Translocation through the endoplasmic reticulum
membrane
Institute of Biochemistry
Benoît Kornmann
Endomembrane system
Protein sorting
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Permeable to proteins but not to ions
 IgG tetramer (16 nm)
 Fully hydrated Ca2+ ion (0.6 nm)
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Benoît Kornmann Institute of Biochemistry ETH Zürich
The signal hypothesis
Blobel, G. & Sabatini, D. D. 1971 in Biomembranes Vol. 2 (ed. Manson, L. A.) 193–195
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Benoît Kornmann Institute of Biochemistry ETH Zürich
The Endoplasmic reticulum
Sheets
Tubules
Nuclear envelope




Sheets and tubules
Rough and smooth
Sheets ~ Rough
Tubules ~ Smooth
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Professional secretory cells
 Plasma cell (activated B lymphocyte) secrete ~500 IgG
molecules per second.
 More than their own dry weight everyday!
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Rough endoplasmic reticulum
 Ribosomes associated to ER
membrane
 Co-translational translocation
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Principal players in protein translocation
 Ribosome
 Signal-recognition particle
(SRP)
 SRP-receptor (SR) on ER
membrane
 Aqueous channel
(translocon)
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Challenges in SRP-mediated targeting
 SRP must recognize nascent
signal peptides and bind
them with high affinity and
selectivity
 Once released, the nascent
polypeptide must engage
with the translocon
 SRP must release peptide
upon binding to SRPreceptor (SR)
 Finally SRP and SR must
dissociate for being recycled
 Therefore energy is needed for completion of the cycle
tember 2013
18.09.13
Benoît Kornmann Institute of Biochemistry ETH Zürich
Signal sequences
 target proteins for secretion and
membrane insertion (PM
proteins, secreted proteins and
proteins of secretory organelles)
 Are located at the N-terminus of
pre-protein
 are typically cleaved off by signal
peptidase
 typical length: 15-25 amino acid
residues
 Bear no sequence homology but
characteristic 3-partite structure
 n-region: hydrophilic, basic
 h-region: hydrophobic, 7-15 amino
acid residues
 c-region: 2-9 polar, small amino
acid residues
(consensus site for cleavage by
signal peptidase)
Signal sequences end-up
inserted in the ER membrane
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Benoît Kornmann Institute of Biochemistry ETH Zürich
SRP is conserved across all three domains of Life
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Eukaryotic SRP pauses translation through its ALU
domain
 The SRP Alu domain competitively inhibits elongation factor
binding by covering the same site on the ribosome
 (eEF2 promotes the translocation step of amino-acyl-tRNA
from A to P site during protein synthesis)
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Signal recognition particle
From Sulfolobus solfataricus (Archea)
(N-terminal)
(methionine-rich)
Interaction with SR
Interaction with
and ribosome
signal peptide
GTPase activity/interaction with ribosome
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Signal recognition in the M-domain
N-Domain
G-Domain
Signal peptide
M-Domain
T. Hainzl, et al., Nature structural & molecular biology. 18, 389-91 (March 2011).
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Binding to the SRP receptor: the N- and G-domains
 Two subunits: alpha and beta (SRα and SRβ)
 SRα resembles SRP54
SRP
N-domain
SRP54 (Mammalian)
N
Ffh (E. Coli)
N
A-domain
SR
SRα (Mammalian)
FtsY (E. Coli)
N-domain
G-domain
C
C
G-domain
N
C
N
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M-domain
Benoît Kornmann Institute of Biochemistry ETH Zürich
C
The SRP-SR complex
 Quasi two-fold symmetrical
heterodimer
 Extensive contacts between
G-domains
 Major rearrangements in Ndomain between monomer
and complex
Ffh
Light: Monomer
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:SR
:SRP
Benoît Kornmann Institute of Biochemistry ETH Zürich
Dark: Dimer
Reciprocal stimulation of GTPase activity
SRP and SR reciprocally stimulate
each other’s GTPase activity  after GTP hydrolysis the complex
dissociates.

tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Reciprocal stimulation of GTPase activity
SRP and SR reciprocally stimulate
each other’s GTPase activity  after GTP hydrolysis the complex
dissociates.

 The two GTPase sites form a
composite active site with the
nucleotides packed in a head-totail manner
 Symmetrical hydrogen bonds
between the 3’OH ribose of one
nucleotide and the γ-phosphate
of the other
 GTP-hydrolysis severs these
connections and leads to
complex dissociation
a.w. attacking water
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Last step of the SRP reaction: the SRP-RNC binds to the
translocon
 Binding of SRP to SR exposes a translocon binding site close
to the peptide exit channel on the ribosome
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
SRP cycle
 SRP M-domain binds to
signal peptide
 SRP-SR interaction liberates
a translocon-binding
domain on the ribosome
 This cause rearrangement
in N- and G-domains
allowing interaction with
SRP receptor
 GTP hydrolysis causes SRPSR complex disassembly
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Next questions:
 How does signal binding
promote SRP-SR complex
formation?
 The answer probably lies in
the RNA moiety of the SRP
 How does binding in Mdomain rearrange NGdomains?
Linker is ordered and
elongated
 How does formation of SRPSR complex cause peptide
release?
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 How does a change in NG
One RNA base is flipped toward GTPase
domain cause a
conformational change in Mdomain?
Ataide et al., Science. 331, 881-886 (February 2011).
18.09.13
Benoît Kornmann Institute of Biochemistry ETH Zürich
RNA may participate in GTPase reaction
Flipped base
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Ataide et al., Science. 331, 881-886 (February 2011).
Benoît Kornmann Institute of Biochemistry ETH Zürich
Animation of SRP
targeting
 Ribosome-bound SRP scan nascent chains for emerging signal
peptides.
 Upon signal sequence binding, conformational changes are
transmitted to the GTPase core, allowing SR binding
 SR binding displace Srp54/Ffh from Ribosomal protein L23
 L3 is now free to bind to translocon
 SRP54/SR complex is free to interact with flipped base on SRP
RNA
 GTP hydrolysis dissociate the complex
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Translocation
 The ribosome translocon complex
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Benoît Kornmann Institute of Biochemistry ETH Zürich
The translocon
 Bacteria:
 SecY
 SecE
 SecG
c
 Eukaryotes:
 Sec61α
 Sec61β
 Sec61γ
 Archea
 Blue: Sec61α
 Red: Sec 61β
 Green: Sec61γ
Sec61 from Methanococcus Jannaschi (Archea)
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Imp0rtant features of the Sec61 channel
 Helix 2a serves as a plug in the closed
state (a)
 Six hydrophobic residues work as a seal in
the open state (b and c)
 These two features likely maintain a
membrane barrier during membrane
protein synthesis
 The pore size of 5-8 Å
would not allow
passage of folded
domains
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Membrane integration requires sideway opening of
the translocon
 The transmembrane helix needs to exit the channel through
a side opening (seam)
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Lateral opening of the translocon
c
Methanococcus Jannaschi
Pyrococcus Furiosus
B. Van den Berg et al., Nature. 427, 36-44 (January 2004).
P. F. Egea, R. M. Stroud, PNAS. 107, 17182-7 (October 2010).
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Topology of membrane proteins
 Membrane topology is established co-translationally in the
ER and can't be changed afterwards
 How does the ribosome know that it has to stop transferring
through translocon when a TM domain happens?
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Topology of membrane proteins: Type I
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Topology of membrane proteins: Type II
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Topology of membrane proteins: Type III (or Type Ia)
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Topogenesis of membrane proteins in the ER
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
What determines the orientation of TMHs?
 Observations:
 Charged residues flanking the hydrophobic core of the signal:
 Positive-inside rule - the more positively charged segment stays in the cytosol
 Hydrophobicity of the signal:
 a. N-terminal signals initially insert in the N exo/Ccyt orientation and then invert based
on their charge distribution
 b. The more hydrophobic the signal, the harder to invert due to higher affinity for the
translocon
 Other possible causes
 Protein folding (internal signals)
 Folding of hydrophilic sequences N-terminal to a signal sterically hinders N-terminal
translocation
 ...but the detailed molecular mechanisms are unknown
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Signal sequence cleavage
 Achieved by signal sequence peptidase
 Co-translational
Blobel, G. & Dobberstein, B. J. Cell Biol. 67, 835–851 (1975).
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Co- and post-translational targeting
 No additional energy source on the cytosolic side
 ATP hydrolysis by BiP (HSP70) in ER lumen
 ATPase activity of SecA pumps protein through the pore of
translocon
tember 2013
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Benoît Kornmann Institute of Biochemistry ETH Zürich
Further reading
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S. F. Ataide et al., Science. 331, 881-6 (February 2011).
T. Hainzl, et al Nat struct mol biol. 18, 389-91 (March 2011).
P. F. Egea, R. M. Stroud, PNAS. 107, 17182-7 (October 2010).
Halic, M. et al. (2004) Nature, 427, 808-814
Egea, P. F. et al. (2004) Nature, 427, 215-221
Shan S. et al. (2004) PloS Biology, 2, 1572-1581
Rosendal, K. R. et al. (2003) PNAS, 100, 14701-14706
van den Berg, B. et al. (2003) Nature, 427, 36-44
Mitra et al. (2005) Nature, 438, 318-324
 Reviews
 Osborne, Rapoport, van den Berg Annu. Rev. Cell Dev. Biol.2005. 21:529–
50
tember 2013
18.09.13
Benoît Kornmann Institute of Biochemistry ETH Zürich