Previously Bio308

Hypotheses for molecular basis of bipolar disorder•Suggest problem lies in protein targetingHow 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 tobecome inserted into the ER (and only the ER)

After insertion non-ER proteins are sorted and deliveredsorting lumenal vs membrane proteins –how?

http://www.udel.edu/Biology/Wags/histopage/empage/ebv/ebv10.gif

Stages of vesicle traffic

3 Stages: Budding, targeting/docking and fusion

DonorDonor

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

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?

Our neurotransmitter receptor need to go ‘through’ 5 cellular compartments before it gets to the post synaptic membrane

Content selection

What goes inside which vesicle?

Lumenal protein:

Transmembrane proteins:

Combination of cytosolic andlumenal proteins determinespecific vesicle content

Budding

Fig 17-58 CBI 13.1 Clathrin

Coat Components

http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/clath.jpg

ClathrinCOPICOPII

Identity determined by whatthe vesicle contains and it’s coat.

Budding IIER vesicle budding

Drin, G, and B. Antonny (2005) News and Views: Helices sculpt membrane. Nature vol: 437

Amino Acid Key

Highly hydrophobic

+ charged - charged

Other

Hydroxylated

Sar1p N-terminal helix

Sar1p-GTP form exposes helix that anchors protein to ER surface by‘floating’ with hydrophobic a.a. interacting with membrane core

Budding III

Drin, G, and B. Antonny (2005) News and Views: Helices sculpt membrane. Nature vol: 437

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.

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:

http://dir2.nichd.nih.gov/nichd/cbmb/sob/in_vivo_dyn.html

What happens after budding?

How do vesicles dock with specific target membrane?

The SNARE hypothesis

Fig 17-59

V-SNARE

T-SNARE

Role of p115Role of Rab proteins

retrograde

Synaptic vesicle fusion

VAMP

SyntaxinSNAP 25

Synaptotagmin

Rab3a

Next: Moving in the other direction: endocytosisTypes: 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?)