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

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 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