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The Secretory Pathway
- Classic Experiments
- ER Translocation
- Membrane budding and fusion
Intracellular Transport
Pathways
Intracellular Transport
Pathways
Vesicular
Transport
Vesicular
Transport
Pulse-chase: Palade and co-workers
3H-Leu pulse to monitor secretion in pancreatic
cells
10’ Golgi
45’ Secretory
Modern version: GFP allows visualization of
transport
1. ts VSV G accumulates in
ER
4. Plasma
2. After lowering T: ER exit sites
3.
Genetics: Schekman and co-workers
Novick et al. 1980: Identification of 23 SEC genes
Genetics: Schekman and co-workers
In vitro assay for vesicular transport: Rothman and coworkers
Balch et al.
In vitro assay for ER translocation: Blobel and co-workers
In vitro assay for ER translocation: Blobel and co-workers
Identification of signal
sequences
SRP: signal recognition
particle
SRP
cycle
SRP
cycle
Site-specific incorporation of modified amino acids to probe ER
translocation
ER translocation can be reconstituted in vitro:
- Sec61 complex: conserved translocation channel
Sec61 subunits (a, b, g)
Sec62/63
TRAM (translocating chain-assoc. membrane prote
- 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 (cotranslat
molecular ratcheting (posttranslational)
Probing of translocation intermediates with fluorescent peptides
From: Liao and Johnson Cell (97)
The Sec61 complex forms a channel….
Menetret et al. Mol Cell (2000) 6:1219
The translocon by EM tomography
From: Beckmann et al. Cell (2001) Vol 107, 361-372
The translocon by EM tomography
From: Beckmann et al. Cell (2001) Vol 107, 361-372
X-ray structure of the prokaryotic translocation channel SecY
From: Van den Berg et al. Nature (2004) 427, 36-44
Model for translocation of soluble proteins
From: Van den Berg et al. Nature (2004) 427, 36-44
Topology of membrane-spanning proteins
Type I membrane proteins have a cleavable signal sequence
Type II membrane proteins have internal signal sequence
Type III membrane proteins have internal signal sequence
Type II+III membrane proteins have internal signal sequences
Model for translocation of transmembrane proteins
From: Beckmann et al. Cell (2001) Vol 107, 361-372
Translocation of proteins with multiple membrane
spanning domains