Tuesday, January 27, 2009
- Our continuation in the theme of movement within the cell – we’ve
been looking at how proteins can move to different compartments in the
cell & we looked at gated transport, transmembrane transport into the
ER, for example & now we’re going to continue on with the 3 rd
mechanism of transporting proteins around the cell called vesicular
transport – compacting proteins into small vesicles & then moving them
from one compartment in the cell to the another.
- We started out looking at what the membranes in a cell looks like – the
lipids & the proteins & we looked at how these membranes are
selectively permeable to certain molecules. Proteins, for example, can’t
freely diffuse across membranes b/c they’re impermeable to a large
hydrophilic or ions, anything with a charge & the further you go away
from hydrophobicity, the less permeable the membrane is those
molecules so you need active mechanisms to transport these ions &
proteins across membranes.
- We looked at how small molecules can be transported by these
transporter proteins or these channels in order to move from one
compartment to another or into and out of the cell.
- Then we also looked at how proteins are sorted to the nucleus by gated
transport, to the ER by transmembrane transport, to the mitochondria.
- Today, we’re going to look at how proteins are transported by vesicle
trafficking.
- This is a continuation of what goes on in the ER.
Found in non-cytosolic leaflet
**Don’t need to know the structures of these lipids**
- The majority of lipids are synthesized in the ER of a cell – these
include phospholipids, cholesterol & another lipid called ceramide.
- A certain subset of these will be modified, in particular ceramide will
be modified in the Golgi Apparatus. Ceramide, which is synthesized in
the ER will be used as a precursor for 2 particular lipids in the Golgi:
sphinogomyelin (SM) where the polar head group from phosphocholine
(PC) is transferred onto ceramide & then glycolipids are also
synthesized in the Golgi & this is when sugars or oligosaccharide chains
will be added onto lipids & this is the synthesis of glycolipids. So both
of these are synthesized in the Golgi from a ceramide lipid precursor &
the ceramide is synthesized in the ER along with these other abundant
lipids in animal cells.
- These ones that are synthesized in the Golgi will be found in the non-
cytosolic leaflet of the plasma membrane since they are synthesized in
the lumen of the Golgi – so the non-cytosolic leaflet is the one exposed
to the extracellular space. These ones in the Golgi are synthesized in the lumen of the Golgi. So this is where the majority of membrane lipids are
synthesized.
by ER expansion
new lipids in cytosolic leaflet of ER.
- New membranes are synthesized by ER membrane expansion which
means that lipids are synthesized & added into the membrane of the ER.
Specifically, the synthesis occurs on the cytosolic leaflet of the ER.
Diagram: all the enzymes that catalyze the synthesis of these lipids have
their active site facing the cytosol – so that means all the lipids are being
synthesized on the cytosolic leaflet of the ER (not going to go into
details of all the enzymes or reactions that occur to synthesize lipids).
All these enzymes have their active site on the cytosolic side of the ER
membrane & the lipids are being synthesized in this leaflet.
- This presents a conundrum to the cell – if the synthesis were just to
occur continuously on this leaflet of the ER membrane, you would just
get expansion of this membrane whereas the other membrane wouldn’t
expand. Recall that lipids don’t tend to flip flop from one leaflet to
another on their own unless there are enzymes there to catalyze that flip-
flop from one leaflet to another. They would just continuously
accumulate in the cytosolic leaflet & then you would get expansion of
one leaflet & the other one would not expand at the same rate. So to
overcome, the cell has specific enzymes that will flip lipids from the
cytosolic leaflet into the luminal or non-cytosolic leaflet of the ER
membrane.
rapidly flipped to other leaflet
scramblase
by vesicular transport
- Diagram: This would be the ER membrane, it’s a lipid bilayer & then
you’re getting new lipids synthesized on the cytosolic leaflet & you can
see that this leaflet would expand & if there was no enzyme to flip it,
this leaflet would expand but this other one would not so there is a
specific enzyme called the scamblase that catalyzes the flipping of
phospholipid molecules. And it will flip specifically lipids to equilibrate
them b/w the 2 leaflets of the membrane so it doesn’t preferentially flip
from one leaflet to another, it just basically equilibrates lipids b/w these
2 membranes & this is not specific so it will do this for almost all
phospholipids that are synthesized in the ER membrane & equilibrate
them b/w the 2 leaflets of the membrane. Scramblase will make sure
that there is a symmetric distribution of lipids in the ER membrane so
only one leaflet doesn’t grow preferentially over the other. This leads to
the symmetric growth of both halves of the bilayer.
- Then from there, the newly synthesized membranes will be distributed
to other membranes in the cell, such as the Golgi, the plasma membrane
& the endosomes – they will be distributed to these ones from vesicular
transport. These vesicles are made up of membranes themselves & they
originate originally from the ER so they’ll go from the ER to the Golgi
to different compartments in the cell & with them they carry along lipids
& distribute them to these other membranes. Flippase (ABC transporter)
energy from ATP hydrolysis
- Recall there are also specific lipids that are found in certain leaflets of
membranes & in particular, the plasma membrane has specific lipids on
its cytosolic leaflet, & these include phosphatidylserine (PS) &
phosphatidylethanolamine (PE) – these are preferentially found on the
cytosolic leaflet of the plasma membrane.
- So if the scramblase is scrambling these lipids from one leaflet to
another, how does the cell preferentially enrich a certain lipid into the
cytosolic leaflet of the plasma membrane? This is done through another
phospholipid translocator & this one flips specific phospholipids to the
cytosolic leaflet of the plasma membrane – called a flippase – it is an
ABC transporter which transports lipids from one leaflet of the
membrane to another.
- Diagram: Here would be a newly delivered membrane going to the
plasma membrane – this would come from the ER – you can see that the
lipid is randomly distributed b/w the 2 leaflets, the yellow & the red
colours but now let’s assume that the red one would be PS, for example
– once it associates with plasma membrane, it needs to be preferentially
enriched on the cytosolic leaflet of the plasma membrane so this is
carried out by this flippase that will flip preferentially these PS to the
cytoplasmic leaflet of this lipid bilayer. This is very important b/c the
distribution of PS to the non-cytoplasmic leaflet of the plasma
membrane can be an indication that a cell is dead so when cells die these
flippases turn off, PS become enriched in non-cytosolic leaflet which is
not their normal situation & this enrichment of PS now in the non-
cytosolic leaflet can be a signal for macrophages to come & digest &
engulf that cell. So it’s very important that a cell that is healthy keeps
the PS on the non-cytosolic leaflet to prevent macrophages from
engulfing & digesting that cell.
- So that’s the way that lipids are distributed in the membranes: 1 st
they’re synthesized in the ER, scrambled b/w the 2 leaflets & then
delivered to the different compartments through vesicular transport &
then if some lipids need to be enriched in one leaflet over another,
flippases will specifically flip those lipids to the appropriate leaflet of
the plasma membrane.
budding with cargo
fusion to target
release cargo
- Vesicular transport occurs from the ER to the Golgi, from the Golgi to
the cell exterior through a process called exocytosis, from the Golgi to
endosomes (intermediate compartments b/w the Golgi & the lysosome
or the Golgi & the cell exterior, for example), & also there’s a process
called endocytosis where vesicles can come from the plasma membrane
& come into the cell to these endosomes & uptake nutrients or other
molecules from the outside of the cell. This movement of proteins
occurs through vesicular transport.
- So whatever is inside a vesicle is referred to as cargo.
- Diagram: There are 3 major components to this process. One of them
is the budding of a vesicle with cargo from a donor compartment so let’s
assume this is where the vesicle is originating from, the vesicle has to
bud off this donor compartment, it could be the Golgi for example, or
the ER, & when it buds off, it needs to contain cargo. This cargo is
usually made up of some proteins & these proteins can either be soluble proteins or transmembrane proteins. So the vesicle needs to bud off the
donor compartment completely & then it needs to fuse with a target
compartment so the donor compartment could be ER & the target
compartment could be the Golgi, for example. So vesicle buds off with
its cargo & fuses with the Golgi & then it needs to release its cargo into
the target compartment. So 3 major steps to delivering a protein to a
new compartment through vesicular transport.
- This is just to show you the different processes where vesicular
transport is important for transporting macromolecules inside the cell.
This is called intracellular vesicular transport & the different arrows
refer to different types of transport that is occurring.
- The red arrows include the secretory pathway & this is usually
movement that originates from the ER early, through the Golgi, through
to the early endosomes or lysosomes & it also includes movement to
outside of the cell through exocytosis. So its movement is from the ER,
through to the outside of the cell through intermediate compartments
such as the Golgi & lysosome & endosomes – that would be the
secretory pathway.
- The green lines indicate the endocytic pathway which is the formation
of vesicles on the plasma membrane & uptaking macromolecules from
outside of the cell & the endocytic pathway goes from the plasma
membrane to the early endosome & usually ends up in the lysosome.
- Now in blue you have retrieval pathways to ensure that lipids don’t
over-accumulate in one area or another so if movement was always
going in this direction, lipids would be lost to the ER & accumulate in
plasma membrane & the plasma membrane would continue to grow
until the cell would be too large for itself. So there needs to be retrieval
pathways to recover membranes that are being moved in a certain
direction so the blue arrows indicate what these retrieval pathways are
so as vesicles are moving through the secretary pathway from the ER to
the Golgi, there are also retrieval pathways to recover some of these
lipids through vesicles also back to previous compartments in the Golgi
& also back to the ER. So retrieval pathways can also come from the
early endosomes, late endosomes & so on & you can see that there are
also retrieval pathways to recover lipids to the plasma membrane – there
is endocytosis occurring so that the lipids are not all lost from the
plasma membrane – lipids are being recovered through a retrieval
pathway from the early endosomes.
- So these are 3 major types of intracellular vesicular transport that are
occurring – all of these use vesicles for transport.
Transmembrane proteins
Soluble proteins
- This is just an outline again to show you how this vesicular transport
occurs. This figure is nice b/c it shows you the lipid bilayer in 2
different colours & you can see how this lipid bilayer is forming into a
vesicle & then note how the luminal side of the donor compartment
forms the inside of the vesicle & then when you fuse to the target
compartment, the luminal side/leaflet of the bilayer also becomes the
luminal leaflet of the new compartment so the orientation of these
leaflets is maintained within this transport.
- If we were to envision this as the plasma membrane so the target
compartment wouldn’t be closed off, this would be the plasma
membrane you can see that the luminal leaflet (the blue one) would
actually become the extracellular leaflet of the plasma membrane so let’s assume this would be the cytosol & this would be the extracellular
space, this blue leaflet now becomes exposed to the extracellular space
& this explains why things that are occurring on the luminal side of the
leaflet end up on the outside of the cell.
- When a vesicle buds, cargo proteins need to be collected into that
vesicle & these can be transmembrane proteins (the green ones in the
diagram) but they can also be soluble proteins & many of these soluble
proteins are actually bound by transmembrane cargo receptors. So these
are transmembrane proteins that will bind to the soluble proteins &
recruit them to the vesicle that is forming.
- Then once the vesicle is loaded with the cargo, it will bud off & then
fuse with the target compartment.
select cargo for vesicle
give curvature to vesicle
- Newly emerging vesicles off a compartment have protein coats over
them. There are 3 major types of protein coats: clathrin coats, COPI
coats, & COPII coats.
- Now clathrin coats are mainly coats that are on vesicles forming from
the plasma membrane or from the Golgi & going to endosomes.
- COPI coats are mainly going within the Golgi so vesicles going from
one compartment of the Golgi to another or vesicles coming from the
Golgi to the ER.
- COPII are on vesicles that are forming on the ER & going to the Golgi.
- The function of these protein coats is to select the cargo for these
vesicles, to give curvature to the vesicle which aids in the formation of
the vesicle as it’s forming off the compartment & to promote the vesicle
budding. These coat proteins play an important role in selecting what’s
going to go into a vesicle & then actually inducing the formation of the
vesicle.
from Golgi to ER (indicated in blue here)
b/w different Golgi cisternae (so within the Golgi from one
compartment of the Golgi to the other)
from ER to Golgi (indicated in red)
from Golgi apparatus (to the endosome) & plasma membrane to
endosome (indicated in green)
- So the different types of protein coats are indicative of where these
vesicles came from & where these vesicles are destined to go. - So how do these protein coats form on the newly emerging vesicles?
One important player in this is these monomeric GTPases so we saw
these GTPases when we looked at gated transport into the nucleus.
GTPases also play an important role in recruiting protein coats to newly
forming vesicles.
- The activity of these GTPases is regulated by 2 classes of protein: GEF
– exchange the GDP bound GTPase ndr GTP so you can think of it as
activating that GTPase & the 2 class of regulators is GAP – activates
the GTPase activity of this enzyme & the enzyme will hydrolyze the
GTP to GDP & then what happens is you can consider that to be turning
off the GTPase. So GEFs activate & the GTPase is bound to GTP &
GAPs inactivate by converting the GTP, inducing the conversion of
GTP-bound form to GDP-bound form.
Vesicle ready for transport to target compartment
- This is a generic overall of how these GTPases actually promote coat
assembly.
- Diagram: There is a GEF (the one that will activate GTPase) that’s
found in the membrane where the vesicle is going to bud & that will
recruit the GTPase to the membrane, exchange the GDP to GTP & this
will activate this GTPase. In this case, when the GDP is exchanged for
GTP, this GTPase will associate with the membrane where the vesicle is
going to form. So basically you have a GTPase that’s recruited to the
membrane where the vesicle is going to form by a GEF that’s found in
that membrane & that will activate the GTPase & the activated GTPase
will now associate with the membrane where the vesicle is forming.
- Once the GTPase is activated, it’s bound to the GTP-bound form & it
will recruit the coat proteins. In this case, this little yellow & brown
structure would be the GTPase & it will recruit the different coat
proteins shown here in red & this would be the outer coat proteins so
basically it’s recruiting this coat to the area where the vesicle is going to
form.
- Once the coat forms, this will induce vesicle formation so the
membrane will start to curve & the vesicle will form & eventually bud
off the membrane. Once the coat is recruited the coat will 1 of all
recruit the cargo to that vesicle, the vesicle will bud off & the final thing
that’s important is that once the vesicle buds off the donor membrane,
the coat will be shed before the vesicle will fuse to its target membrane
so the vesicle buds off, the vesicle is un-coated so the protein coat falls
off & for COPI & COPII coated vesicles, this involves GAPs & for the
clathrin coat, there is a different mechanism that we’ll look at.
- Once these vesicles have shed off their coat, then they’re ready for
transport to their target compartment but remember that the coat has to
be shed off the vesicle before it can fuse to its target compartment.
ARF GTPase
Sar1 GTPase
recruits Sar1
recruit coat protein subunits
- So let’s look at a specific example here.
- For COPI & for clathrin coated vesicles, the GTPases are called ARF
GTPases & these are the ones responsible for recruiting these 2 types of
coats, the COPI & the clathrin coats to these vesicles.
- For COPII coats, it’s the Sar1 GTPase that recruits these COPII coats
& this is shown on the right diagram how this occurs. So here is the
Sar1 GTPase in the cytosol, it’s not associated with the membrane & it’s bound in its GDP-bound form so this would be considered the inactive
form. It actually has an amphipathic helix that allows it to associate with
the membrane where the vesicle is going to form but it’s hidden inside
the protein & not exposed in the GDP-bound form.
- There is a Sar1 GEF that’s found in the membrane where the vesicle is
going to form that recruits the Sar1 GTPase to the appropriate
membrane where the vesicle is going to form. The GEF will exchange
the GDP for GTP on the Sar1 GTPase & the binding of GTP to the Sar1
GTPase exposes this amphipathic alpha-helix & that allows it to
associate with the membrane through this amphipathic helix so the
GTP-bound form will bind to the target membrane.
- So just to over in more detail, the Sar1 GEF is in the ER membrane.
Recall that COPII vesicles are moving from the ER membrane to the
Golgi, so in the ER membrane, this GEF has to recruit this Sar1 GTPase
to initiate the vesicle formation. So the Sar1 GEF recruits Sar1 (as
shown in the slide) – it will activate Sar1 by exchanging the GDP for
GTP & once this happens, an amphipathic helix is exposed & allows it
to interact with the membrane. And once Sar1 GTP is in the membrane,
it can recruit coat proteins to that membrane & then the coat proteins are
the ones that will recruit the cargo & induce the vesicle formation.
cargo (these will be transmembrane proteins)
transmembrane cargo receptors (the ones that will bind to soluble
cargo proteins)
SNAREs
- Diagram: Inner layer is shown here, can see the Sar1 GTPase but it
also recruits these coat proteins that are in the inner layer – this inner
layer is mainly responsible for binding to the membrane of the donor
compartment & also, it will select the cargo that’s going to be in the
vesicle.
-The outer layer of the vesicle coat associ
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