The Biogenesis of E. coli Inner Membrane Proteins Dr. Joen Luirink

Transcription

1 The Biogenesis of E. coli Vrije Universiteit Amsterdam, The Netherlands 1 Talk SRP-mediated targeting to the mammalian ER and to the bacterial inner membrane Insertion of membrane proteins at the Sec complex Biogenesis of two model E. coli IMPs YidC: chaperone and insertase? 2 Protein targeting 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Protein targeting and secretion in E. coli FtsY SRP Trigger factor Folding/ modification SecB FtsY SecYEG SecA Leader peptidase ABC transporter TAT TolC Gsp secretion 4 Wickner and Schekman, 2005 SRP- mediated targeting 5 Egea et al., 2005 Conservation of the SRP/SR 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Cryo-EM structure of ribosome-srp complex 12Ǻ Alu domain blocks contact site for EFs S domain is close to L23/35 near nascent chain exit site SRP54 has different conformation ( open ) on ribosome than in solution ( compact ) 7 Halic et al., 2006 Co-translational targeting by the SRP 8 Halic et al., 2005 Specific aspects of E. coli SRP Relativ ely simple, no Alu domain (no elongation arrest?) Specific for inner membrane proteins (IMPs) SRP binds specifically to nascent IMPs (cross-linking) Depletion specifically affects insertion of IMPs (genetics) Competition with the chaperone trigger factor at NC exit site 9 Ferbitz et al., 2004 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Trigger factor PPIase In proximity to most NCs; May remain bound to elongating chain Cooperates with downstream chaperones (DnaK/J/GrpE) to assist folding Docks at L23/L29 (like SRP) SRP and TF can interact simultaneously with ribosomes SRP preferentially binds to hy drophobic targeting signals 10 Protein targeting and secretion in E. coli FtsY SRP Trigger factor Folding/ modification SecB FtsY SecYEG SecA Leader peptidase ABC transporter TAT TolC Gsp secretion 11 Wickner and Schekman, 2005 Crystal structure of the Sec-translocon Front TM 6-10 α TM 1-5 Plug Cytosol γ β Back Hinge Signal sequence Pore ring Plug Closed Nascent chain Open Perpendicular and lateral Open Perpendicular Ribosomebinding site Sec61αβγ from M. jannaschii; 3.2 Ǻ Sec61αβγ unit forms narrow channel, hourglass shape Channel is plugged Large cy tosolic loops Clamshell conformation, lateral opening for TM exit? Narrow pore; Down to 5 Ǻ Oligomeric organization? Plug Closed SecYEG1 SecYEG2 TMS Pore ring Plug TICB 14, Oct. 2004, page 570 Open Plug No nascent chains Closed PCC Polypeptide chain TMS release Hairpin insertion into membrane Open PCC Half-open PCC 12 Driessen et al., 2005 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Talk SRP-mediated targeting to the mammalian ER and to the bacterial inner membrane Insertion of membrane proteins at the Sec complex Biogenesis of two model E. coli IMPs YidC: chaperone and insertase? 13 Model proteins In vivo dependencies Membrane integration requires SRP, SecY, SecE, SecA (Urbanus et al., 2001; Wolfe et al.,1985; de Gier et al., 1996; de Gier et al., 1998) Consecutiv e interactions of FtsQ/Lep during biogenesis? 14 In vitro translation and cross-linking 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Protein targeting and secretion in E. coli FtsY SRP Trigger factor Folding/ modification SecB FtsY SecYEG SecA Leader peptidase ABC transporter TAT TolC Gsp secretion 16 Wickner and Schekman, 2005 Cross-linking 108FtsQ 17 Scotti et al., 2000 Evidence for a dual role of YidC in IMP integration YidC specifically interacts with the TM segment of nascent IMPs such as FtsQ, Lep and MtlA YidC can be co-purified with the Sec-translocon Depletion of YidC mildly affects integration of Sec-dependent IMPs like Lep Depletion of YidC strongly affects integration of Sec-independent IMPs like M13 procoat Lep FtsQ M13 18 Yi and Dalbey, 2005 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Essential poly topic inner membrane protein of 60 kda (Saaf et al., 1998) Member of the Oxa1p/YidC/Alb3 family Oxa1p is inv olv ed in membrane integration of both nuclear and mitochondrial encoded IMPs (Hell et al., 2001) Alb3 is inv olv ed in integration of LHCP into the thy lakoid membrane (Moore et al., 2001) YidC Alb3 and Oxa1p can compensate for the loss of YidC (Jiang et al., 2002; van Bloois et al., 2005) 19 Sequential interactions during membrane integration of FtsQ (1) The TM segment of FtsQ interacts first with SecY, then with YidC Lipids are close to the TM early during membrane integration 20 Urbanus et al., 2001 Sequential interactions during membrane integration of FtsQ (2) YidC functions in the lateral diffusion of TM segments from the Sec-translocon into the lipid bilay er? 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Membrane integration of Lep H1 (1) Scanning cross-linking on 50Lep Model of 50Lep Role for YidC in reception of TM? Houben et al., Membrane integration of Lep (2) Interactions of H1 with SecY (position 15) and YidC (position 10) during further insertion? And H2? 23 Membrane integration of Lep H1 (3) Position 10 Myc X-YidC Position 10 in H1 has mov ed away from YidC at a nascent chain length of 102 Position 15 in H1 has mov ed away from SecY at a nascent chain length of Rapid mov ement of H1 into lipid bilay er Houben et al., 2004 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Membrane integration of Lep H2 (4) H2 inserts at SecY H2 is near YidC after a further sy nthesis of 20 to 40 amino acids H2 has mov ed away from YidC at a nascent chain length of 222 amino acids Houben et al., Membrane integration of Lep (5) The TMs of Lep mov e one by one from a Sec/YidC insertion site into the lipid bilay er (consistent with sequential insertion model) 26 Substrates of YidC? Weak effect X FtsQ Lep MalF MtlA X M13 PF3??? Strong effect Many other 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Effects of YidC depletion on the biogenesis of F 1 F o ATPase YidC depleted membrane v esicles have a reduced ATPase activity Decrease in ATPase activ ity correlates with reduced amount of F 0c subunit Inside van der Laan et al., 2003 Outside ab2 Stator Cn=? Rotor 28 Effects of YidC depletion on the biogenesis of cytochrome o oxidase YidC depleted membrane v esicles have a reduced cy tochrome o oxidase activity Decrease in cy tochrome o oxidase activity correlates with reduced amount of functional cy tochrome o oxidase complex α CyoA van der Laan et al., Properties of F 0 c and CyoA 2 closely spaced TMs in N out, C out topology Participate in oligomeric integral membrane complexes (F 0a1b2c10 and Cy oabcd) Homologues of F oc and Cy oa in mitochondria (ATP9 and CoxII) strongly depend on Oxa1p for proper membrane assembly F 0c and Cy oa might be important natural substrates of the YidC only pathway in E. coli 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Membrane assembly of F o c In vivo analy sis (protease mapping) Membrane assembly of F oc requires SRP and YidC but not SecE In vitro analy sis (x-linking) TM1 of nascent F oc interacts with SRP in the cy tosol and primarily with YidC in the membrane Targeting of Foc by SRP to YidC (Sec-independent)? 79F0cTAG15 31 van Bloois et al., 2004 Membrane assembly of CyoA Protease mapping: SRP and YidC required for assembly of N-terminus; Sec required for C-terminus X-linking: SRP x-linked to TM1 Targeting of Cy oa by SRP Protease mapping: Sec-independent assembly (only YidC required) Distinct requirements for assembly of the N- and C-terminus of CyoA 32 van Bloois et al., 2006 Biogenesis of E. coli membrane proteins TF 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 YidC: molecular chaperone and insertase? Function of YidC? In conjunction with the Sec-translocon - Lateral diffusion of TMs - Folding (Nagamori et al., 2004) - Quality control As a separate entity - Membrane insertion - Translocation of periplasmic loops Oligomeric organization of YidC? In conjunction with the Sec-translocon As a separate entity Targeting to YidC? 1. Targeting 2. Insertion 3. Folding/assembly/quality control Substrates of YidC Structure of YidC? 34 X-ray structure of SecYEG Y I D C van den Berg et al., People and projects Halic, Nature 427: 808, 2004 Ribosome/SRP complex Edith Houben Ferbitz, Nature 431: 590, 2004 Ribosome/TF complex TF Sec-translocon Edwin v. Bloois Malene Urbanus Edith Houben Van den Berg, Nature 427: 36, The screen versions of these slides have full details of copyright and acknowledgements 12

13 Acknowledgements Stockholm University Jan-Willem de Gier RUG, Groningen Martin v an der Laan Arnold Driessen ETH, Zürich Josef Brunner Weizmann Inst., Rehovot Raz Zariv ach The screen versions of these slides have full details of copyright and acknowledgements 13