Thursday, January 22, 2008
- Today we continue with sorting of proteins to the endoplasmic
reticulum, another form of transmembrane transport.
- Recall there are 2 types of ER: the rough ER (actively carrying out
protein synthesis with ribosomes associated with it – the ribosomes are
the things that give it the rough appearance) & smooth ER. We are going
to focus on the rough ER today.
- Proteins are sorted to the ER through transmembrane transport – recall
this is the 2 type of transport that we covered in the last lecture – the 1
type is gated-transport & then we covered transmembrane transport. Once
proteins enter the ER from the cytosol & from there, they can be further
sorted to other compartments such as the Golgi, lysosomes or even
secreted to the cell exterior by vesicular transport.
- Today we’re going to look at how proteins get from the cytosol into the
ER by transmembrane transport.
- ER is not a static structure, it is actually changing all the time in terms
of its location in the cell & its composition in the cell. This video nicely
- Video: This would be an ER network here & just take a look at how that
network changes just as you’re looking at it over time.
- Description: The ER is a highly dynamic network of interconnected
tubules that span the cytosol of the eucaryotic cell, like a spider’s web.
The network is continually reorganizing with some connections being
broken while new ones are being formed. Motor proteins moving along
microtubules can pull out sections of ER membranes to form extended
tubules that then fuse to form the network.
- This is just to demonstrate how dynamic this whole process – ER is
very dynamic & proteins are coming into it at multiple locations where
there is the rough ER with ribosomes associated with it. Synthesis & modification of proteins (covered in this lecture)
Synthesis of lipids (covered in a later lecture)
Have ER signal sequence
- The proteins that are sorted to the ER all have a signal sequence which
is present on the protein so this would be the mRNA here (in the
diagram), a ribosome associates with it & starts to translate that protein &
the ER signal sequence comes out at the N-terminus – the 1 part of the
protein that comes out of the ribosome so it’s an N-terminal endoplasmic
reticulum signal sequence – ER signal sequence.
- The types of proteins that have an ER signal sequence are both soluble
proteins & transmembrane proteins & these are many proteins destined
for the Golgi, secretion outside the cell & also to the lysosomes. Also
some proteins are destined to stay in the ER.
Hydrophobic AAs at N-terminus
- mRNA comes out of the nucleus & then ribosomes associate with the
mRNA & to translate that mRNA into protein. Normally, what occurs is
that multiple ribosomes will bind to an mRNA molecule & you’ll get
polyribosomes in the cytosol associated with that mRNA & each one of
these ribosomes can be translated into protein from this one mRNA
molecule. That’s where all proteins start, most of them, in the cytosol.
Some of them have an ER signal sequence that emerges at the N-
terminus. This sequence that emerges is a hydrophobic AA sequence
without any characteristic specific AA sequence, it’s mainly this
hydrophobicity that gives it its characteristic ER signal sequence. For an
ER protein, as it’s being translated from the ribosome, the ER signal
sequence will emerge 1 at the N-terminus.
- Once the ER signal sequence emerges from the ribosome, this whole
complex is then directed to the ER membrane. As soon as the ER signal
sequence emerges, it’s directed to the ER membrane, then the protein that
is destined to go into the ER is directed through transmembrane transport
in a co-translational process into the ER so the ribosome continues to
translate the protein as it’s being funneled into the ER.
- How this occurs is that there is actually this signal recognition particle
cycle that occurs, that recognizes this ER signal sequence as it’s
emerging from the ribosome so there’s a signal recognition particle that
will bind to the ER signal sequence that will pause translation & then that
whole complex with the signal recognition particle will be directed to a
signal recognition particle receptor that’s on the ER membrane then
translation will resume & the protein will be integrated into the ER in a
co-translational process as the protein is being translated off the
- There are 2 major components to direct the emerging peptide with the
signal sequence to the ER: the signal recognition particle also referred to
SRP & the single recognition receptor SRP receptor.
- Diagram: Here is the ribosome, the mRNA & the protein that’s destined
for ER is being translated – the first thing to emerge is this N-terminal ER
sndnal sequence – this will be bound by the SRP shown in the diagram &
2 this SRP will bind to the SRP receptor that is found in rough ER
membranes – that will bring this entire complex to the ER. Both of these
proteins have GTPase domains & they’re normally bound to GTP in this phase of the process where they’re bringing the ribosome & the newly
synthesized protein to the ER. So they are both bound to the GTP so both
the SRP & the SRP receptor have GTPase domains & are bound to GTP
at this state.
- Normally the SRP & the ribosome have a very low affinity for each
other. When the ribosome is free without a protein coming out of it with
the signal sequence, there is very low affinity b/w the 2 but when the SRP
encounters a ribosome that has a protein with a ER signal sequence
coming out of it then that becomes a very high affinity interaction & the
SRP will bind to the signal sequence & then it can guide the protein &
ribosome to the ER by binding to this SRP receptor.
Prevent diffusion of ions, small molecules
- In the ER, there is this protein translocator through which the newly
synthesized protein will be funneled through. So you can think of it kind
of like a tube of tooth-paste where the protein coming out would the
toothpaste coming out of this ribosome which would kind of be the tube
of toothpaste squeezing the protein out & injecting it into this protein
translocator into the ER.
- Diagram: The ribosome is brought to the protein translocator once it’s
bound to the SRP receptor. Here is the ribosome with the newly
synthesized peptide, the ER sequence here has bound to the SRP, SRP is
bound to SRP receptor & now this entire complex is brought to this
- The translocator channel is also called the translocon & it’s a gated
channel meaning that it’s not always open – it’s kind of shown in the
diagram as a plug but this will actually open once the ribosome & the ER
signal peptide binds to the translocator. So what happens then is these 2,
the SRP & the SRP receptor, bring the ribosome & the newly synthesized
peptide to the translocator & the binding of the ribosome & the ER signal
peptide to the translocon opens it up & allows the protein to be
synthesized through the translocon. Now the ribosome forms a very tight
seal with the translocator & this is important so it prevents the diffusion
of ions & small molecules out of the ER.
- Once this happens, how does this dissociation occur? What’s known is
the SRP & the SRP receptor hydrolyze their GTP to GDP & inorganic
phosphate & this somehow releases the complex so the SRP will be
released into the cytoplasm & the SRP receptor will be released back into
the ER membrane. It’s not known exactly what triggers this GTP
hydrolysis at this time but what’s known is that GTP hydrolysis precedes
the dissociation of the entire complex.
Cleaves ER signal sequence
Translocator gated in the 2 direction
- Examples of how proteins are inserted into the ER.
- Example of a soluble protein – this has no transmembrane domains &
the 1 thing is that these proteins have an ER signal sequence at the N-
terminus (again). All the translation is occurring in the cytosol & what is
not shown here is the SRP & the SRP receptor but what you have to
assume what has just happened is that the SRP & the SRP receptor have
brought this newly synthesized protein to the translocon. - And in this case, the ER signal sequence acts as a start-transfer
sequence. When the translocon binds to this N-terminal ER signal
sequence, it actually signals this to start translocating that protein into the
ER so this will bind directly so this signal sequence will bind to the
translocon & that signals to the translocon to start transferring this protein
through the translocon into the ER. So you can imagine up here (and it’s
not shown either) is there is a huge ribosome that is actively translating
this protein & as it’s translating, it’s being funneled into the ER.
- So the start-transfer sequence or the ER signal sequence at the N-
terminus binds to the translocator & then the protein is transferred into
the ER. And then there is the signal peptidase & this is a protease that
will cleave off the signal peptide off the protein so a mature soluble
protein does not have its signal peptide associated with it b/c the signal
peptidase will cleave the ER signal sequence once the protein gets inside
the ER. What that does is it releases mature protein into the ER lumen &
then the signal sequence that’s been cleaved off actually moves laterally
into the lipid bilayer & that’s b/c this translocon is gated in a 2 direction
so not only can it allow proteins to go through in this direction but it can
also gate in the other direction also & allow proteins or this signal peptide
to diffuse out into the membrane so it’s gated in 2 directions – one to
allow the protein to enter the ER lumen & then the 2 direction to move
things into the ER membrane. So the signal sequence will be cleaved off
& then will be released into the ER membrane & the mature protein will
be released into the ER lumen so this is for soluble proteins.
- Answer: C
- Answer: D
- Animation shows how this transmembrane transport into the ER occurs.
- This is for a soluble protein. Here is the ER, the signal sequence, signal
peptidase cleaves it off then you have your protein soluble in the ER
lumen. Sorted out during translocation
- Recall that there are different types: there are single-pass & multi-pass
transmembrane proteins & this just means how many times the protein
goes through the membrane – so a single pass will only go through the
membrane once, while the multi-pass goes through the membrane
- Here are 3 examples of single-pass transmembrane proteins that are
going through the ER membrane. Notice that in each of these 3 cases,
there’s something different about the way they go into it – there are 3
different ways of inserting a single-stss trdnsmembrane protein into the
ER membrane. In these 2 cases (1 & 3 in the diagram), the carboxy
terminus of the protein ends up in the cytosol as compared to the 2 case
where the carboxy terminus of the protein ends up in the ER lumen – this
is due to the way in which the protein is inserted into the membrane.
COOH in cytosol
- This example is very similar to soluble protein in that the protein has an
ER signal sequence at its N-terminus.
- Here is the ER signal sequence in the diagram & that will act as a start-
transfer sequence – but in this case, unlike the soluble protein, there is
another domain that acts as a stop transfer sequence – this is also a
hydrophobic domain but since it comes after this start-transfer sequence
as it’s being transferred through this hydrophobic domain will act as a
stop-transfer & will actually compose the transmembrane domain. So this
transmembrane domain is actually a stop-transfer sequence so what
happens is the start-transfer sequence will start translocating the
translocation of the protein, through the ER membrane, through the
translocon & then this 2 hydrophobic domain is actually a stop transfer
sequence – the transfer will stop there, the synthesis of the peptide will
continue into the cytosol, the ribosome (not shown) continues to
synthesize it on the cytosol side then the signal peptidase cleaves off the
signal peptide & then the whole thing laterally diffuses into the ER
membrane – so both the signal peptide & the mature transmembrane
protein are laterally gated into the ER membrane – so both of these
laterally diffuse into the lipid bilayer & what happens is in this case, the
carboxy terminus ends up in the cytosol & the amino terminus ends up in
the ER lumen.
- This is the 1 way of getting a single-pass transmembrane protein into
the ER membrane.
- Animation: signal peptide there, there is the stop transfer stops there,
continues into the cytosol, signal peptidase cleaves it & this time the
lateral diffusion allows this to stay in the membrane. This is much more
effective b/c you can see how the ribosome, the SRP, the SRP receptor
are coming together to do this event. Note in this case, the carboxy
terminus ends up in the cytosol & the amino terminus in the ER lumen. Internal start-transfer sequence
(+) amino acids on cytosolic face
COOH inndR lumen
- This 2 example, in this case, you don’t have an N-terminus start-
transfer sequence, it’s an internal start-transfer sequence. So the protein
starts to be synthesized in the cytosol, the N-terminus comes out but it’s
not right at the N-terminus that the start-transfer sequence occurs but
- Now one of the differences is this will not be cleaved off of the protein
– an internal start-transfer sequence is not cleaved but this will also guide
this protein to the ER membrane so the SRP will bind to this, bring it to
the SRP receptor & then will start the transfer.
- Once the transfer is complete, the translocon again gates in the lateral
direction & allows this protein to laterally diffuse into the lipid bilayer.
- In this case, the orientation is important here – what happens is the N-
terminus is in the cytosol this time & the carboxyl terminus is in the ER
lumen (reverse of what we just saw). What’s important is that there are
positively charged AAs on one side of the start-transfer sequence &
negatively charged AAs on the other side of this internal start-transfer
sequence & the positively charged end of this internal start-transfer
sequence always orients itself on the cytosolic face of the ER membrane
so you can see here on the diagram that this end of the start-transfer
sequence would be positively charged & would be facing the cytosol –
that orientation means that the N-terminus will face the cytosol so what
happens is as the protein is being synthesized, this internal start transfer
sequence is exposed & will contortion itself in this way to make sure that
these positively charged residues end up on the cytosolic face of the ER.
Then what happens is the rest of protein is synthesized & pushed through
the translocon until the carboxy terminus ends up in the ER membrane
then the translocon will laterally gate to allow this transmembrane protein
to move into the ER membrane.
- So the orientation: the positively charged AAs of this internal start-
transfer sequence are always on the cytosolic face & what this means in
this case is that the carboxy terminus is in the ER lumen & the amino
terminus is in the cytosol.
internal start-transfer sequence
N-terminus in lumen*
COOH on the inside*
- Now we look at another example where we flip the charges on this
internal start-transfer sequence – again there is an internal start-transfer
sequence – this is the 3 example in terms of single-pass transmembrane
proteins. There is an internal start-transfer sequence but this time the way
it orients itself, note that the positive charges are towards the carboxy