Topic 7: Endomembrane System Flashcards
What is the endomembrane system?
the set of organelles interconnected via vesicles
How are proteins targeted to each of the destinations in the endomembrane system?
translation of all proteins starts in cytoplasm
sorting signals that all localization
What are sorting signals?
all translation begins in the cytoplasm on free ribosomes
a continuous stretch of amino acid sequence, typically 15-60 amino acids long
What is the ER signal sequence?
generally at N terminal, contains block of hydrophobic amino acids
What is the nuclear localization signal sequence?
one or two short sequences with positively charged amino acids
What is the mitochondria/chloroplast signal sequence?
organelle specific signals at N terminal
What is the ER lumen “retention” signal sequence (KDEL)?
this four amino acid sequence causes proteins to be kept in the ER lumen “retention signal”
What is the secretory pathway?
proteins translated by bound ribosomes are either destined for the ER or for other compartments within the endomembrane system
one such destination is the plasma membrane for secretion
a good example of this type of transport is neurotransmitter release into the synapse after an action potential
once a protein is in the endomembrane system they never see they cytoplasm again
What are the steps in the secretory pathway?
translation by bound ribosomes in ER
from ER, vesicular transport moves cargo and membranes along the secretory pathway
proteins destined for secretion move: ER –> Golgi –> PM
What is the pulse-chase experiment?
method for determining location of radioactively labelled compounds
fed living cells radioactive amino acids “the pulse”
fixed the cells for TEM, cut sections, after various timepoints after pulse
dipped sections on grids into photographic emulsion, wait while decay events expose film
develop photographic emulsion
see pots on areas containing radioactivity
do “pulse-chase”, give pulse of radioactivity then a time period (“chase”) without radioactivity
What are Sec mutants?
yeast (Saccharomyces cerevisiae) with mutations in key proteins
mutants blocked in different steps of ER-Golgi-PM transport
GFP-secretory protein fusions tracked with confocal “green fluorescent protein” ER resident fusion or thin section TEM
GFP cargo fusions were put into sec mutants = mutant had accumulated of GFP in different compartments
What is the structure and functions of the ER?
controls calcium levels
site of lipid synthesis
site of protein translation and folding
cargo synthesized at ER moves to the cis-Golgi
How does cargo travel from organelle to organelle?
use vesicles
coat proteins drive the formation of vesicles
cop II, cop I, clathrin
What are Cop II coated vesicles?
ER to cis-Golgi, “coatomer”, soluble protein complex that forms coat is recruited from the cytoplasm
in vivo: Cop II molds the vesicle
spontaneously self-assemble into cage-like structures
coat proteins use adapters to associate with membrane and regulate assembly
What is the major cargo of Cop II proteins?
vesicles that move from ER –> cis-Golgi use Cop II (anterograde transport)
What are Cop I coated vesicles?
Golgi to ER recycling
possibly used in intraGolgi traffic - not known for sure
What is the major cargo of Cop I coated vesicles?
proteins that are supposed to stay in ER may accidentally be transported to Golgi (Cop I vesicles recycle back to ER)
ER resident proteins contain KDEL sequence, therefore there must be KDEL receptors in both ER and Golgi to read KDEL sequence, KDEL receptors link via adapter protein to Cop I coats
What are clathrin coated vesicles?
PM to endosomes and Trans Golgi to lysosome
clathrin has a distinctive morphology
coatomer coated vesicles (COPII and COPI) are hard to see in thin section/TEM, look like fuzzy coats instead of spiky like clathrin
once a vesicle is formed and uncoated, it must fuse with the correct target membrane
What is the major cargo of clathrin coated vesicles?
receptor-mediated endocytosis (RME)
moves cargo from membrane to endosomes (transient membrane band compartment before lysosome)
What are the steps of the formation of clathrin coated vesicles?
ligand binds to membrane associated receptors
adaptor proteins cluster receptors on a specific area of membrane
clathrin associates after ligand binding to shape the vesicle
vesicle fission needs other assoc. proteins, eg. dynamin/dynactin (wraps around neck and stretches)
clathrin dissociates once vesicle forms
What is vesicle fusion?
targeting of vesicles to specific organelles requires G proteins (Rab)
What are Rab GTPases?
docking and tethering vesicles to target membrane
many different kinds of Rab GTPases (on mature vesicle), associate with different vesicles and help direct them to the appropriate destination (not sufficient for specificity)
activated by GEF’s on the donor membrane
What are the steps of Rab GTPase docking and tethering?
mature vesicle = no coat proteins
SNARE’s allow membrane fusion
part of destination specificity Rabs can exist in GTP or GDP bound forms
tethering protein allows Rab specificity
specificity protein at target membrane
vSNARE/tSNARE combo gives more specificity
proximity of vesicle to target allow vesicle fusion
How is membrane fusion is selective?
SNARE proteins: transmembrane proteins
specificity and catalyzing vesicle fusion with target membrane
How do signal sequences on cargo proteins aide in direction and localization?
sequence tags
there is information on cargo that facilitate localization
e.g. M6P: mannose-6-phosphate tag (sorting signal for lysosome)
What are SNARE proteins?
bring membranes close together, displace water and promote membrane fusion
helical domains: v and t SNARE form twisted complexes that pull membranes together
What are v-SNARE proteins?
associated with vesicle membranes
large cytoplasmic domain
many different varieties
What are t-SNARE proteins?
on target membranes
specific to v-SNARES
What is the structure of the Golgi?
Golgi apparatus refers to all the stacks in a cell
each flattened membrane disk is called a cisterna, plural cisternae (cis/medial/trans cisternae)
each cisterna has a unique complements of proteins associated with it
facing the Golgi lumen are many membrane-bound enzymes that make and modify polysaccharides
each stack has a cis (forming) face and trans (leaving) face with a network of membranes at each face
What are the two directions of movement of material through the Golgi?
anterograde: forward movement, cis to medial to trans
retrograde: backwards movement, believed to be for recycling of escaped residents
What are the two models of how materials move through Golgi?
vesicle trafficing model
cisternal progression model
What is the vesicle transport model?
receive vesicles from ER to the cis Golgi
moves to the medial Golgi using vesicles
uses vesicles to move
observed vesicles interacting with Golgi and cisternal proteins
What is the evidence for the vesicle transport model?
there are cisternae specific enzymes
vesicles are observed associating with Golgi
What is the cisternal progression model?
each cisterna forms from the fusion of vesicles, then “matures”, along with its cargo as it moves through the stack cis to trans
whole of medial Golgi matures into trans Golgi
cargo stays inside, no vesicles
Golgi is “moving” in a way
molecules may be too big to fit in vesicles
What is evidence for the cisternal progression model?
large molecules (too big to fit in vesicles associated with Golgi) pass through Golgi (eg. procollagen)
How do both models contribute to movement of materials through the Golgi?
anterograde: use cisternal maturation, ER –> Golgi
retrograde: go back because KDEL was read wrong, use vesicles, vesicles from ER form new cis Golgi
What is the function of the Golgi?
glycosylation, packaging and sorting of glycoproteins
What is glycosylation?
covalent addition of sugars
function: cell-cell recognition, protection from dehydration and form recognition by pathogens
glycosylation starts in ER and completes stepwise through Golgi cisternae
therefore glycosylation is only found on extracellular surface or facing lumen of endomembrane organelles
How does glycosylation add oligosaccharides to proteins?
it is rare to have glycoproteins in cytoplasm
oligosaccharide processing occurs in ER and Golgi
first step: sugar tree is added in ER as the oligosaccharide is transferred off a glycolipid
What is constitutive secretion?
unregulated secretion, “housekeeping”
steady stream of vesicles to PM that every cell has all the time for housekeeping (modify, turn over PM proteins, growth and secretion of some proteins)
What is regulate exocytosis?
contents made (hormones, mucus, etc.) and bud from TGN then vesicles stored full of material close of PM
when signal comes, exocytosis occurs
secretory granules dock at the cell surface and release their contents when they are stimulated to do so (due to rise in intracellular calcium concentration)
What is the lysosomal pathway?
lysosome contain hydrolytic enzymes
proteins destined for lysosome contain M6P tag (glycosylation)
M6P receptors in TGN (collect and concentrate M6P containing cargo)
late endosome = low pH, causes activation of M6P cargo/receptor (receptors recycled back to TGN)
late endosome matures into lysosome where endocytosed cargo is degraded by M6P-containing hydrolytic enzyme
What is the endocytosis pathway?
endocytosis is the process where vesicles bud off the plasma membrane and internalize membrane and extracellular fluid contents
pinocytosis and receptor-mediated endocytosis
What is pinocytosis?
cell drinking
also known as bulk-phase endocytosis
brings about uptake of extracellular fluids without recognition by surface of plasma membrane
What is receptor-mediated endocytosis?
brings about uptake of specific macromolecules (ligands) following their binding to receptors on plasma membrane
a ligand is any molecule that can bind a receptor
What are endosomes?
network of membranous tubules
What are early endosomes?
associated with periphery of cell
neutral pH
M6P (-)
What are late endosomes?
are located more interior of the cell
lower pH
M6P (+)
What are coated pits?
sites on membrane where receptors for receptor-mediated endocytosis are concentrated
pits contain clathrin and adaptin that associated with intracellular domain of receptor
coat protein cluster receptors and forms vesicle
What are the steps of receptor mediated endocytosis?
receptor on cell surface binds to molecules to be taken up (ligand)
fuse with endosome; acidic interior causes dissociation of ligand from receptor
ligand degraded following transfer to lysosome
receptor can be recycyled
What organelles do materials pass through in the secretory pathway?
ER –> Golgi –> PM
What organelles do materials pass through in the lysosomal pathway?
ER –> Golgi –> lysosome
What organelles do materials pass through in the endocytosis pathway?
PM –> endosome –> lysosome
