BIOMG 1350
Professor Huffaker
Fall 2016
Week 7: Lecture 2 of 2
Wednesday, October 5th
Lecture: Vesicular Transport
Prelecture:
iclicker question: Imagine that you have engineered a gene that encodes a protein containing two
signal sequences, one for import into the nucleus and one for import into the ER. If this gene is
expressed in a cell, where do you predict that the protein will localize? Answer: B) In the ER
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The ER is the entry point for proteins destined for a variety of locations ●
“Roadmap” of vesicle traffic
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ER is throughout the entire cytosol
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proteins travel through the Golgi apparatus and can go to two places, through endocytosis (lysosome) or exocytosis (transport vesicles)
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transport between ER and Golgi, etc., is mediated by transport vesicles that are small,
membrane bound organelles that transport material
Topology of vesicle budding and fusion ○
start with donor compartment (red dots are soluble proteins) (green lines are transmembrane proteins)
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the circular shape represents budding, eventually will bud off the vesicle with the proteins it needs
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it will then fusion with target compartment and put membrane bound proteins into membrane ○
Soluble proteins (red dots) are never in cytosol and same goes for transmembrane proteins (green lines)—the lumen and cytosolic sides will always remain there
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Lumen of the ER, Golgi, endosomes and lysosomes are topologically equivalent to the
outside of the cell Lecture: (3 questions to consider):
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How do vesicles bud and fuse? ●
How is correct cargo taken up by each vesicle?
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How is the correct target membrane identified? ●
Coat proteins help shape membranes into vesicles
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fuzzy shapes suggests that there are proteins surrounding the vesicles ○
coated pits refer to the indentation ○
see table 15-4 for table of some types of coated vesicles ■
Clathrin-coated vesicle formation □
1) Selection of cargo to be transported is mediated by transport receptors
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2) Adaptins capture cargo receptors by binding their cytoplasmic tails
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3) Coat proteins bind adaptins to start formation of a vesicle (called coated pits)
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4) Dynamin wraps around the vesicle neck to complete pinching off □
5) Vesicle is then released from mebrane □
6) Soon after, coat comes off after vesicle release (they will create a nake transport vesicle) □
So, the entire result is to transport the soluble proteins.
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Transmembrane proteins can interact directly with adaptins because they do not
need cargo receptors to be transported
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There are proteins that are on the vesicles that complement the target proteisn
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2 main important ones that direct transport vesicles ●
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Rab proteins
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SNARES play a central role in membrane fusion
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interacts with tethering proteins □
a Rab protein with GTP bound associates with vesicles □
target membrane contains a tethering protein and a t-SNARE
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the vesicle includes a v-nare □
v-SNARE interacts with t-SNARE on target membrane □
There is a specific Rab protein/tethering proteins and v-Snare/t-SNARE for each
transport step
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SNARES play a central role in membrane fusion
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the 2 bilayers must come within 1.5 nm of each other to allow membrane fusion
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water must be displaced from hydrophilic groups—energetically unfavorable □
they move like wenches □
after fusion, the SNARES are pried apart in a reaction that requires ATP because
they are originally so tightly attached to each other ○
iClicker Question: Your friend has just joined a lab that studies vesicle budding from
the Golgi and has been given a cell line that does not form mature vesicles. He wants to
start designing some experiments but wasn’t listening carefully when he was told about
the molecular defect of this cell line. He’s too embarrassed to ask and comes to you for
help. He does recall that this cell line forms coated pits but vesicle budding and the removal of coat proteins don’t happen. Which of the following proteins might be lacking
in this cell line?
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Answer: C) Dynamin
The secretory pathway
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many proteins are glycosylated in the ER
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Lipid-linked oligosaccharides are attached to dolichol by 2 phosphates
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this lipid is transferred (via oligosaccharyl transfererase) ■
this is then called N-linked oligosaccharide
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protein-linked oligosaccharides never reside in the cytosol
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The Golgi apparatus is made of a stack of flattened, membrane enclosed sacs called
cisternae ■
cis refers to where it comes in, trans refers to where it goes out. cis also refers to
regions that have lots of tubule structures ■
oligosaccharide processing (when proteins from ER go through Golgi) ■
Golgi is responsible for sorting to 2 places: plasma membrane or lysosome via endosome ■
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the ones that go to lysosome have a particular sugar □
requires a particular oligosaccharide modification that occurs in the Golgi. this
happens to both soluble and membrane lysosomal proteins
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plasma membrane sorting does not require any signal
exocytosis—constitutive and regulated pathways □
regulated: one form of regulation can refer to extracellular signal such as hormone or neurotransmitter □
example: release of insulin from cell ■
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receptor-mediated endocytosis □
example: LDL which enters cells via receptor-mediated endocytosis □
LDL is surrounded by a protein lipid layer that can be transported into blood
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in a clathrin-coated vesicle, it then uncoats, fuses with endosome, and the low
pH of the endosome causes LDL to dissociate from its receptor □
return of LDL receptors to plasma membrane □
mutations in the LDL receptor cause hypercholesterolaemia = too much LDL
in the bloodstream
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some issues that cause this include:
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some receptors that can’t bind LDL
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receptors that can bind LDL but can’t take it up
iclicker question: You are working in a biotech company that has discovered a
small-molecule drug called A275 that inhibits the activity of the endosomal VATPase, the proton pump that maintains the acidic pH of the endosome. When
cells are treated with A275, the pH of the endosome is neutralized. In this situation, what do you expect to happen to the LDL receptor after it binds LDL on the
cell surface?
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Answer: D) It will become stuck in the early endosome.