protein targeting and export 3 Flashcards
formation of microsomes
5 points
start with intact cell and pulse with radiolabelled aa
homogenize, separate out microsomal fractions
allow protein synthesis to go out to completion
microsomal membrane can be destroyed with detergents
labelled proteins in intact microsomes are resistant to protease digestion
sucrose gradient is seporatedby
density
of different conc
how to fractionate cellular components
place at the top of gradient and spin
denser particles sediment the furthest
free or bound differences
how do we extract ribosomes from the microsome
treat with detergent
free or bound differences
how to make stripped microsomes
adding EDTA (chelates Mg2+)
or mix KCL and puromycin
causes premature chain termination
mix KCL and puromycin
how does it cause premature chain termination
mimicking aa-tRNA
free or bound differences
what can we do with the componets
reconstruct them in vitro
both free and bound ribosomes can
translate the same mRNA
and attach to the microsome and translate while bound
if both ribosomes can do the same job
message (mRNA) differentiates what happens
N-terminal signal sequence
ER what do the localised proteins contain
core of 6-12 hydrophobic AA, preceded by one or more basic aa
mature proteins what happens when they are in the ER
signal sequence by cleavage between the residue
we can change the order of components and add them together to reconstruct the experiment what happens
mRNA have been translated into protein with the right signal
cannot enter microsome after they are made
also if the dont enter the signal sequence is not removed
what is used to recognise the signalsequence protruding from the elongating ribosome
the signal recognition particle SRP
what is the SRP composed off
6 proteins
and a 300 specific nucleotide RNA molecule that binds the ribosomal RNA
what does the p54 protein contain
hydrophobic patch of methionine that interact
