Translocation through the endoplasmic reticulum membrane. Institute of Biochemistry Benoît Kornmann

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1 Translocation through the endoplasmic reticulum membrane Institute of Biochemistry Benoît Kornmann

2 Endomembrane system Protein sorting

3 Permeable to proteins but not to ions IgG tetramer (16 nm) Fully hydrated Ca2+ ion (0.6 nm)

4 The signal hypothesis Blobel, G. & Sabatini, D. D in Biomembranes Vol. 2 (ed. Manson, L. A.)

5 The Endoplasmic reticulum Sheets Tubules Nuclear envelope Sheets and tubules Rough and smooth Sheets ~ Rough Tubules ~ Smooth

6 Professional secretory cells Plasma cell (activated B lymphocyte) secrete ~500 IgG molecules per second. More than their own dry weight everyday!

7 Rough endoplasmic reticulum Ribosomes associated to ER membrane Co-translational translocation

8 Principal players in protein translocation Ribosome Signal-recognition particle (SRP) SRP-receptor (SR) on ER membrane Aqueous channel (translocon)

9 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

10 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: 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

11 SRP is conserved across all three domains of Life

12 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)

13 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

14 Signal recognition in the M-domain N-Domain G-Domain Signal peptide M-Domain T. Hainzl, et al., Nature structural & molecular biology. 18, (March 2011).

15 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 M-domain C

16 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 :SR :SRP Dark: Dimer

17 Reciprocal stimulation of GTPase activity SRP and SR reciprocally stimulate each other s GTPase activity after GTP hydrolysis the complex dissociates.

18 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

19 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

20 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

21 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? 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, (February 2011).

22 RNA may participate in GTPase reaction Flipped base Ataide et al., Science. 331, (February 2011).

23 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

24 Translocation The ribosome translocon complex

25 The translocon Bacteria: SecY SecE SecG c Eukaryotes: Sec61α Sec61β Sec61γ Archea Blue: Sec61α Red: Sec 61β Green: Sec61γ Sec61 from Methanococcus Jannaschi (Archea)

26 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

27 Membrane integration requires sideway opening of the translocon The transmembrane helix needs to exit the channel through a side opening (seam)

28 Lateral opening of the translocon c Methanococcus Jannaschi Pyrococcus Furiosus B. Van den Berg et al., Nature. 427, (January 2004). P. F. Egea, R. M. Stroud, PNAS. 107, (October 2010).

29 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?

30 Topology of membrane proteins: Type I

31 Topology of membrane proteins: Type II

32 Topology of membrane proteins: Type III (or Type Ia)

33 Topogenesis of membrane proteins in the ER

34 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

35 Signal sequence cleavage Achieved by signal sequence peptidase Co-translational Blobel, G. & Dobberstein, B. J. Cell Biol. 67, (1975).

36 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

37 Further reading S. F. Ataide et al., Science. 331, (February 2011). T. Hainzl, et al Nat struct mol biol. 18, (March 2011). P. F. Egea, R. M. Stroud, PNAS. 107, (October 2010). Halic, M. et al. (2004) Nature, 427, Egea, P. F. et al. (2004) Nature, 427, Shan S. et al. (2004) PloS Biology, 2, Rosendal, K. R. et al. (2003) PNAS, 100, van den Berg, B. et al. (2003) Nature, 427, Mitra et al. (2005) Nature, 438, Reviews Osborne, Rapoport, van den Berg Annu. Rev. Cell Dev. Biol :529 50