Williams BCH 444 2014 Lectures 3-6

2004 nature 42736 front pore size without plug is

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Unformatted text preview: nococcus jannaschii [Van den Berg et al. (2004) Nature 427:36] γ Front α Pore size without plug is very narrow ~5-8 Å. Too narrow to explain translocation of polypeptides folded into αhelices (~12 Å). Some flexibility of the pore ring is required. Back β Tom Rapoport Translocon pore is blocked by a mobile plug . Cysteine mutants introduced into the plug and the γ subunit show that the plug can move towards γ to form disulfide bond. Such a movement would open the pore. top view seen by ribosome side view This is at odds with other estimates of pore size by biophysical means controversy A ring of hydrohobic amino acids (yellow) surround the pore. Proposed to act as a gasket surrounding the translocating polypeptide to prevent leakage of small molecules such as Ca2+ across the open pore. 23 Structure of the SecY (Sec61) translocon - 2 How is the channel gated? - binding of the N-terminal signal sequence is known to open the pore - signal sequence binding site is located between TM2b and TM7 of the α subunit - the signal sequence-TM interactions induce a major conformational change, shifting the plug and opening a “lateral gate” to create the translocation pore Model for different stages in the translocation of a secretory protein into the ER lumen Cryo-EM structure of E. coli translocon in closed and open states - Park, E. et al (Dec. 2013) Nature, in press - Closed state made with purified ribosomes and purified SecYEG (Secα,γ,β) - Open state purified from E. coli expressing a “stalled” 100 residue nascent chain disulfide linked to SecY translocon opens like a clamshell to create the pore and lateral gate plug (in gold) only shifts modestly Plug movement not as dramatic as depicted in this model. Pore ring may provide barrier to small molecules during translocation 24 Structure of the SecY (Sec61) translocon - 3 How does the cleaved signal leave the translocon? How do transmembrane (TM) segments leave the translocon? - the signal sequence binding site between TM2b and TM7 is the only point where a helical polypeptide (like a signal or TM) could move laterally out of the translocon into the lipid bilayer - as shown on previous slide, during translation, the translocon opens like a clamshell to create a “lateral gate” to allow exit of signal or TM seqments into the surrounding lipid. side view Structure of nascent chain with looped signal sequence trapped within the translocon Park, E. et al (Dec. 2013) Nature, in press ribosome cutaway view top view E. coli SecYEG translocon (blue) Signal sequence (beige spacefill or green ribbon) can readily exit the translocon after cleavage into surrounding lipid bilayer via “lateral gate”. TM segments can do the same. 25 CryoEM reconstruction of the ribosome-Sec61 translocon Sec 61 purified from detergent-solubilized yeast or dog rough microsomes and reconstituted with 80S ribosomes translating a nascent 120 aa polypeptide - Becker et al. (2009) Science 326:1369 Note: sec61 binding sites on ribosome overlap with SRP binding sites – SRP must leave before ribosome can dock on translocon sec61 (Protein-Conducing Channel) cutaway view of complex showing nascent chain (NC) exit tunnel through large subunit and filling Sec61 pore 12-15 Å gap between ribosome and Sec61 important for release of cyto domains of TM proteins (see later) sec61 Ribosome - Sec61 contacts (C1-C4) - note how Sec61 loops L6/7 and L8/9 engage the ribosome exit site 26 Model of Ribosome - Sec61 – nascent chain complex Translocation of membra...
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This note was uploaded on 03/27/2014 for the course BCH 444 taught by Professor Mccallan during the Spring '14 term at University of Toronto- Toronto.

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