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The organelles that populate a cell allow the eukaryotic cell to concentrate critical
reactants to promote specific chemical reactions and other biological processes. This
degree of spatial specialization also presents a challenge since the various proteins
required in each organelle compartment must cross that organelle membrane from
the cytosol. Don’t forget that protein translation occurs in the cytosol. In addition,
there must be some mechanism by which proteins are specifically targeted to their
cellular destinations. There is not one simple way cells move from organelle to
organelle, or cytoplasm to organelle. We’re going to discuss three.
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Proteins are imported into organelles by different mechanisms, three of which we’ll discuss in this
lecture.
1. Proteins and nucleic acids move into (and out of) the nucleus through channels in the membrane
called nuclear pores. These are protein-lined structures that span both the inner and outer
membranes of the nuclear envelope. Nuclear pores are large enough that ions and small molecules
(e.g. metabolites) can freely diffuse through them, but proteins and nucleic acids cannot move
through the pore without assistance. This assistance takes the form of specialized proteins (a.k.a
transport receptors) that bind to cargo and facilitate transport through the nuclear pore.
Interestingly, proteins do not need to unfold to pass through the nuclear pore and can therefore
cross while maintaining their higher order 3-dimensional shape. In contrast to nuclear transport,
proteins that move into chloroplasts, the ER, and mitochondria must do so in an unfolded state, i.e.
as linear chains of amino acids. The different channels that allow this kind of transport are much
smaller than nuclear pores and can only accommodate linear polypeptide chains. We will soon see
how this is achieved for those proteins that must cross the membrane of the endoplasmic reticulum.
On the other hand, proteins move within the secretory pathway - between the ER, Golgi, plasma
membrane, and lysosomes - inside lipid vesicles. As we will see, vesicles are loaded with cargo proteins
from the lumen (interior space) of one compartment, and discharge their cargo into a second
compartment. We will explore an example of vesicle transport by discussing how proteins traffic from
the ER to the Golgi and on to the plasma membrane.
Let’s now turn to the issue of how proteins are targeted to the correct compartment.
Signal sequences are stretches of amino acids within protein sequences that play an
essential role in targeting proteins to their correct destinations within cells. Typical
signal sequences act like an address to specify the correct destination for a protein,
and can either be contiguous stretches of amino acids or they can consist of amino
acids distributed throughout the protein sequence, which are clustered together in
the folded structure of the protein to form a signal patch. While the signal sequences
that target to a particular compartment are generally not identical, there are
chemical characteristics of particular classes of signal sequence. For example, ER
signal sequences tend to include a stretch of hydrophobic amino acids, while nuclear
signal sequences tend to have stretches of positively charged amino acids.
Signal sequences are typically recognized by so-called transport receptors and at least
one transport receptor exists for each compartment. These free floating receptors
are involved in targeting proteins typically because the receptor protein binds a
another protein on the destination compartment that is unique to that compartment.
Some proteins have more than one signal sequence and these can function
sequentially. For example, one sequence can dictate entry into the secretory system
i.e. to cross into the ER, while an independent sequence can dictate retention in the
endoplasmic reticulum.
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Here is an example where an ER retention signal is used for quality control. Some
proteins as they fold and assemble into their mature form cover up an ER retention
signal. Once in the ER, if the protein has undergone appropriate assembly into the
mature form, they can exit the ER and move to their final destination (e.g. the dimer
on the hypothetical example here). On the other hand, if proteins don’t fold properly
or don’t assemble with their component parts, the ER retention signal is exposed and
the protein is not trafficked to its final destination – it remains in the ER.
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Here is an example: potassium channels! These are multimers e.g. this one is a
tetramer, meaning it has four subunits per functional channel. If the tetramer does
not assemble correctly, an ER retention signal is exposed and the channel is not
trafficked to the membrane. It remains in the ER.
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Signal sequences are both necessary and sufficient for proper protein targeting. We
know this from experiments in which mutant proteins are expressed in cells either
lacking their appropriate signal sequence or having an inappropriate signal sequence.
For example, a protein that is normally targeted to the ER lumen will fail to get there
and will remain in the cytosol if it lacks the appropriate signal sequence. Since the
only change to that protein is the absence of the signal sequence, this experimental
result indicates that the signal sequence is necessary for proper targeting. In a
second experiment, a protein that normally resides in the cytosol has an ER signal
sequence added and as a result it targeted to the ER lumen. This result indicates that
the signal sequence is sufficient for targeting since that is the only
addition/modification of the protein.
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Let’s take a look at the nucleus. The photo of the left is a picture of a cell with the
nuclear pores stained green and red. You can see that the nucleus is absolutely
covered in pores, which is necessary to handle the large amount of traffick in and out
of the nucleus.
As an aside, this photo was taken by Stefen Hell, who was one of three recipients of
the 2014 Nobel Prize in Chemistry for developing approaches to see higher resolution
structures under the microscope.
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Nuclear pore looks like this. These are protein-lined structures that span both the inner and outer
membranes of the nuclear envelope. Nuclear pores are large enough that ions and small molecules
(e.g. metabolites) can freely diffuse through them, but proteins and nucleic acids cannot move through
the pore without assistance. This assistance takes the form of specialized proteins (a.k.a transport
receptors) that bind to cargo and facilitate transport through the nuclear pore. Interestingly, proteins
do not need to unfold to pass through the nuclear pore and can therefore cross while maintaining their
higher order 3-dimensional shape.
Proteins and nucleic acids move into (and out of) the nucleus through channels in the membrane called
nuclear pores. These are protein-lined structures that span both the inner and outer membranes of the
nuclear envelope. Nuclear pores are large enough that ions and small molecules (e.g. metabolites) can
freely diffuse through them, but proteins and nucleic acids cannot move through the pore without
assistance. This assistance takes the form of specialized proteins (a.k.a transport receptors) that bind to
cargo and facilitate transport through the nuclear pore. Interestingly, proteins do not need to unfold to
pass through the nuclear pore and can therefore cross while maintaining their higher order 3dimensional shape.
In contrast to nuclear transport, proteins that move into chloroplasts, the ER, and mitochondria must do
so in an unfolded state, i.e. as linear chains of amino acids. The different channels that allow this kind of
transport are much smaller than nuclear pores and can only accommodate linear polypeptide chains.
We will soon see how this is achieved for those proteins that must cross the membrane of the
endoplasmic reticulum.
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http://www.stickyworldwide.com/wp/wpcontent/uploads/2012/06/ica_train_of_thought_rev.jpg
Want Some Meat on the Green Line was actually the Haymarket stop, where one
butcher in particular would constantly intercept shoppers and passers-by with his
toothy sales pitch, “Want some meat?”
Now Cough on the Orange Line Was the stop for the New England Medical Center.
Ask Not on the Red Line was the JFK Museum and Library
Gooey Center on the Blue Line replaced Government Center.
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hydrophobic interactions between Phe-repeats are relatively weak,
flexible, filamentous Phe-Gly nucleoporins
Sieve like structure that helps keep out proteins and RNA.
Phe residues are sufficiently exposed to allow rapid binding of transport receptors.
The aggregates in fact display an organization into parallel or antiparallel substructures,
comprising between two and four monomers each, which are connected by β-bridges. Our
simulations thus suggest that FG repeat aggregation is not governed by hydrophobic Phe-Phe
interactions alone, but rather that the formation of secondary structural elements via
backbone-backbone hydrogen bonding also plays an important role in the aggregation process.
hydrophobic Phe side chains indeed drive initial aggregation, but that the final structure of FG
repeat aggregates is largely determined by interrepeat backbone hydrogen bonds that bring the
cluster into a proto-β strand state.
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K+ channel
- Highly selective for K+
- Very small: one ion passing through the membrane
- Single membrane and 4 identical channel protein.
Nuclear pore
- HUGE: 450 proteins!
External diameter of about 120 nm (30 times the size of a ribosome)
Channel diameter 25 nm
0.1 nm (nanometer) diameter of a hydrogen atom
0.8 nm Amino Acid
2 nm Diameter of a DNA Alpha helix
4 nm Globular Protein
6 nm microfilaments
7 nm thickness cell membranes
20 nm Ribosome
50 nm Nuclear pore
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Quasi selective
The problem: small proteins don’t cross the nuclear pore but large proteins do!!
Answer:
active (selective/ regulated) passage of macromolecules, proteins and RNAs
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K+ channel
- Very small: one ion passing through the membrane
- Single membrane and 4 identical channel protein.
- Single ion = < 1nm
Nuclear pore
External diameter of about 120 nm (30 times the size of a ribosome)
Channel diameter 25 nm or more = 100s time wider than K+ channel.
-
Quasi selective
Metabolites can move.
Proteins and nucleic acids can’t move on their own.
A small protein like a histone cannot move through – but other small proteins like
GFP can.
- Ribosomes come out and mRNA comes out but a tRNA does not go in.
The problem: small proteins don’t cross the nuclear pore but large proteins do!!
Answer:
active (selective/ regulated) passage of macromolecules, proteins and RNAs
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Answer: active (selective/ regulated) passage of macromolecules, proteins and RNAs
Pore is not empty
Proteins line the the pore and have long
flexible, filamentous Phe-Gly nucleoporins
Sieve like structure that helps keep out proteins and RNA.
hydrophobic interactions between Phe-repeats are relatively weak,
Phe residues are sufficiently exposed to allow rapid binding of transport receptors.
The aggregates in fact display an organization into parallel or antiparallel substructures,
comprising between two and four monomers each, which are connected by β-bridges. Our
simulations thus suggest that FG repeat aggregation is not governed by hydrophobic Phe-Phe
interactions alone, but rather that the formation of secondary structural elements via
backbone-backbone hydrogen bonding also plays an important role in the aggregation
process.
hydrophobic Phe side chains indeed drive initial aggregation, but that the final structure of FG
repeat aggregates is largely determined by interrepeat backbone hydrogen bonds that bring
the cluster into a proto-β strand state.
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flexible, filamentous Phe-Gly nucleoporins
Sieve like structure that helps keep out proteins and RNA.
hydrophobic interactions between Phe-repeats are weak,
backbone-backbone hydrogen bonding also plays an important role in the
aggregation process.
a proto-β strand state.
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Phenylalanine side chains exposed, it also facilitates and probably accelerates binding
to transport receptors
Speed up movement through the pore 100x at least.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877321/#!po=46.8750
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Proteins are imported into organelles by three different mechanisms.
Proteins and nucleic acids move into (and out of) the nucleus through channels in the membrane called
nuclear pores. These are protein-lined structures that span both the inner and outer membranes of the
nuclear envelope. Nuclear pores are large enough that ions and small molecules (e.g. metabolites) can
freely diffuse through them, but proteins and nucleic acids cannot move through the pore without
assistance. This assistance takes the form of specialized proteins (a.k.a transport receptors) that bind to
cargo and facilitate transport through the nuclear pore. Interestingly, proteins do not need to unfold to
pass through the nuclear pore and can therefore cross while maintaining their higher order 3-dimensional
shape.
In contrast to nuclear transport, proteins that move into chloroplasts, the ER, and mitochondria must do so
in an unfolded state, i.e. as linear chains of amino acids. The different channels that allow this kind of
transport are much smaller than nuclear pores and can only accommodate linear polypeptide chains. We
will soon see how this is achieved for those proteins that must cross the membrane of the endoplasmic
reticulum.
Finally, proteins move within the secretory pathway - between the ER, Golgi, plasma membrane, and
lysosomes - inside lipid vesicles. As we will see, vesicles are loaded with cargo proteins from the lumen
(interior space) of one compartment, and discharge their cargo into a second compartment.
We will explore an example of vesicle transport by discussing how proteins traffic from the ER to the Golgi
and on to the plasma membrane. As you will see later on, all of this information is critical for understanding
how HIV co-opts the host cell to produce functional viral particles during an infection.
If signal sequences are addresses then ER and golgi are perhaps the PO and the mail
trucks.
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Insulin from the pancreas – cells to take up glucose, cells to make fat.
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Love. Trust. social Bonding. Parenting
Milk to feed an infant.
plasma, salivary, or urinary oxytocin and brain oxytocin activity,
Made in the brain e.g. pituitary and hypothalamus.
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Patterning
Build an animal
Limbs
plants
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If signal sequences were like addresses then ER and golgi
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Break Out followup:
Cytoplasmic extracts can be made which contain ribosomes, tRNA, ATP, GTP, and all the
requisite amino acids. With the addition of purified mRNA, these extracts will support protein
translation in vitro.
When cells are broken up or gently homogenized, the endoplasmic reticulum disperses into
small closed membrane sacs called ER microsomes. In this experiment we can assume that the
ER microsomes maintain the orientation of the membrane in terms of the luminal (inside)
versus cytoplasmic (outside) faces.
The above two procedures are combined to assess the targeting of a secreted protein to the
lumen of the ER. You synthesize mRNA encoding a large protein that is normally secreted from
the cell fused with green fluorescent protein (GFP). The GFP fusion protein takes about twenty
minutes to be translated and is detectable in the microscope based on GFP fluorescence. In
Sample A, fusion protein mRNA is added to a cytoplasmic extract without any membrane
components. After one hour, ER microsomes are added to the extract, which is then incubated
for another hour. In Sample B, the same experiment is repeated except the mRNA and ER
microsomes are added to the cytoplasmic extract at the same time and incubated for one hour
total. In the microscope we can see that Sample A has diffuse GFP fluorescence in the extract
with no signal in the microsomes. In contrast, Sample B exhibits significant fluorescence in the
microsomes and none in the extract.
So what of the three possible interpretations?
Option A is not likely since in Sample B the GFP fusion protein clearly crosses the
membrane of the ER microsome to accumulate in the lumen.
Option B is a reasonable interpretation since in Sample B the presence of the ER
microsomes while translation is occurring correlates with transport into the lumen.
In Sample A, proteins that have already been translated appear to be incapable of
crossing into ER microsomes added later.
Option C is not likely since Sample A does exhibit GFP fluorescence in the extract
indicating that translation has occurred, but no signal is present in the microsomes.
Furthermore, in both samples the same amount of time is spent in the presence of
microsomes, so if transport occurred in Sample B, there was enough time for it to
occur in Sample A by a post-translational mechanism.
If we take a closer look at what occurred in each of the samples, we see that in
Sample A complete fusion proteins were successfully translated by free ribosomes in
the extract, but these intact proteins were not able to cross the ER membrane posttranslationally. Furthermore, since this is a secreted protein that is meant to enter
the ER lumen, the appropriate N-terminal signal sequence is present but still fails to
support transfer across the ER membrane after complete translation has occurred.
In Sample B, translation begins on free ribosomes in the extract, which are then
targeted to the ER membrane (via the signal sequence), where translation continues
together with simultaneous transport across the membrane. This is the process of cotranslational transport where the ribosome is docked with a translocation channel
that allows the newly synthesized polypeptide chain to enter the ER lumen as it is
being made. We will look at this process in greater detail in a few slides.
In both diagrams you will note that multiple ribosomes are translating the same
mRNA simultaneously. This structure is called a polyribosome and results from the
fact that after one ribosome has initiated translation and moved down the mRNA,
another ribosome can initiate while the first is still translating. This ultimately results
in several ribosomes moving down the mRNA and translating the same protein in a
staggered row.
In the cell, translating ribosomes can exist free in the cytosol or associated with the
ER membrane. Whether a ribosome falls into one category or another entirely
depends on the protein that it is synthesizing. In the case of cytoplasmic proteins,
such as actin, there is no targeting sequence for the ER membrane and the entire
translation process occurs in the cytosol where the completed protein is released. If
the mRNA encodes a secreted protein or a transmembrane protein, then an ER signal
sequence is present which targets the translating ribosome to the ER membrane -resulting in a membrane-attached ribosome. Membrane-attached ribosomes are
what give the rough ER its unique appearance in the electron microscope. Ribosomal
subunits (small and large) are recycled after each translation event and depending on
which mRNA they happen to initiate translation on, will become either free or
membrane-attached. Thus, the two categories of ribosomes are functionally
equivalent and draw from a common pool of ribosomal subunits in the cytosol.
Let’s consider targeting of a protein to the ER. This protein can be either a free floating protein that ends up in the ER
lumen or a transmembrane protein that is inserted into the ER membrane. In the case of a free floating protein it
simply crosses the ER membrane during co-translational transport and is released into the ER lumen. Later we will
discuss how a transmembrane protein ends up being inserted into the ER membrane.
As is the case with all proteins, the translation process begins in the cytosol with the initiation of ribosome assembly
and the start of translation. The signal sequence is often at the N-terminus or close to the N-terminus and is
therefore translated early in the process. The signal sequence is recognized by a signal recognition protein (SRP) in
the cytosol that binds to both the sequence as well as to the ribosome. The binding of the signal recognition protein
halts translation, and the stalled complex of the mRNA, ribosome, nascent protein chain, and the SRP then binds to
proteins embedded in the ER membrane. These proteins include a translocation channel, and when the SRP leaves
the complex, the signal sequence is transferred to the wall of the translocation channel. This relieves the block to
translation, and as translation resumes the polypeptide chain passes through the channel and enters the lumen of
the ER (in the case of a free floating protein).
The signal recognition particle displays three activities in the process of cotranslational targeting: (I) binding to signal
sequences emerging from the translating ribosome, (II) pausing of peptide elongation, and (III) promotion of protein
translocation through docking to the membrane-bound SRP receptor (FtsY in prokaryotes) and transfer of the
ribosome nascent chain complex (RNC) to the protein-conducting channel
Translation proceeds with the synthesis of the first transmembrane domain (TM), which exits laterally as an α-helix
into the lipid bilayer with retention of the positively charged N-terminal domain (according to the positive-inside rule)
on the cytoplasmic side of the membrane
SRP has a large hydrophobic pocket lined with methionine (unbranched, flexible side chains) which makes it flexible
enough to bind many different signal sequences. (Dowhan and Bogdanov, Ann. Rev. Biochem., 2009, 78, 515)
Movie of SRP
1. SCANNING - Survey ribosome
2. TARGET - Binds SS and arrests translation
3. TRANSFER - SRP receptor & Brings protein to txlocon.
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driving force for passage through the translocon comes from the ribosome; the translocon is
an inert channel.
Signal sequence
Charge and charge distribution
What would you predict for charge and hydrophobicity effects?
Start transfer sequences. These are of two types:
N-terminal signal peptide sequence - a cluster of about 8 hydrophobic amino acids at
the N-terminal end of a protein. This sequence remains in the membrane and is
cleaved off of the protein after transfer through the membrane.
Internal start transfer sequence. Similar to a signal sequence, but located internally
(not at the N terminal end of the protein). It also binds to the SRP and initiates
transfer. Unlike the N-terminal signal sequence, it is not cleaved after transfer of the
protein.
Stop transfer signal. This is also a sequence of about 8 hydrophobic amino acid residues. It
follows either a N-terminal signal sequence or a start transfer sequence. The stop transfer
signal is a membrane crossing domain. It remains in the membrane. The peptide is not
cleaved.
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Now that we know how proteins are targeted to the ER with signal sequences, we need to understand how they
move from the ER to other compartments. Many proteins in the ER travel next to the Golgi apparatus. These so
called cargo proteins consist of both free floating proteins in the ER lumen as well as transmembrane proteins in the
ER membrane.
How do proteins move from one membrane-bound compartment to another? Remember, it is energetically
favorable for transmembrane proteins to remain in the ER membrane and not in water; likewise it is favorable for
soluble proteins to remain free floating in the ER lumen. Thus, there is no energetically favorable way for these
proteins to cross the membrane of one compartment and enter or cross the membrane of another on their own. The
answer is to use transport vesicles that bud off of from the membrane of one compartment and fuse with the
membrane of another. So, vesicles carry cargo from the ER to the Golgi, and from the Golgi to various destinations
including endosomes, lysosomes, and the plasma membrane.
Let’s now turn to the issue of how proteins are targeted to the correct compartment. Signal sequences are stretches
of amino acids within protein sequences that play an essential role in targeting proteins to their correct destinations
within cells. Typical signal sequences act like a zip code to specify the correct destination for a protein, and can
either be contiguous stretches of amino acids or they can consist of amino acids distributed throughout the protein
sequence, which are clustered together in the folded structure of the protein to form a signal patch. While the signal
sequences that target to a particular compartment are generally not identical, there are chemical characteristics of
particular classes of signal sequence. For example, ER signal sequences tend to include a stretch of hydrophobic
amino acids, while nuclear signal sequences tend to have stretches of positively charged amino acids.
Signal sequences are typically recognized by so-called transport receptors and at least one transport receptor exists
for each compartment. These free floating receptors are involved in targeting proteins typically because the
receptor protein binds a another protein on the destination compartment that is unique to that compartment.
Imagine translation via SRP and the translocon to make a txmembrane protein.
Let’s trace the fate of the protein.
Vesicles do not form spontaneously
‘coat’
Adaptor
cargo
Transport vesicles clearly bud from one compartment and fuse with another. As they do so they carry
soluble proteins, transmembrane proteins and other membrane components like lipids from the donor
compartment to the lumen and membrane of the target compartment.
During the process of budding and fusion the topology (orientation in the membrane) of the protein is
maintained. For example, for transmembrane proteins that begin the transport process with a domain
in the lumen of the donor compartment (the domain that starts out exposed to the lumen is shown in
red; the transmembrane region is shown in black), end up displaying that domain on the cell surface. In
this manner, the result of various enzymatic processing steps that occur in the ER lumen (e.g. the
addition of carbohydrates a.k.a. glycosylation) ends up displayed on the surface of the cell. This is
important for the appropriate function of many proteins, including some HIV proteins. This also means
that any part of a protein that is exposed to the cytosol will remain exposed to the cytosol for the entire
vesicle transport process.
We know that protein targeting depends on specific signal sequences of amino acids that bind to
transport receptors in the cytosol, which in turn facilitate the specific association of the protein with its
target organelle or membrane. It is also clear that some proteins are subject to a series of targeting
events before they reach their final destination. Such proteins include all secreted proteins, plasma
membrane receptors, and some proteins destined for lysosomes. These proteins are first targeted to
the ER, but are then undergo a secondary targeting to vesicles moving proteins from the ER to the
Golgi, and then on to their final destinations. The ER is the most extensive membrane system in the
cell. Unlike other organelles, it also serves as the entry point for proteins destined for other organelles,
as well as the plasma membrane. Proteins that are targeted to lysosomes, the Golgi apparatus, and the
plasma membrane must all enter the ER first.
Three kinds of proteins are transferred from the cytosol to the ER:
(1) Proteins destined for secretion from the cell, all of which are eventually targeted to the plasma
membrane in vesicles.
(2) Transmembrane proteins that are partially translocated across the ER membrane and end up
embedded in it.
(3) Free floating proteins that will either remain in the ER lumen or end up in lysosomes.
All three kinds of proteins are directed to the ER by signal sequences. As we discovered in the breakout
question, unlike proteins that enter other organelles, most proteins that enter the ER do so before they
are completely translated. As the polypeptide is being synthesized it moves across the ER membrane
through a specialized protein channel.
If signal sequences were like addresses then ER and golgi
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