Jada Cumberbatch

816010507

BIOC2162

Secretory and Circulatory Systems

Tutorial #2

1). Explain the differences between signal sequences and signal patches.
Directs signal are provided via the protein's movement through the system, and thus
govern its eventual position in the cell, are limited in each protein's amino acid sequence.
To be sorted to the correct organelle information proteins must be encoded within the
polypeptide.
Existing are two varieties of sorting signals – signal patch and signal sequences.
Signal sequences are three dimensional arrangements of molecules that form when the protein
folds up.
The amino acids that form the patch can be either from a single stretch of amino acids that are
exposed on folding or exist in regions of the polypeptide chain that are distinct from one another,
but which come together as the protein folds.
These patches generally persist in the finished folded and sorted protein.
Are continuous stretches of amino acids from approximately 15-60 amino acids in length.
These sequences are often removed once the sorting process has occurred.
2). Describe the structure of the Nuclear Pore Complex (NPC)
The double-membrane envelope is penetrated by nuclear pore complexes and is continuous with
the endoplasmic reticulum.
The ribosomes that are normally bound to the cytosolic surface of the ER membrane and outer
nuclear membrane are not shown.
The nuclear lamina is a fibrous meshwork underlying the inner membrane
Nuclear pore complexes seem to have four structural building blocks: column subunits, which
form the bulk of the pore wall; annular subunits, which are centrally located; lumenal subunits,
which contain transmembrane proteins that anchor the complex to the nuclear membrane; and
ring subunits, which form the cytosolic and nuclear faces of the complex.
In addition, fibrils protrude from both the cytosolic and the nuclear sides of the NPC. On the
nuclear side, the fibrils converge to form basketlike structures. Disordered domains of the core
proteins (not shown) are thought to extend toward the centre of the NPC, blocking the passive
diffusion of large macromolecules.
note that the inner and outer nuclear membranes are continuous at the edges of the pore
Note that some of the NPCs contain material in their centre, which is thought to be
macromolecules in transit through these NPCs.
Unstructured regions of the proteins lining the central pore form a tangled meshwork, which
blocks the passive diffusion of large macromolecules.
During active transport, however, even particles up to 39 nm in diameter can pass through
NPCs.
Data suggests that the NPC contains a pathway for free diffusion equivalent to a water-filled
cylindrical channel about 9nm in diameter and 15nm long.
Such a channel would only occupy a small fraction of the total volume of the pore complex.
Many cell proteins are unable to pass by diffusion through the NPC and therefore the nuclear
compartment and the cytosol can maintain different protein compliments
These proteins and RNA molecules must bind to specific receptor proteins that ferry them
actively through the NPCs

3). Explain how proteins are targeted and imported into the nucleus.
The selectivity of the nuclear import process residues in nuclear localization signals which are
present only in nuclear proteins.
These signals can be either patches or sequences
Nuclear import of proteins begins when the nuclear import receptors (NIR) recognizes
localization signal in the proteins
The NIRs are soluble cytosolic proteins and they drift around the cytosol looking for the nuclear
targeting sequence to latch unto and recognize and initiate the transport process
The NIRs generally have an S-shape and binds specific sequences at one site
Many other nuclear import receptors bind both to the NPC proteins and to a nuclear localization
signal on the cargo proteins that they transport
There are multiple types of NIRs binding multiple sequences
Some being signal patches so different tertiary structures would be present and must contain the
stretch of positive residues that generally indicate that the protein must be transported into the
nucleus (large run of positively charged amino acids indicate that the protein should be
transported to the nucleus)
Some proteins require adaptor proteins in order to bind to its NIRs
The adaptors are structurally related to the nuclear import receptors and recognize nuclear
localization signal that binds them to a nuclear import receptor
The import receptor also binds to nucleoporins some of which form fibrils that extend into the
cytosol from the rim of the nuclear pore complexes (cytosolic fibrils provide a pathway to the
complex allowing the receptor to enter)
NIRs are a family of related gene products
The import receptors are soluble cytosolic proteins.
Numerous nuclear import receptors attach to NPC proteins and to a nuclear localization signal on
the cargo proteins that they transfer.
Cargo proteins 1, 2, and 3 possess various nuclear localization signals, resulting in each binding
to different nuclear import receptors.
Cargo protein 4 needs an adaptor protein to attach to its nuclear import receptor. The adaptors
are physically associated to nuclear import receptors and identify nuclear localization signals on
cargo proteins. They possess a nuclear localization signal that joins them to an import receptor.
The import receptors attach to the protein to be transported as well as to nucleoporins which
form the fibrils that spread into the cytosol from the rim of the nuclear pore complexes
The fibrils contain many short amino acid repeats that contain Phe and Gly and are therefore
called FG-repeats.
FG recurrences aid as binding sites for the import receptors and line the path through the nuclear
pore complexes taken by the receptors and the cargo proteins.
The NIRs bind transiently moving along repeatedly binding and disassociating to adjacent
repeats leading proteins down into the nucleus
Once in the nucleus the receptor disassociates from the cargo protein and return to the cytosol

4). Explain the role Ran plays in the import and export nuclear proteins.
The nucleus is small compared to the rest of the cell, hence, anything being moved is being
concentrated as result the import and export of proteins in the nucleus results in different
concentrations in different areas of the cell and require an input of energy(+ ∆G )

The energy requirements are met by GTP hydrolysis via the monomeric GTPase, Ran
Ran is active when GTP is bound (Ran-GTP) and inactive when GDP is bound (Ran-GDP)
They are like molecular switches, GAP and GEF are used to switch between both phases
GAP-GTPase activating protein increases the rate of hydrolysis converting the bound GTP
to bound GDP-GEF exchanges with GDP with GTP
Ran-GAP is in the cytosol while Ran-GEF is associated with chromatin so that it can be in the
nucleus
Across the membrane Ran is in different states in different places
Ran-GEF would exchange GDP for GTP in the Nucleus when it goes into the cytosol, the Ran-
GAP would inactivate GTPase and convert the GTP to GDP forming Ran-GDP (seen in the
cytosol)
Ran-GDP is brought into the nucleus by its own import receptor which is specific for GDP-
bound conformation of Ran
The Ran-GDP receptor is structurally unrelated to the main family of nuclear transport receptors;
however, it binds to FG-receptor in the NPC proteins and bounces down the NPC bringing the
cargo protein into the nucleus
Upon arrival into the nucleus Ran-GTP binds to the NIR displacing the cargo protein but
changes the conformation of the NIR causing the protein to fall off (unloaded state)
The receptor contains repeated alpha-helix motifs that stack on top of each other forming a
flexible spring like structure that can adopt multiple conformations in response to binding of
cargo proteins and Ran-GTP
The Ran-GTP and cargo proteins bind to various regions of the coiled spring
The Ran-GTP is partially covers a cargo binding in the Ran-free state (the protein falls off when
it is bound)
The Ran-GTP bound NIR then exits the nucleus (leaving potential) bouncing up the FG-repeats
up the nuclear pore changing the GTP to GDP causing Ran to fall off as it only binds to the GTP
bound state
The Ran is now free to bind to another cargo protein to transport it to the nucleus and repeat
cycle
NIRs are loaded in the cytosol and unloaded in the nucleus in a repeating cycle
Localization of Ran-GDP in the cytosol and Ran-GTP in the nucleus results from the localization
of two Ran regulatory proteins: Ran GTPase-activating protein (Ran-GAP) is located in the
cytosol and Ran guanine nucleotide exchange factor (Ran-GEF) binds to chromatin and is
therefore located in the nucleus.
Movement through the NPC of loaded nuclear transport receptors occurs along the FG-repeats
displayed by certain NPC proteins. The differential localization of Ran-GTP in the nucleus and
Ran-GDP in the cytosol provides directionality (red arrows) to both nuclear import and nuclear
export .
Ran-GDP is imported into the nucleus by its own import receptor, which is specific for the GDP-
bound conformation of Ran.
The Ran-GDP receptor is structurally unrelated to the main family of nuclear transport receptors.
However, it also binds to FG-repeats in NPC proteins and hops across the NPC.
5). Explain why nuclear proteins retain their signal sequences/patches.
In mitosis nuclear proteins that are not bound to membranes or chromosomes intermix entirely
with the cytosol of the dividing cell.
In late anaphase the nuclear envelope reforms and the NPCs reassemble and start actively re-
importing proteins that contain nuclear localization signals.
Nuclear localization signals are not cleaved off after transport into the nucleus.
Nuclear proteins need to be imported repeatedly once after every cell division.
In contrast once a protein has been imported into any of the other membrane-enclosed organelles
it is passed on from generation to generation within that compartment and need never be
translocated again
The signal sequence on these molecules is often removed following translocation.

6). Explain how proteins are imported into the mitochondrial matrix from the cytosol.
Include details of the energetic requirements of the process.
The information required for transport of proteins into the mitochondrial matrix is encoded in the
primary sequence of the protein given at the N-terminus
The first 18 amino acids of the cytochrome oxidase (COX) which is a large multiprotein
complex located in the inner mitochondrial membrane where it functions as the terminal enzyme
in the electron transport chain serves as a signal sequence for import of the subunit into the
mitochondrion
The signal tends to form an amphipatic alpha helix with the side chain being positively charged
The two complexes required are TOM and TIM
TOM contains a translocation channel that proteins move and receptors that are known as (three)
TOM 20, TOM 22 AND TOM 17
TOM 22 and TOM 20 moves the vast majority proteins and TOM 17 moves a subset of proteins
There is a protein complex called SAM in the outer membrane that plays a role in outer
membrane protein assembly
There are three complexes in the inner membrane TIM 23 that has two extensions that come out
into the inner membrane space that mediate interaction between TIM 23 and TOM complex
It contains an import ATPase associated of the matrix side of the membrane
The main part of the ATPase is an Hsp70 (chaperon)
There is a TIM 22 complex that arranges a subset of proteins into inner the membrane and the
OXA complex
The protein interacts with the complexes depending on where they are going
The protein is produced, it has the N-terminal amphipatic alpha helix, this is a post translational
event, so the protein is made completely and is released into the ribosome before it is moved
anywhere
The targeting sequence is recognised, and it binds to the receptor
After it binds to the receptor it threads through the space in the TOM complex
As it goes into the matrix the TIM 23 assists it to move across the membrane
Transient Association of TIM 23 and TOM 20 allows the protein to move across them at the
same time
The protein then arrives at the matrix where the signal sequence (sorting sequence is removed) of
the peptide is cleaved by the proteases (two proteases that sit in the matrix) and the protein then
folds (or the protein can fold before they cleave)
Moving from the cytosol to the matrix requires energy as the protein moves against its
concentration gradient (the protein is snaking through in an unfolded state)
The protein moves in an import competent conformation (unfolded state) that requires energy
Cytosolic Hsp70 holds the protein in an unfolded state via ATP hydrolyses by clamping down
with the ATP and releasing when hydrolyses to ADP as the protein threads through the TOM
complex
Due to respiration there is a proton motive force across the inner membrane at TIM 23 complex
(respiration occurs with the pumping of protons in the mitochondria)
This creates an electric field for the protein to pass through causing electrophoresis to occur as
the amphiphilic alpha helix signal sequence is positively charged and would move to the
oppositely changed pole
The intermembrane space is positively charged, and the matrix space is negatively charged so the
signal sequence electrophoreses through the TIM complex
The rest of the protein does not have a positive change and so would not undergo electrophoresis
As a result, more energy is required and is provided by a mitochondrial Hsp70 that is associated
by a mitochondrial Hsp70 that is associated with the TIM complex
As the protein enter the TIM complex the Hsp70 binds to it pulls and releases repeatedly in an
animal an ATP dependent manner (conformational change driven via the binding of ATP via
Hsp70)
When the protein is completely in the matrix the signal sequence is then cleaved and the protein
is folded
Energy may also need to refold those proteins that do not fold properly done by a protein
chaperoning complex that sits and waits in the mitochondrial matrix.
7). Explain how proteins are sorted to the mitochondrial outer and inner membranes
Porins (porins) go the outer membrane where it fully folds to produce an indirect route, coming
into the mitochondrial intermembrane space before it heads out into the outer membrane space
The protein is recognized by TOM but it does not allow it to be recognized via TIM complex but
rather interact with chaperones in the intermembrane space (Hsp70)
The Hsp70 provide the driving force for TOM to bring it in holding them in the IMS an import
competent conformation
The SAM complex recognizes then recognizes it and threads it into the outer membrane to
produce the porin
There is a stop transfer sequence that is hydrophobic in nature that is about 20 amino acids in
length (can form a membrane spanning helix)
The signal sequence pokes into the matrix as it electrophoresis across as it is cleaved it is
discarded, the rest of the protein is not transported because this hydrophobic sequence behind it
stops the process
A process called lateral gating which causes the complex to open in the membrane and throws
the protein out which is the stop transfer sequence that moves into the membrane as a
transmembrane helix
The process is the same for the inner membrane, but the protein is instead recognised by the
matrix side of the OXA complex and assemble into the inner membrane and forms the inner
membrane protein
This route is also taken by the proteins in the mitochondrial genome
Also, the two pathways illustrated before they are released into intermembrane space by a second
signal peptidase, which has active site in the intermembrane space and removes the hydrophobic
signal sequences
Some of the intermembrane proteins come in through SAM and go via chaperones while others
come in as stop transfer proteins before the helix goes into membrane there is a protease located
in the intermembrane space that cuts between the stop transfer sequence and the mature protein
releasing the mature protein into the intermembrane space
The metabolite (ANT) transporters contain internal signal sequence and snake through the TOM
complex as a loop
TOM does not associate with TIM 22 and TIM 23
The protein moves through TOM into the inter membrane space where it interacts with
chaperones that hold it in an unfolded state
Binding to the chaperones in the intermembrane space guides the protein to the TIM 22 complex
that is specialized for the insertion of the multipass inner membrane proteins (TIM 22 recognized
internal signal sequence grabs a hold of the protein and threads into the inner membrane)
This requires the membrane potential as well as but not the mitochondrial Hsp70 or ATP
hydrolysis
However, the Hsp70 is needed both in the cytosol and the intermembrane space to hold the
proteins in their unfolded conformations

8). Describe the structure of the signal recognition particle (SRP). What signal does it
recognize and how is it able to accommodate a wide array of protein sequences?
Recognizes proteins that are destined for the ER
SRP (mammalian) is an extended complex comprising of six protein subunits as well as a single
RNA molecule (SRP RNA). Methionine resides are found plentifully within the binding pockets
which causes it to be flexible so a variety of sequences can fit into different binding pocket to
recognize lots of different combinations of amino acids
However, it recognizes long runs of hydrophobicity about 20 amino acid in length
The RNA in the particle may mediate an interaction with ribosomal RNA
It is flexible with two domains (a translational pause domain and signal sequence binding
pocket)
There is a ribosome start making the protein and the protein has a signal sequence on the N-
terminus that says go to the ER and is recognized by SRP
One end of the SRP binds to the ER signal sequence on the growing polypeptide chain, bends on
the hinge region while the translational pause domain binds to the ribosome itself then pauses
translation (translation used as the driving force for import)
It holds the process until there is reassemble on the ER and we restart the process once the SRP
is released
There is a signal sequence that is recognized (long stretch of hydrophobicity) via the ribosome
causing it to move it to move to the membrane and associate with the translocase and start
pushing the protein through as it is made
SRP recognizes the proteins that are destined for the ER

9). Explain the mechanisms by which proteins are targeted to and inserted into the
membrane of the E.R. Include details of orientation and multi-pass membrane protein
insertion.
This is co-translational as the protein is made while coming off the ribosome (no driving force as
the protein is made and pushed through via the addition of amino acids, it’s in an unfolded state)
This occurs on the rough ER (it’s transient)
The ribosomes associated with the rough ER are moving between the ER and the cytosol, they
move to the ER when they start to translate a protein that is directed to the ER
This is not a special set of ribosomes associated with the ER as general ribosomes provides them
start to make a ribosome, it will move the ER and associate with it
A signal sequence is recognized, the ribosomes move to the membrane associates with the
translocase and starts pushing protein through as it makes it cleave off signals and it release
proteins into the ER
Upon arrival to the ER, the SRP receptor protein as it is looking for the SRP bound to the signal
sequence
These two come together and you recognize that the ribosome is making a protein and it should
go to the ER
The entire process is assembled unto the translocase (SEC 61)
It sits on the translocase releases the SRP, SRP receptor, translation begins and the driving the
protein through
The signal sequence is not only recognized by the SRP it also plays a role in assemble on the
transporter
When the signal peptide comes in and fits into, opens it, removes the plug and the growing chain
keeps growing, the growing protein disassociates
It’s a very tight association when the ribosome sitting on top of the SEC 61
translocated
There are post translational events, that make the protein and move it through, this is done a lot
by yeast and by a small amount in humans
To do this a lot of complexes are required as they hold it in an unfolded conformation outside
and inside PIP is required as it hydrolyses ATP and pulls the protein in (Hsp70)
For a single span protein the process begins as before until it gets to the stop transfer, then it gets
to lateral gating, the SEC 61 opens side ways into the membrane, it throws it out, the signal
peptide cleaves off, this will be degraded rapidly
The signal transmembrane protein, alpha helix with the N-terminal on the inside and the C-
terminal on the outside
Multipass proteins have a shorter transfer and stop transfer internal sequences
The orientation in which the start transfer goes is determined by changes around it
Once the start sequence is in the protein feeds through until the stop transfer sequence is met,
causing the membrane passes to be present leading to lateral gating where the protein is released
into the membrane
More than one pass would have multiple start and stop sequences
The first start sequence would set the orientation based on changes present (negative to the
lumen and positive to the cytosol)
The protein is threaded multiple times as each start and stop sequence is read
There is no difference between the start and stop sequence ( they are both long stretches of
hydrophobicity it is just the order that they are read in that makes them different)