectly inactivates Sar1p because it helps it get rid of its bound GDP so it can bind to a GTP
What are Sec23/24 and Sec13/31?
sets of heterodimers which are structural coat complexes
What is Sec23 also?
Sar1p-GAP (GTPase activating protein)
What is Sec23?
stimulates Sar1p to hydrolyze its bound GTP to GDP, causes uncoating of the vesicle and release of the coat subunits for recycling
What does v-SNARES bind to?
membrane-bound forms of Sar1p and Sec23/24
Where are ER-export signals in?
C-terminus
What does the ER-export signals bind to?
sec23/24
How does vesicular tubular clusters (VTCs) form?
Scissions from the ER, COPII vesicles cluster which fuses with each other at ER export sites
What signal is involved to bring back proteins caught up in bulk flow?
retrieval signal KDEL (Lys-Asp-GLu-Leu)
Where is KDEL found at?
c-terminus of most soluble ER-resident proteins
Where are KDEL receptor proteins found in?
in compartments of VTCs and CGN
What does binding of a KDEL receptor to a KDEL sequence trigger?
formation of a COPI vesicle
What does ER resident membrane-bound proteins have?
dibasic retrieval signals at their cytoplasmic tails (C or N terminal)
Where are type I transmembrane proteins?
N-terminus in the lumen (non c-face)
Where are type Ii transmembrane proteins?
C-terminus in the lumen
Signal of type I transmembrane protines
dilysine
Signal of type II transmembrane proteins
diarginine
What is COPI vesicles necessary for?
retrieval of escaped ER proteins
COPI vesicles recycle SNARE proteins from what 2 places
Golgi back to ER; TGN to CGN
What transport is COPI vesicles involved in?
CGN to TGN transport
What is the GTPase that helps coat formation for COPI vesicles?
ADP-ribosylation factor (ARF)
What is ARF-GDP?
soluble cytosolic protein that is activated by ARF-GEF1
What does ARF-GDp help?
helps GDP associate from ARF so GTP can bind
What does conformational change of ARF-GDP expose?
exposing a myristic acid at ARF’s N-terminus, which enable it to anchor into the c-face of the Golgi membrane
What does membrane-bound ARF-GTP recruit?
COPI coat complexes (coatomers)
Function of bound coatomers
helps deform the membrane during vesicle budding analogous to the action of Sec23/24 and Sec13/31 for COPII vesicle formation
What are proteins that are recycled back to the ER?
a.Type I membrane proteins with a dilysine retrieval signal in their cytoplasmic tail
b. soluble proteins with KDEL, bound to KDEL receptor, bound to coatomer proteins
What does ARF-GAP bind to and stimulate?
ARF-GTP; stimulates hydrolyzing of GTP to GDP, leading to uncoating of the vesicle
Where does retrieval of ER-resident proteins occur?
from the VTCs, CGN, and points farther along the secretory pathway
Function of selective retention
It allows a given protein to be anchored in place at its final destination
Where does selective retention of proteins operate?
Golgi
What proteins are many Golgi enzymes?
Type II transmembrane proteins
Where are N-terminus and C-terminus for type II transmembrane proteins?
N-terminus on c-face
C-terminus in Golgi lumen
What are the two models for Golgi retention?
a. Kin recognition model
b. Bilayer thickness model
How does kin recognition model explain golgi retention?
Golgi recognize each other by enzymes, which creates aggregates too large to enter a COPI vesicle
How does the bilayer thickness model explain Golgi retention?
length of membrane-spanning domain keeps protein in the Golgi because later parts of the secretory pathway have increasing cholesterol which result in increased membrane thickness
A family that are monomeric GTPases
Rab/Ypt family
Location of GDP-bound form of Rab
Cytosolic
Location of GTP-bound form of Rab
IMP with C-terminal anchor of two phenyl (geranylgeranyl) chains
Rab-GDP exists in the cytosol complexed with what?
Guanine nucleotide Dissociation Inhibitor (GDI)
The complex of Rab-GDP and GDI result in what 2 things?
a. phenyl group is isolated
b. Rab is prevented from being an IMP
What helps exchange GDP to GTP?
Rab-GEF
What helps exchange GTP to GDP?
Rab-GAP
tethers that bound-Rab-GTP binds to
Rab collectors
Steps of vesicle targeting (4)
- Rab-GEF stimulates GDP-GTP exchange; GDP is released
- Rab-GTP is incorporated into budding vesicle
- Vesicle docks and fuses with target membrane
- Rab-GAP stimulates GTP hydrolysis; GDI binds RAB-GTP
What protein mediates docking?
SNARE proteins
What two IMPs are SNAREs with the N-terminal cytoplasmic domains?
t-IMPS or acyl or phenyl-anchored IMPs
How many v-SNARE helix interact with how many t-SNARE helices?
1:3
What catalyzes the unraveling of SNARE?
NSF
NSF binds indirectly to SNAREs via?
SNAP (soluble NSF attachment protein)
The best-characterized endocytic vesicles have what two?
a. coat protein clathrin
b. 1 or more adaptor complexes
Clathrin-coat structures form a 3-legged complex aka
triskelion
What does each leg of triskelion consist of?
one heavy chain and one light chain of clathrin
The clathrin cages contain what 2 lattices?
hexagonal and pentagonal lattices
Each side of pentagon or hexagon constist of how many legs from different triskelion?
4
Structure of hexagonal and pentagonal clathrin lattices
hexagon - flat
pentagon - curvature
Scission requires what two?
GTP energy and GTPase dynamin
Function of dynamin
release clathrin-coated vesicles from the plasma membrane
What mutation helped the researchers elucidate the function of dynamin?
Shibire Drosophila mutation
What 2 chaperone proteins are involved in uncoating?
auxilin and hsp70-type of uncoating ATPase = Hsc7
Adaptors are part of what family?
AP family
AP-2 binds to what/where?
phsophatidylinositol-4,5-bisphosphate (PI4,5P or PIP2)
AP-3 binds to what organelles?
early and late endosome, trans-Golgi network, and specialized lysosome
AP-1 binds to what organelles?
late endosome and trans-Golgi network
Structure of adaptors
heterotetramers with a trunk domain
What does ears of adaptors do?
interact with clathrin and other proteins
What happens to adaptors during coat assembly?
adaptors are phosphorylated, exposing binding sites for phosphoinositide and for cytoplasmic tails of pm receptors
Cytoplasmic tails of cargo proteins are what based?
tyrosine-based or dileucine-based
Cell’s entire pm internalized in clathrin-coated vesicles every ____ hours
1-2
Within 1 min after coated vesicle formation, what happens?
scission and removal of the clathrin coat occurs
The tethering, docking, and fusion of uncoated vesicles with early endosomes is similar to that of the exocytic pathway in what way?
it requries Rabs, tethers, and SNAREs
What are the two main scenarios for internalized receptors and ligands?
a. Internalized receptors discharge their ligands in early endosomes and ligan moves to late endosomes and to lysosome
b. Internalized receptors and ligands move together to late endosomes and to lysosomes
What are the 3 examples of internalized receptors + ligands?
a. Transferrin receptor
b. LDL receptor
c. Insulin receptor (EGF receptor)
Transferrin receptor
- Internalization of iron complexed with transferrin
- Iron is released to cytoplasm due to low pH in early endosomes
- Apotransferrin (w/o iron) attached to the receptor moves to a recycling tubule off of the early endosomes
- transported in a vesicle to the cell surface
LDL receptor
- Internalization
- LDL released into the endosomal lumen
- cholesterol is released and LDL is degraded
- receptor gets into endosomal tubular extensions and is recycled to the pm
EGF receptor
- endocytosis
- ligan and receptor move to late endosomes for degradation
MVB
multivesicular bodies
What are MVBs?
late endosomes with internal vesicles
What is the maturation process?
Early endosomes to late endosomes to lysosome
Transport vesicles with new lysosomal enzymes and membrane components coming from TGN fuses to where and what happens?
fuses with late endosomes, converting them into lysosomes
Early endosomes bind to microtubule tracks accumulate in where?
perinuclear cytoplasm next to the MTOC (microtubule organizing center)
Why does ubiquitin ligase covalently attach a ubiquitin?
to mark the specific receptors for down-regulation and not for recycling
Soluble lysosomal enzymes and lysosomal t-IMPs are translated where?
in ER
Where do soluble lysosomal enzymes and lysosomal t-IMPs get to and what happens there?
get to the TGN where they are sorted via their lumenal M6P signal
Where was M6P signal generated?
in Golgi
A phosphotransferase adds the M6P to what?
selected terminal mannose residues
Terminal mannose residues are part of what?
the original N-linked oligosaccharide which was added in the ER
What is the signal for the M6P addition?
signal patch formed when protein folding brings together non-contiguous aa residues
What 2 signals do cytoplasmic tails of M6P receptors have?
tyrosine signal and dileucine signal
Tyrosine signal is recognized by
AP-1 clathrin adaptor complexes
Dileucine signal interacts with
GGA protein (golgi/gaba-adaptin/ARF)
After dileucine signal is recognized, GGA hands over what?
hands over M6P receptor and clathrin to AP-1 complex on vesicle
What keeps lysosomal enzymes from degrading molecules prior to their arrival in late endosomes/lysosomes?
a. inactive at higher pH
b. some synthesized as proenzymes, requiring proteolytic cleavage to be activated
c. once activated by lower pH, some are phosphatases that cleave M6P sorting signal so they cannot go retrograde to the TGN along with the M6P receptors
TGN is responsible for sorting proteins into vesicles destined to where?
apical and basolateral membranes
What signals do basolateral use in their cytoplasmic domains?
tyrosine and dileucine-based signal
What signals do apical membrane-destined proteins use in their cytoplasmic domains?
No specific signal
What do apical membrane-destined proteins have in their lumenal domain?
N- or O- linked sugars and GPI anchors
What is recycling endosomes capable of?
sorting molecules
What does regulated secretion use from the TGN?
secretory granules
What is the signal for secretion?
secretagogue
A Ca2+ binding protein that is a key player
synaptotagmin
What does synaptotagmin regulate?
regulates vesicle release from synaptic vesicles by binding to SNARE pairs and holding them in an inactive configuration
A nerve impulse triggers the release of Ca2+, which causes
change in conformation of synaptotagmin, causing to let go from SNARE complexes, allowing membrane fusion
Secretion of proteins enter the ER or Golgi without the ER-Golgi pathway via what?
ABC transporter
ABC transporter binds protein on where?
c-face
What are some proteins that move by ABC transporter?
interleukins, fibroblast growth factor (FGF)
GRASP
Golgi-membrane associated protein
GRASP grabs protein from where?
c-face
Overexpression of MDR protein in some cancer cells allows them to be
resistance to several cytotoxic drugs
What resistance is the chief cause of malaria?
chloroquine-resistance in Plasmodium falciparum
What causes a spastic paralysis?
tetanus toxin
What causes flaccid paralysis?
botulinum toxin
