ER targeting and ER folding

Question 1

What is needed to enter ER?

Answer 1

Entry to ER needs a signal peptide

When immature protein is cleaved from its signal peptide then it becomes mature

Question 2

Describe signal peptides

Answer 2

Signal peptides are very variable and tend to have positive AA and the N terminus and a hydrophobic stretch which is recognised by SRP
The signal sequence of a soluble protein has two signalling functions. It directs the protein to the ER membrane, and it serves as a start-transfer signal (or start-transfer peptide) that opens the pore.

Question 3

Overview of how proteins are secreted

Answer 3

1. From the cytosol, proteins cross (translocate) the
   ER membrane and exit the ER in vesicles.
2. These vesicles then fuse with the Golgi, and the
   proteins are transported through the Golgi into
   secretory vesicles.
3. After fusion of the last vesicles with the plasma
   membrane, the protein content (cargo) is secreted
   to the outside of the cell.

Question 4

How to proteins go from
CYTOSOL to
ER membrane to
ER LUMEN?

Answer 4

An emerging ER signal peptide’s is captured by SRP (recognition step)
Which directs the SRP/SP complex (targeting step) to the α subunit of SRP receptors in the ER membrane allowing recruitment of a closed translocon.
SP folds into loop by SRP, signal cleaved in translocon, releasing SP
Even after it is cleaved off by signal peptidase, the rest of the protein is threaded continuously through the membrane as a large loop. Once the protein has passed through the membrane, the translocated protein is released into the ER lumen

Question 5

Describe the SP cycle

Answer 5

SRP/SRP receptor complex is
dismantled and SRP is recycled.
Signal peptidase removes the SP, which is
released into the ER membrane. There are
millions of active translocons in the ER
membrane, so at any one time there could
be millions of cleaved signal peptides – which
could disrupt the ER membrane.

Question 6

How is translation terminated?

Answer 6

Higher eukaryotes utilise signal peptide
peptidase (SPP), which cuts the released SP
in the plane of the membrane, allowing easy
removal of two half SPs. As translation
terminates, the secretory protein is freed
into the ER lumen and folds, the translocon
closes, and the ribosome subunits are
recycled.

Question 7

What do N-glycans do?

Answer 7

N-glycans act as flags for folding and ER Quality Control
They increase protein solubility
And influence folding rates and final protein conformation.

Question 8

How are disulphide bonds formed?

Answer 8

They are formed in oxidising conditions catalysed by PDI

PDI recognises unstable proteins and bind + shuffles S-S until stable conformation reached.