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Translocation through the endoplasmic reticulum membrane
Institute of BiochemistryBenoît Kornmann
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Protein sorting
Endo
mem
bran
e sy
stem
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Permeable to proteins but not to ions
IgG tetramer (16 nm)
Fully hydrated Ca2+ ion (0.6 nm)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
The signal hypothesis
Blobel, G. & Sabatini, D. D. 1971 in Biomembranes Vol. 2 (ed. Manson, L. A.) 193–195
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
The Endoplasmic reticulum
Sheets and tubules
Rough and smooth Sheets ~ Rough Tubules ~ Smooth
Tubules
Sheets
Nuclear envelope
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Professional secretory cells
Plasma cell (activated B lymphocyte) secrete ~500 IgG molecules per second. More than their own dry weight everyday!
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Rough endoplasmic reticulum
Ribosomes associated to ER membrane Co-translational translocation
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Principal players in protein translocation
Ribosome Signal-recognition particle
(SRP)
SRP-receptor (SR) on ER membrane
Aqueous channel (translocon)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Finally SRP and SR must dissociate for being recycled
SRP must release peptide upon binding to SRP-receptor (SR)
Therefore energy is needed for completion of the cycle
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
SRP is conserved across all three domains of Life
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Signal recognition particle
(N-terminal)
Interaction with SR
and ribosome
GTPase activity/interaction with ribosome
(methionine-rich)
Interaction with
signal peptide
From Sulfolobus solfataricus (Archea)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Signal recognition in the M-domain
Signal peptide
N-Domain
G-Domain
M-Domain
T. Hainzl, et al., Nature structural & molecular biology. 18, 389-91 (March 2011).
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Binding to the SRP receptor: the N- and G-domains
Two subunits: alpha and beta (SRα and SRβ) SRα resembles SRP54
N-domain
G-domainN-domain
G-domain
M-domain
A-domain
N
N-domain
N
CN
C
N
N C
C
SRP54 (Mammalian)
Ffh (E. Coli)
SRα (Mammalian)
FtsY (E. Coli)
SRP
SR
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
The SRP-SR complex Quasi two-fold symmetrical heterodimer
Extensive contacts between G-domains
Major rearrangements in N-domain between monomer and complex
Ffh
Light: Monomer
Dark: Dimer:SRP:SR
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Reciprocal stimulation of GTPase activity
SRP and SR reciprocally stimulate each other’s GTPase activity -
after GTP hydrolysis the complex dissociates.
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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-to-tail 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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
SRP cycle
SRP M-domain binds to signal peptide
SRP-SR interaction liberates a translocon-binding domain on the ribosome
GTP hydrolysis causes SRP-SR complex disassembly
This cause rearrangement in N- and G-domains allowing interaction with SRP receptor
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Next questions:
How does signal binding promote SRP-SR complex formation? How does binding in M-
domain rearrange NG-domains?
How does formation of SRP-SR complex cause peptide release? How does a change in NG
domain cause a conformational change in M-domain?
The answer probably lies in the RNA moiety of the SRP
Linker is ordered and
elongated
One RNA base is flipped toward GTPase
Ataide et al., Science. 331, 881-886 (February 2011).
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
RNA may participate in GTPase reaction
Flipped base
Ataide et al., Science. 331, 881-886 (February 2011).
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Translocation
The ribosome translocon complex
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
c
The translocon
Bacteria: SecY SecE SecG
Eukaryotes: Sec61α Sec61β Sec61γ Archea
Blue: Sec61α Red: Sec 61β Green: Sec61γ
Sec61 from Methanococcus Jannaschi (Archea)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Imp0rtant features of the Sec61 channel
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Membrane integration requires sideway opening of the translocon
The transmembrane helix needs to exit the channel through a side opening (seam)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Lateral opening of the translocon
c
Methanococcus Jannaschi Pyrococcus Furiosus
P. F. Egea, R. M. Stroud, PNAS. 107, 17182-7 (October 2010).B. Van den Berg et al., Nature. 427, 36-44 (January 2004).
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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?
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Topology of membrane proteins: Type I
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Topology of membrane proteins: Type II
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Topology of membrane proteins: Type III (or Type Ia)
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Topogenesis of membrane proteins in the ER
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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 Nexo/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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Signal sequence cleavage
Blobel, G. & Dobberstein, B. J. Cell Biol. 67, 835–851 (1975).
Achieved by signal sequence peptidase Co-translational
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
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
Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13
Mittwoch, 18. September 2013
Further reading
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–
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