ER translocation Flashcards
(48 cards)
Who is Gunter Blobel, what did he discover and examples of it?
Nobel prize for the signal hypothesis
- Functions of cells depends on molecules in the right place at the right time
ExAmple- Insulin needs to be transported out of the cells the molecules are packaged into vesicles which deliver their cargo
Randy schekman- What did he do?
Discovered genes encoding proteins that are key regulators of vesicle traffic
looked at mutant cells
discovered genes that control transport
James rothman- what did he do?
found protein complex which enabled vesicles to fuse with their target membrane at the right location so cargo is delivered
Thomas C. suldhof- what did he do?
Found the role of proteins, signal transmitted and what senses calcium iron channels
What is the role of signals in ER translocation?
- mediate protein sorting
- sequence
- patching
they can be linear and patch
Difference between patched and linear
in the unfolded proteins linear only has signal regions the the end of the protein whereas patch has them dispursed throughout
ER retrieval signals- 2 types
KDEL- resident ER soluble proteins found in the lumen of the ER
KKXX- resident ER membrane proteins
Resident ER protein t
Have to be targeted to many different organelles
What does the signal sequence contain ?
positively (Arg/lys) and negatively (glu/Asp) charged amino acids
What is the endoplasmic reticulum?
Rough- ribosomes closely associated with the membrane
Smooth- site of lipid synthesis
ER extends throughout the cell
Isolation of ER
Homogenization- RER more dense so stops sedimenting and floats at high sucrose conc (bottom)
SER- less dense so floats higher at low sucrose level
Isolation of signal sequence
- transfection approach for defining signal sequence
2. biochemical approach
transfection approach for signal sequence
Plasmid used to transfect cells
3 different biochemical approaches
- labelling protein co-fractionates with the organelle during centrifugation
- the signal sequence is removed by a specific protease that is present inside the organelle-
- protein is protected from digestion when proteases are added to the incubation medium but its susceptible if a detergent is first added to disrupt the organelle membrane
What is the genetic approach of studying protein translocation?
- grow yeast on histamine
WT yeast- enzyme in cytosol, cell lives without histadine as a nutrient
Engineered yeast- enzyme targeted to ER, cell dies without histamine as nutrient
Engineered mutant- not all enzymes taken up into ER, cells live without histamine as nutrient
What is the nurture of the ER signal sequence
variable
At least 8 non polar aa at the centre
What are the early experiments of lysomes establish
Signal hypothesis- newly made protein should end up in the lumen of the ER
How is the signal sequence interpreted
Contains
- translational pause domain
- GTPase and SRP receptor binding site
- signal sequence binding pocket
What does binding of SRP ensure
Coupling of translation to translocation
1- SRP binds to signal sequence in polypeptide
2- causes pause in translation
3- SRP ribosome attaches to SRP receptor in ER membrane
4- Translation continues
5- SRP and receptor displaced and recycled
6-translocation continues
Pore- what is led through and how is it opened
Non polar aaa (sec61) led through
the complex sits in the pore when the ribosome is absent the pore has plug in it
What is the purpose of Sec61?
facilitates transfer of nascent chain across the ER membrane
released into ER and folds up
Translocation of soluble proteins
- inactive protein
- active translator
- mature protein in the lumen
Integration of double pass transmembrane protein
2 sequences- start and stop transfer
- the mature transmembrane proteins gets into the ER membrane but not fully through into the lumen as signal sequence not cleaved off arrest anchor
- stops in membrane
What is the ER lumen rich in?
- chaperones and gylcosylating enzymes
- Glycosylation- membrane asymmetry and quality control
- chaperones-secreted and membrane proteins
