Transcript Getting things where they need to go: Protein Targeting

Previously Bio308
Hypotheses for molecular basis of bipolar disorder
•Suggest problem lies in protein targeting
How are proteins targeted and delivered?
Sorting places proteins in membrane and in lumen of organelles
PM (and other) proteins use Sec or SRP mediated translocation to
become inserted into the ER (and only the ER)
After insertion non-ER proteins are sorted and delivered
sorting lumenal vs membrane proteins –how?
http://www.udel.edu/Biology/Wags/histopage/empage/ebv/ebv10.gif
ER proteins
ER
Where can a protein end up in the ER? How does it get there?
Lumenal proteins
Single transmembrane span proteins
Multipass transmembrane proteins
What category do our neurotransmitter and
neurotransmitter receptor fall in?
How do you
get soluble lumenal
proteins vs ‘Type III’
What’s different?
How does it happen?
Getting out of the ER
Golgi enzyme involved in glycosylation
Neurotransmitter
receptor
ER
Lysosomal acid hydrolase
Now what?
Vesicular traffic
Secretory pathway: also method for delivering new PM proteins
ER to Golgi to trans-Golgi network
 then constitutive or regulated exocytosis
Constitutive and Regulated Exocytosis
Constitutive= constant, sometimes called ‘bulk flow’
Constitutive does not mean ‘un-regulated’
Regulated= needs additional signal to initiate fusion of vesicle with PM
Stages of vesicle traffic
3 Stages: Budding, targeting/docking and fusion
Donor
Donor
Target
Target
Consequences of unregulated vesicular traffic
Mixing of organelle contents ( won’t function correctly)
Mislocalization of proteins ( won’t function correctly)
Inappropriate levels of secretion (too hi or too lo)
A Dead Cell
Vesicular traffic control
Our neurotransmitter receptor need to go ‘through’
5 cellular compartments before it gets to the post synaptic membrane
How does a vesicle ‘know’ what components it should contain?
How does it ‘know’ which membrane it should go to?
How does it fuse when it gets there?
Content selection
What goes inside which vesicle?
Lumenal protein:
Transmembrane proteins:
Combination of cytosolic and
lumenal proteins determine
specific vesicle content
http://biology-animations.blogspot.com/2009/10/clathrin-animation.html
Budding
Fig 17-58
Coat Components
Clathrin
COPI
COPII
Identity determined by what
the vesicle contains and it’s coat.
http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/clath.jpg
Budding II
ER vesicle budding
Sar1p N-terminal helix
Amino Acid Key
Highly hydrophobic
+ charged
- charged
Hydroxylated
Other
Sar1p-GTP form exposes helix that
anchors protein to ER surface by
‘floating’ with hydrophobic a.a.
interacting with membrane core
Drin, G, and B. Antonny (2005) News and Views: Helices sculpt membrane. Nature vol: 437
Budding III
ER vesicle budding
Floating many Sar1p in top leaflet makes it ‘bigger’
than the bottom one.
Results --> bulge that can more easily interact with
coat proteins.
Drin, G, and B. Antonny (2005) News and Views: Helices sculpt membrane. Nature vol: 437
Fission
ER vesicle budding….fission
Ring of parallel helices at neck might aid fission.
New data for ER; had seen a protein (epsin) help
deform PM for clathrin coated vesicles.
May suggest that using a helix to deform
membrane is common mechanism for
budding/fission
Targeting/Docking:
What happens after budding?
How do vesicles dock with specific target membrane?
http://dir2.nichd.nih.gov/nichd/cbmb/sob/in_vivo_dyn.html
The SNARE hypothesis
V-SNARE
T-SNARE
Role of p115
Role of Rab proteins
retrograde
Fig 17-59
Synaptic vesicle fusion
VAMP
Syntaxin
SNAP 25
Rab3a
Synaptotagmin
Next: Moving in the other direction: endocytosis
Types: Phagocytosis– specialized cells
Pinocytosis– all cells
Connection– perhaps the # of our receptor’s on PM is
controlled by endocytosis
Pinocytosis ‘problem’
rate of pinocytosis internalizes 100% of PM per hour
?
(How can this be?)