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The Secretory Pathway - Classic Experiments - ER Translocation - Membrane budding and fusion
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Intracellular Transport Pathways
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Vesicular Transport
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Pulse-chase: Palade and co-workers 3H-Leu pulse to monitor secretion in pancreatic cells 10’ Golgi45’ Secretory Granules
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Modern version: GFP allows visualization of transport 1. ts VSV G accumulates in ER 2. After lowering T: ER exit sites 3. Golgi 4. Plasma membrane
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Genetics: Schekman and co-workers Novick et al. 1980: Identification of 23 SEC genes
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Genetics: Schekman and co-workers
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In vitro assay for vesicular transport: Rothman and co- workers Balch et al. 1984
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In vitro assay for ER translocation: Blobel and co-workers
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Identification of signal sequences
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SRP: signal recognition particle
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SRP cycle
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Site-specific incorporation of modified amino acids to probe ER translocation
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ER translocation can be reconstituted in vitro: - 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 ratcheting (posttranslational)
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Probing of translocation intermediates with fluorescent peptides From: Liao and Johnson Cell (97)
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The Sec61 complex forms a channel…. Menetret et al. Mol Cell (2000) 6:1219
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From: Beckmann et al. Cell (2001) Vol 107, 361-372 The translocon by EM tomography
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From: Beckmann et al. Cell (2001) Vol 107, 361-372 The translocon by EM tomography
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From: Van den Berg et al. Nature (2004) 427, 36-44 X-ray structure of the prokaryotic translocation channel SecY
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From: Van den Berg et al. Nature (2004) 427, 36-44 Model for translocation of soluble proteins
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Topology of membrane-spanning proteins
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Type I membrane proteins have a cleavable signal sequence
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Type II membrane proteins have internal signal sequence
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Type III membrane proteins have internal signal sequence
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Type II+III membrane proteins have internal signal sequences
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From: Beckmann et al. Cell (2001) Vol 107, 361-372 Model for translocation of transmembrane proteins
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Translocation of proteins with multiple membrane spanning domains
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