Modified tRNAs allowed isolation (cross-linking approach) and analysis (fluorescence quenching ) of translocation channel.

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Presentation transcript:

Modified tRNAs allowed isolation (cross-linking approach) and analysis (fluorescence quenching ) of translocation channel

In vitro reconstitution of ER translocation: - Sec61 complex: conserved translocation channel Sec61 subunits (a, b, g) Sec62/63 TRAM (translocating chain-assoc. membrane protein) - phospholipids (proteoliposomes) and luminal chaperones (BIP) - SRP/SRP receptor only required for co-translational translocation not for post-translational translocation (e.g pre-pro-alpha factor). - energetics of translocation: protein conducting channel (cotranslational) molecular ratchetting (posttranslational)

From: Beckmann et al. Cell (2001) Vol 107, 361-372

From: Beckmann et al. Cell (2001) Vol 107, 361-372

Topology of membrane-spanning proteins -> Topogenic sequences determine orientation

Type I membrane proteins have a cleavable signal sequence Stop transfer sequence (hydrophobic): when mutated -> secretion

Type II membrane proteins have internal signal sequence Internal signal is not cleaved. Direction is determined by residues outside of hydrobhobic helix (++-outside rule: postive aa face cytoplasm)

From: Beckmann et al. Cell (2001) Vol 107, 361-372

Translocation of proteins with multiple membrane spanning domains

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Summary ER translocation: SRP-dependent and -independent pathways; translocation occurs through Sec61 complex; topogenic sequences determine overall orientation. ER function Compartmental identity: maturation versus fixed compartments Identification of components: combination of genetics, biochemistry... Vesicular coats: COPI ~ retrograde: Golgi->ER COPII ~anterograde: ER->Golgi CCV post-Golgi, various adaptors