Adaptin acts as adapter, links cytosolic side of exit receptor to clathrin proteins
Clathrin is a triskelion molecule due to its structures, 3 large and 3 small subunits. Amino termini point inward, so when formed together, form hexagonal or pentagonal shape that forms a skeleton around a vesicles like a soccer ball. Stitching is clathrin and the coating is the PM
We need to bring in cholesterol to cell . The LDL that contains cholesterol
Core of esterified molecules linked to fatty acids surrounded by phospholipid monolayer with cholesterol embedded. Small protein wraps around core as well as PL monolayer. Protein carries recognition sequence for LDL binding receptor.
Dalton protein that wraps around core and it carries a recognition sequence signal for LDL binding receptor
Cholesterol from blood, receptors recognize in membrane and bring in LDL via endocytosis
LDL binding site on LDL receptor recognizes LDL in EXCSpace
Adaptin protein AP-1 will recruit clathrin to localization of receptors.
Auxillin and heatshock uncoat and then will fuse with endosomes
In endosomes the ph is different enough (more acidic slightly) just enough to dissociate receptor from LDL. LDL is trafficked to lysosomes where hydrolases break it down and release cholesterol into cytosol to be reused by cell and receptor to be reused in the PM
This process of endocytosis happens all the time whether LDL Is present or not.
How is Manose-6-phosphate generated – slide 30
ER signal sequence that puts proteins into the ER as they are made
Once in ER and folded, N-glycosylation puts oligosaccharaides on that lysosomal protein
Once protein moves in golgi, in cis golgi, we see there is phosphorylation of this sugar that is place on N-terminus of asparagine,
Form a structure that is a sugar plus the phosphate
GlcNAc-P stands for N-acetylglucosamine phosphate
Recognition enzymes cleave off some of the sugar to leave the M6P present
As it moves through cis/medial/trans golgi, the phosphorylation acts as a recogntion sequence for enzymes that are gonna come in and cleave spme sugar away, exposing just M6P component of oligosaccharide tag and M6P is recognized as signal sequence that will be trafficked via the receptor to the lysosome.
Once fused with lysosome the M6P hydrolase is released into them due to high acidity ph5-5.5
Dissociation leads to removal of Pi from enzyme so it’s functional
The Pi just acts as a tag to get hydorlase to lysosome
M6P receptors are recycled back to golgi and used again for trafficking
This phosp. Event is the beginning of this transformation of protein to M6P
Slide 31 – how do vesicles go to where they need to go?
Motor proteins carry vesicles once budded form donor membrane to wherever they need to be
MP can recognize signal sequences on cargo itself, or recongize target signals within membrane of vesicle itself.
Phosphoinositides can be recognized by MPs in membrane and carried to vesicles
Centrosomes and mictrotubules extend out with + ends toward plasma membrane
Kinesin and dynein family MPs bind to vesicles as they bud and carry to certain places. Kinesin moves +, dynein moves –
GTP hydrolysis takes place between association and fusion of vesicle
2 family of proteins come in and dissociate v and t snares from each other
SNAPs and NSF proteins work to break apart associate between v and t SNARES
Slide 34 – How proteins are moved into mitochondria/specifically matrix
Begins in cytosol because the proteins to be trafficked into mitochondriaare synthesized by free ribosomes in cytosol
Once translated, immediately bound by heat shock proteins (Hsp70)
Heatshock protein 70 recognizes N-terminus alpha helix domain that is specific targeting to the mitochndria
TOM protein binds to recognition sequence, alpha helix, and links it to the pore in the mitochondira to allow for movement through the memrane (post translational transport)
Once protein moves to pore after recognized by receptor, TOM protein fuses with TIM protein (trans. Protein. Of inner mito membra) and makes a single large pore to bring protein to matrix of mitochondria.
Process requires energy because the heatshock proteins that keep the protein from folding in cytoplasm, requires the hydrolysis of ATP to be stripped off so protein can be moved through membrane
As moves through membran, signal peptidase cleaves signal and protein moves through
Hsp70 helps bind and pull protein through TIM and then the Hsp70 needs to be removed to be folded properly
Another HSP helps fold protein again using ATP and now we have a properly folded protein in matrix of mitochondria (Hsp60 folds to final conformation)
N-terminus alpha helix is positively charged amino acids that have hydrolxylated amino acids in between but can be recognized by this TOM receptor to begin post-translational import