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Lecture 4 Endoplasmic reticulum
Now what we are going to talk about is the methodology of how people go about studying membrane
protein
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) one of the methods is called solubilization by detergents
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Both of these can disrupt the
membrane and break it up into little pieces and in doing so releasing all of the cytoplasm and that makes
it
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Triton X 100 you
have a polar end here a hydroxyl group and a nonpolar end
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If you look at these detergent molecules they have a hydrophilic end and a
hydrophobic tail
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But when you add these type of compounds in with membranes what happens is that you
will break them up
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What it does is that it mixes in with the
phospholipid molecule and forms this type of hybrid miclle but in doing so the membrane protein will be
broken up and they will be released from the membrane
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Say you are going to study a membrane protein, say sodium potassium pump, very very important
pump
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A lot of the ATP that is generated in the cell is put into making this membrane protein
function
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If you want to study its 3-dimensional structure you
have to isolate enough of the protein and then crystallize it for x-ray crystallography study
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So the first thing is to add the detergent and it will form a hybrid micelle and the
protein will be released from the membrane
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Beside the sodium potassium protein you
have other proteins in the membrane
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Then after you purify it you
still have a lot of detergent molecules and phospholipid molecules still attached so you want to get ride
of it and this can be by dialysis
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It comes in
one micron to ten microns and you have your protein in here
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The protein will remain in place, only water molecules and other small molecules can
get out
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The nice thing is that the volume doesn’t change
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When you put this phospholipid into a solution it will form its most energetically stable

structure and in this case it is a lipposome
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And using this sphere you can do your test
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When you deliver drugs just like that
some of the chemicals can get broken down by enzymes and acidic and alkaline condition of your GI
tract
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There is another way is that you can create a single lipid membrane, for example if you have two
chambers
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It is just
phospholipid you can study its function, but if you have protein molecules also within this artificial
membrane then you have a way of studying how the protein affects the transport of molecules between
these two chambers
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So these are the methods of isolating important membrane proteins
for study
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I mentioned that if you
have an integral membrane protein it does not flip because it is covalently anchored to the two polar
sides of the lipid bilayer
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The next topic will be about the
movement of the membrane protein in the membrane
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It
demonstrates that membrane protein can move freely in the membrane it is not restricted in any
particular area of the cell
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In mouse cell you
have peculiar membrane protein and in humans you have membrane proteins that can only be found in
human
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There are two methods of
creating this hybrid cell: 1
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If two cell are pressed against each
other in the presence of this small molecule they will fuse
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) The second method is to use a Sendhi
virus—they found out in the early 60’s that the viral coat proteins of these viruses can promote the
fusion of cells
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if the membrane proteins are restricted to certain regions of the cell you will
only have the mouse protein aggregation to the mouse membrane and the human proteins aggregating
to the human membrane
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But this is not true, after the fusion
you find that the mouse protein and the human proteins are mixed
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What you see as a result
is a cell with green and red flourscence mixed up together
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Now how fast does it move? The movement of the protein is also important in the function of the cell
because some of the proteins are important like the receptor proteins
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For example hormone receptor will
have less of a chance to meet up with the ligand molecule in this case the hormone
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What is a flourscent
molecule? It is a small molecule but if you shine UV Light on it, the UV light will energize it and
subsequently the molecule will release that UV light in the form of a flourscence
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So this is a cell
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Then you remove the lazer
beam and the watch for the flourscence molecule to return to that area
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This recovery reflects how fast the other flourscent proteins in
other areas move into this area
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The
movement of the protein turns out to be very fast
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You have again these flourscent tag
proteins and you pick one area and start shooting this area continuously
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So as the molecules in this area is bleached it will move out of this area
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First
there is no bleaching, but eventually the bleaching will spread over to this area until the bleaching is just
like the area where it is irradiated
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It depends on the size of the protein, how it is anchored to the membrane
and all that
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A lot of
time it is restricted
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If we look at the
epithtelial cell
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Remember that
material cannot get through from outside to inside the cell, however molecules can move from this cell
through the…
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In the apical side they found that there is one kind of protein that is restricted to
only this to this side and there are membrane proteins that are restricted to the basal and basolateral
side
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So this is one point on the restriction of proteins
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If you look at the sperm if the protein are
allowed to move from the head all the way to the tail, but this is not the case
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The anterior head of the cell, certain proteins can only be found in this region and certain
types of proteins can only be found in the mid region and tail region
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So what are some of the mechanisms restricting the
movement of these proteins? There are four possibilities : 1
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They tend to
stay together so you always find them together
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) The second one is that you have the membrane
proteins is teethered to extracellular protein and they have affinity to these membrane proteins
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This teethering could be covalent it could be
noncoavlent, it depends
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) By teethering to intracellular proteins--- So you can have inside cytosolic

proteins that can be teethered to a group of membrane proteins and restrict them in space
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) proteins
can interact with each other---so you have a group of similar membrane proteins here and when you
put them together in the extracellular domain they stay together
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These are four methods that a
particular membrane protein can be restricted in a particular area
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Red blood cells have no nucleus it is a disc shape
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If you look at inside the cell
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These are the cytoskeleton that gives the red blood cell its biconcave shape
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Something is
very interesting, if you heat up the red blood cell up a little bit this cytoskeleton become separated from
the cytosolic side of the red blood cell membrane and as a result that biconcave shape becomes
rounder
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So the red blood cell ceases to work
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(36:00)



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