to Cytosol ER Transport
Reading Assignment: For 5th Ed: 723-732
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Cytosol-to-ER Transport
Structure and function of ER
How is a cargo protein transported into the ER?
What is the machinery mediating cytosol-to-ER transport?
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Structure of ER
a lipid bilayer membrane enclosing lipid bilayer membrane enclosing a space called ER lumen existing in the forms of tubes and sacs in the forms of tubes and sacs throughout the cytosol tubes interconnected via three-way junctions constituting more than 50% of the total membrane in most cells a dynamic structure
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Dynamics of ER ER is a highly dynamic organelle (e.g. a potential role in neuronal signaling)
Movie 12.4 12.4
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Two types of ER membrane domains
polyribosomes attached where the biosynthesis t h bi of proteins takes place no ribosomes attached where the biosynthesis t h bi of lipids takes place
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Rough appearance of the rough ER is due to the presence of ribosomes on the ER membrane from the cytoplasmic side
Note the presence of ribosomes on the nuclear outer membrane as well (remember that the nuclear outer membrane is connected to the ER)
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Polyribosomes
Polyribosomes: multiple ribosomes translating a single mRNA at the same time. Polyribosomes are generated because another ribosome can start translation of a mRNA molecule before the previous ribosomes finish the translation (a common feature of mRNA translation) polyribosome
nascent polypeptide chains of different lengths
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Different localizations of two pools of polyribosomes
Depending on the nature of the translated proteins, there are two pools of polyribosomes in cell pools of polyribosomes in a cell.
Cytoplasmic polyribosomes: Polyribosomes translating cytosolic proteins remain in the cytosol
ER-associated polyribosomes: Polyribosomes translating a protein targeted to ER are recruited to the ER membrane from the cytosol (to be discussed later)
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homogenization: the process of breaking up a cell & making the lysate uniform microsomes: small vesicles generated from fragmented ER following homogenization sucrose gradient centrifugation: a type of centrifugation used to separate the subcellular structures based on their densities.
Rough & smooth ER can be separated by their different densities
increasing density density
Rough ER has a higher density due to its associated polyribosomes
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Functions of ER
1. biosynthesis of many proteins, including a. proteins secreted to the cell exterior b. transmembrane proteins on the PM c. Transmembrane or lumenal proteins of vesicular transport organelles, including ER, Golgi, secreotory vesicles endosomes secreotory vesicles, endosomes, & lysosomes. lysosomes 2. biosynthesis of most lipids 3. intracellular calcium storage (signaling) (remember that the ER lumen is one major Ca2+ store)
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This slide will be included in the next handout
all proteins of these vesicular compartments, regardless whether membrane or lumenal proteins, are first synthesized in the ER and delivered to their final destinations via the destinations via the vesicular transport pathway
Figure 12-6 Molecular Biology of the Cell ( Garland Science 2008)
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Cytosol-to-ER Transport
Structure and function of ER
How is a cargo protein transported into the ER?
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Cytosol to ER Transport
General features:
bi-direction transport (although it seems uni-direction from the diagram) the diagram) co-translational import in most animal cells
imported as unfolded polypeptide chains ER signal sequence required (often cleaved after translocation)
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overview of cytosol-to-ER transport (a co-translational event in most animal cells)
polyribosomes
docking of polyribosomes to the ER (docking occurs during translation)
translocation (newly synthesized proteins move into ER)
folding (translocated proteins are folded)
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How do we know ER translocation occurs co-translationally?
Which microsomal membrane should one use, smooth or rough?
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Co-translational docking of polyribosomes to the rough ER requires ER signal sequence (on a cargo), SRP (in the cytosol), as well as SRP receptor (on the rough ER membrane)
Docking of polyribosomes to the ER ER signal sequence SRP SRP receptor
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ER signal sequences (also called ER signal peptides) SRP (signal recognition particle), and SRP receptor
ER signal sequence: a short stretch of hydrophobic residues (usually 6-12 a.a. long) located at the N-terminus of a cargo protein targeted to the ER the of cargo protein targeted to the ER SRP: a complex (containing both RNAs and proteins) recognizing the ER signal sequences of a cargo protein SRP receptor: an ER integral membrane protein composed of two subunits, & . It It recognizes SRP. SRP Both SRP and SRP receptor are GTPases.
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ER Signal Sequence Different types of transport signals of intracellular transport
Comparison of Signal Sequences
nucleus import nucleus export
anywhere anywhere
no no
1 or 2 clusters of basic a.a. leucine rich sequence
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Question:
Why is the signal sequence often cleaved in ER and mitochondrial transport, but not in the nuclear transport?
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(1) SRP binds to the ER signal sequence
Due to its N-terminal location, the signal peptide is recognized by SRP soon after its synthesis (before the translation is completed).
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(2) Once bound to ER signal sequence, SRP interacts with the ribosome to cause a translational pause
nascent polypeptide chain exit site
to block translation
another example of protein conformational change
In addition to binding to the signal sequence, SRP also interacts with the ribosome to cause a pause in translation. This assures the co-translational transport
Figure 12-39b Molecular Biology of the Cell ( Garland Science 2008)
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(3) Interaction between SRP & SRP receptor (ER membrane protein) recruits polyribosomes to the rough ER membrane polyribosomes recruits to the rough ER membrane
The paused complex containing mRNA, the ribosomes & nascent polypeptides is then recruited to the rough ER membrane via the SRP-SRP receptor interaction in a co-translational manner.
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(4) SRP & SRP Receptor also function to proof-read ER signal sequences
Translation will not occur until SRP leaves the polyribosome complex, which in turn is controlled by the GTP-binding
Both SRP and SRP receptor are GTPases, and they can proof-read ER signal SRP SRP GTP th ER sequences. A bad signal sequence will dissociate with the SRP before the hydrolysis of GTP. Thus, SRP/SRP receptor function as a molecular clock to allow sufficient time to check the quality of the signal sequences.
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(5) SRP communicates with the translocon to initiate the translocation
translocation initiation
If the signal sequence is good, the mRNA-ribosome-nascent polypeptide chain complex is the signal sequence is good the mRNA polypeptide chain complex is transferred from the SRP/SRP receptor to the translocon and the GTP hydrolysis of SRP/SRP receptor occurs, translation resumes and the translocation begins. (signal sequence opens up the translocon and functions as a signal to start the translocation)
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Multiple functions of SRP
interacts with the signal sequence (to recruit the polyribosomes to the ER) interacts with the ribosome (to stop the translation) interacts with the SRP receptor th SRP (to dock the polyribosomes to the ER) (to proof-read the signal sequence) communicates with the translocon (to initiate the translocation process)
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Why is the SRP structure so complex, compared to other types of signal receptors such as importins?
SRP is a ribonucleoprotein particle (containing RNA + protein) (no need to memorize the names of subunits)
SRP also interacts with SRP receptor (not shown here)
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Translocation and cleavage of signal sequence
polyribosomes
docking of polyribosomes to the ER ER signal sequence signal sequence SRP SRP receptor
translocation translocon signal peptidase ribosomes
folding chaperones other proteins
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Translocation of the nascent polypeptide chain via an open translocon (translocator)
After attaching to the ER membrane, the ribosomes and nascent chains are transferred to the translocon. As translation resumes, the elongating chain passes directly from the large ribosomal subunit to the central pore of the translocon
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To maintain the permeability barrier of the ER membrane, the translocon must close in the absence of translocation
3-D Structure of ER Translocon (Translocator) in the closed state
Three subunits, Sec61, Sec61 and Sec61, form a Sec61 translocon complex w/ a central pore. In its closed state, part of the Sec61 protein forms a plug which closes the pore from the lumenal side
Figure 12-42a Molecular Biology of the Cell ( Garland Science 2008)
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Conformational change between the closed and open states of ER translocon
Signal sequence interacts with the translocon and induces a conformational th change, which leads to the removal of the plug to allow the passage of nascent polypeptide chain into the lumen. The pore can probably open to the lateral side as well to allow the signal sequence and the transmembrane domains to be released to the lipid bilayer where it can be subsequently cleaved or inserted
Figure 12-42 Molecular Biology of the Cell ( Garland Science 2008)
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How to maintain the permeability barrier while the translocon is open (since the plug is removed)?
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The ribosome forms a tight seal with an open translocon
tight seal on the cytoplasmic side (permeability barrier)
After attaching to the ER membrane, the ribosomes and nascent chains are transferred to the translocon. As translation resumes, the elongating chain passes directly from the large ribosomal subunit to the central pore of the Translocon (w/o be exposed to the cytoplasm)
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Evidence for a tight seal on the cytoplasmic but not the lumenal side
Pore closes to the cytoplasmic side closes to the cytoplasmic side
recall that the iodide ion is membrane impermeable
Pore opens to the lumenal side
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Two modes of gating of the Sec61 translocon
plug
tight seal
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Folding of the translocated nascent polypeptide chain
polyribosomes
docking of polyribosomes to the ER ER signal sequence SRP SRP receptor
translocation translocon signal peptidase ribosomes
folding chaperones other proteins
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Folding of the translocated polypeptide requires ER chaperones
Chaperones are needed in ER lumen to facilitate the folding of a nascent chain. During the translocation, no chaperones are needed in the cytoplasmic side.
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Question:
Considering that proteins to be transported are unfolded in both cases, why is cytoplasmic chaperone only needed in the mitochondrial import?
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chaperones are required on both cytosolic and lumenal (matrix) sides during cytosol-to-matrix transport
For the import into mitochondria, cytosolic chaperone hsp70 is required to maintain the unfolding status of the transported protein
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Why are chaperones needed only in the lumenal side during the ER translocation?
During a co-translational translocation, the length of nascent chain is short on the cytoplasmic side during the translocation. As a result, it cannot fold before being pushed into the lumen and thus there is no need of chaperones there
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Question:
What is the energy source for the directional transport during the ER translocation? (compared to the mitochondrial case)
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ER-to-cytosol retrograde transport
1. ER to cytosol retrograde transport of misfolded proteins (will be discussed later) 2. ER to cytosol retrograde transport of pathogens
surface
for Ricin to enter the cytosol: probably via Sec61 translocator
endosomes
from Sandvig & van Deurs (2002) FEBS Letters 529(1): 49-53
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