KJ2 THE SIGNAL HYPOTHESIS

Post translational translocation

Proteins targeted for mitochondria, nucleus, and chloroplasts that are translated in the cytosol and contain a targeting sequence for delivery after translation

Co translational translocation

Membrane, luminal, and secreted proteins complete translations on ribosomes on RER

Pulse-chase labelling

Introduction of radiolablled aa into cell culture then chased with normal aa which can then be recorded using autoradiography

Microsomes

Small, closed vesicles formed from fragmented ER with ribosomes attached during cell fractionation

Introduction of protease w/o detergent

No digestion of secretory protein resulting in a bold line in gel electrophoresis

Introduction of protease w/ detergent

Digestion of secretory protein resulting in scattered lines in gel electrophoresis due to protease breaking down proteins indiscriminately

Readout experiment

In vitro translation IgG mRNA microsomes disrupted w/ detergent resulting in longer proteins if microsomes disrupted or a smaller mature proteins if not. They are the same protein but with an additional sequence of aa at the NTD

The Signal Hypothesis

Highly conserved signal sequence were found at the N terminus and started w/ + aa followed by 6 - 12 hydrophobic aa

Sec 61 translocon

Conserved protein (yeast → humans) that allow proteins to go form cytosol → ER. Contains a pore with a plug forming a channel in ER membrane that translates the mRNA then immediately elongates and translocates it into ER lumen

Steps in Co translational translocation

N’ signal sequence → SRP binds sequence and ribosome stops translation → SRP targets complex to ER by SRP-receptor on membrane and GTP binding → SRP-receptor hydrolyses GTP and polypeptide transferred to Sec 61 → Polypeptide elongates and translocates through channel into lumen and signal sequence cleaved by signal peptidase → Polypeptide folds, ribosomes dissociate and channel closes

Integral membrane proteins

Proteins targeted for ER, Golgi, PM, lysosome and all synthesised on rough ER and remain embedded as it translocates

Topogenic

Orientation and number of times a polypeptide crosses the membrane and orientation remains same as when it enters ER

Stop-transfer anchor sequences

Hydrophobic regions that embed into membranes

Signal anchor sequences

Embed into membranes

Type 1 membrane protein

ER SS initiates translation in ER and stop-transfer anchor sequence stops polypeptide from transferring into the lumen and anchors protein in membrane. N terminus in lumen and C terminus in cytosol

Type 2 membrane protein

No ER SS instead has signal transfer sequence which anchors proteins in membrane and reverse topology so starts translation in free cytosol then finishes in ER

Getting proteins into membrane

Polypeptide-ribosome complex associates with translocon, translocates, SS cleaved by signal peptidase → Stop-transfer anchor sequence translated and enters translocon → Prevents further translocation into lumen → Stop-transfer anchor moves laterally into membrane via cleft in translocon subunits → Anchored in membrane, translocon closes, and synthesis is finished in cytosol → Ribosome dissociates leaving protein in membrane