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-workers3H-Leu pulse to monitor secretion in
pancreatic cells
10’ Golgi
45’ Secretory Granules
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
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 co-workers
Balch et al. 1984
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
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 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)
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
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
The translocon by EM tomography
From: Van den Berg et al. Nature (2004) 427, 36-44
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
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
From: Beckmann et al. Cell (2001) Vol 107, 361-372
Model for translocation of transmembrane proteins
Translocation of proteins with multiple membrane spanning domains