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Lecture 7: Intracellular vesicle trafficking

We started to talk about protein glycosylation
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Now the glycosylation, the oligiosaccharide complex is very specific
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You have a branching
going one
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It is always an amino acid, followed by any amino
acid followed by either serine or threionine
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Now where does it bind? Now before
binding this oligiosaccharide is actually attached to the membrane through a membrane lipid
called Dolichol
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This
transfer is made possible by another protein by the name of Oligosaccharyl transferase
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Now this example is an example of a membrane protein, but what I want to
tell you is that it is the same for a soluble protein
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Another thing is a protein that is linked to a very important membrane lipid, phosphotydl
inosotil
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Now this protein is also important in linking completed
proteins from the ER to the membrane
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So the first
one is inositol and it is followed by glucosamine(suagar) flowed by 3 mannose
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Obviously this particular protein will end up linking with
glycosylphosphatidyl inositol at the COOH segement of the peptide
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This protein is called GPI anchored protein
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What we are talking about now is the folding of the protein after the soluble protein is
completed in the ER lumen
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You have all of these amino acids NH2 group,
OH group, COOH group associated with all of the amino acids
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One of the possibilities is that the protein can fold by
itself to its most stable conformation
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So it is able to promote the linking of two cystein

amino acids
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Third is that some proteins need help
in the ER to fold to a proper most stable structure
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) Calnexin
2
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) ERP 57

We will only be talking about Calnexin
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Now you have an unfolded protein but you have an nlinked oligiosaccharide attached to this soluble protein
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First you
have an enzyme that will get rid of the glucose
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This process is called glucose trimming
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This is the reason these two proteins interact
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Unless this interaction occurs the protein will not fold properly
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This becomes the properly folded form, if it succeeds
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This incompletely folded protein will be recognized by a specific enzyme called
glucosyl transferase that will add another glucose back to the oligiosaccharide complex
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This is a very fast and efficient
reaction
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Hopefully the second time it succeeds, but if it fails it will go again and try again
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But what if it is not properly folded after all this
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This translocator is
different from the translocator that brings that growing peptide into the ER
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Once this protein is in the
cytosol there will be an enzyme called N-glycanase that will turn off and remove the
oligiosaccharide group all together
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Ubiquitin is a protein that is present in large amounts in the cytosol and one of
its most important functions is to direct the folded protein towards destruction
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When this
complex is completed, it will find its way toward a complex called a proteosome, where there is
a lot of enzyme activity that will chew the misfolded protein to its original amino acids
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So secretory vesicles are the means
where proteins get transported from one place to the other
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Now where do you find these vesicles and
where do they function? This is ER and then you have the Golgi and then you have the plasma
membrane
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As the protein gets
processed clathrin coated vesicles are formed in the trans Golgi
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A lot of the proteins that are specific to the ER lumen are
carried along by COP11 vesicles, but those vesicles that originally belonged to the ER must be
returned to the ER
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Three types of coated vesicles
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Before we do this lets look how the COP 2 vesicles are formed
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What is Sar1-GTP? GTP- is guanidine diphosphate
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Sar1 is actually an ATPase molecule that is covalently linked to a small molecule GTP
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This means that there is a small sequence of nucleotides that has a
hydrophobic region and hydrophilic region
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The ER membrane has a protein called Sar1-GEF(Guanine Exchange Factor)
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What happens when they bind together, it is the
function of Sar1-GEF to exchange GDP to GTP
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It is the function of
GEF
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It is a very
important reaction
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Now when
the Sar-1 is successfully imbedded, you have proteins floating around one called Sec 23, sec 23
is a cytoplasmic protein that will bind to Sar-1 and when it binds it will cause conformational
change and then it will bind to another protein referred to Sec 24
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Now if a protein is going to be transported out of the ER, if it is a
soluble protein it is called a cargo protein and in the ER membrane you will have…you will have
a domain that recognizes the cargo protein
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All of the sudden with the bind
of the protein the membrane protein takes on a different function
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Your formation of the Sar1-GTP brings in sec 23, causes some changes
in this protein causes it to bind to Sec 24 which brings it to close proximity to the ER
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Once bound it will activate
the receptor, then it will bind to a specific cargo
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So this is the start of the COP 2 formation, so
what happens is when this reaction is starting, the curvature of the cytoplasmic membrane
starts to bud
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This protein forms the outer coat of the COP2 protein
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We said
that the binding site of Sec 24 will bind to the cargo receptor but also in turn the receptor pick
up the cargo protein
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So as
the protein is formed not only are you pulling in and not only do you have the soluble protein
inside the vesicle, but you also have the transmembrane proteins as part of the vesicle
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COP 2 is just the name
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So in the ER you have this formation of a coated vesicle
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What must happen first? You must get decoating, meaning the sec 31 is removed and
these two will come together
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With these vesicles there are proteins like little filaments protruding out that are responsible
for fusion between membranes
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Normally
before fusion you have these strings of proteins wrapped around each other but with the
presence of NSF, they will disentangled and will become separated
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COP 2 vesicles fuse with each other and not just two but another one can fuse with
this one and so forth
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It is a structure found between the
ER and the cis Golgi
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