2.1 - Transmembrane Transport (Protein Target to ER) Flashcards
ER
protein synthesis organelle within cell comprised of branching tubules and flattened sacs and lots of ribosomes
ER function
synthesis of (almost) all proteins that are secreted to extracellular space is followed by transport to ER lumen
microsomes
small vesicles produced upon breaking ER by sonication
- like small versions of ER
ER signal sequence
- short N-terminal AA sequence (~20-25)
- vary greatly in AA
- structure: positively charged N-terminal, 7-15 NP AA, peptidase cleavage 3-7 AA after hydrophobic sequence
what is the AA sequence of the ER signal sequence
none, the physical properties matter much more than the exact amino acid sequence
is the ER signal sequence interchangeable
yes
does anything happen when you put the ER signal sequence on a cytosolic protein
yes, it will be redirected to the ER
why are ER proteins that are synthesized in vitro without a microsome larger?
usually, the ER signal sequence is cleaved off after the protein is transported
how is the ER signal sequence cleaved off
- recognized by translocons (complementary receptors)
- signal peptidases removes the signal sequence
how are microsomes purified
equilibrium sedimentation on sucrose gradient
- smooth microsomes (low density) stop sedimenting and float at low [sucrose]
- rough microsomes (high density) stop sedimenting and float at high [sucrose]
leader peptide
the last peptide of the of the ER signal sequence
what happens when microsomes are added after protein synthesis with proteins that have an ER signal sequence
they are not transported into the microsome and the
explain the 1970 experiment with protein synthesis (insert s7) where microsomes were add after
P=precursor protein
M=mature protein (leader peptide cleaved off)
lane 1+2: addition of microsomes after protein synthesis does not transport protein inside
lane 3+4: addition of protease digests the proteins outside of the microsome. addition of a non-ionic detergent digests the proteins inside the vesicles
explain the 1970 experiment with protein synthesis (insert s8) where microsomes were add during protein synthesis
lane 1: only p proteins synthesized
lane 2: some mature proteins synthesized and transported to the microsome and the signal sequence is cleaved off
lane 3 : the p proteins that were not transported to the microsome are digested by the o protease
lane 4: the proteins that were in the microsomes have been digested by the proteases as well because the detergent has broken down the microsome bilayer
in protein synthesis of ER proteins, how are the ribosomes targeted to the ER?
with the help of a signal recognition particle (SRP)
for ER proteins, how is the signal sequence inserted into the channel
ER signal sequence inserted as a hairpin loop into the channel through the translocation channel
what state is the ER protein in when it traverses the ER membrane
in the unfolded state
SRP structure
elongated particle made by six different polypeptides bound to small RNA molecule
what is the function of the RNA molecule in SRP
acts as a scaffold to organize the SRP proteins
how do the ends of the SRP interact with the ribosome
- one end blocks the amino acid+tRNA entry door of the ribosome (stops protein synthesis)
- one end interacts with emerging signal sequence
how does the SRP guide the ribosome to the ER?
- SRP bound to the ribosome attaches itself to the SRP receptor protein in the rough ER membrane
- SRP and SRP receptor released from the ribosome regulated by GTP
why is the specific sequence of the SRP so important
revents protein misfolding/aggregation/activation in the cytosol
