Part 2: Intracellular trafficking

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Modified tRNAs allowed isolation (cross-linking
approach) and analysis (fluorescence quenching )
of translocation channel
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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)
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From Beckmann et al. Cell (2001) Vol 107, 361-372
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From Beckmann et al. Cell (2001) Vol 107, 361-372
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Topology of membrane-spanning proteins
-gt Topogenic sequences determine orientation
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Type I membrane proteins have a cleavable signal
sequence
Stop transfer sequence (hydrophobic) when
mutated -gt secretion
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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)
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From Beckmann et al. Cell (2001) Vol 107, 361-372
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Translocation of proteins with multiple membrane
spanning domains
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Formation of a glycosylphosphatidylinositol
(GPI)-anchor
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ER function
- Proper folding of proteins (chaperones,
lectins, petidyl-prolyl-isomerases) -
Formation of disulfide bonds (PDI) GSH prevents
oxidation in cytosol GS-SG NADPH H ltgt 2
GSH NADP - Proteolytic cleavages - Addition
processing of carbohydrates - Assembly into
multimeric proteins - Ca2 storage - Lipid
synthesis - Detoxification (liver!)
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Folding of Influenza hemagglutinin (HA)
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Ser/Thr
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Movies
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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... Vesicula
r coats COPI retrograde Golgi-gtER
COPII anterograde ER-gtGolgi CCV
post-Golgi, various adaptors