2.1 - Transmembrane Transport (Protein Target to ER)

Question 1

ER

Answer 1

protein synthesis organelle within cell comprised of branching tubules and flattened sacs and lots of ribosomes

Question 2

ER function

Answer 2

synthesis of (almost) all proteins that are secreted to extracellular space is followed by transport to ER lumen

Question 3

microsomes

Answer 3

small vesicles produced upon breaking ER by sonication

- like small versions of ER

Question 4

ER signal sequence

Answer 4

• 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

Question 5

what is the AA sequence of the ER signal sequence

Answer 5

none, the physical properties matter much more than the exact amino acid sequence

Question 6

is the ER signal sequence interchangeable

Answer 6

yes

Question 7

does anything happen when you put the ER signal sequence on a cytosolic protein

Answer 7

yes, it will be redirected to the ER

Question 8

why are ER proteins that are synthesized in vitro without a microsome larger?

Answer 8

usually, the ER signal sequence is cleaved off after the protein is transported

Question 9

how is the ER signal sequence cleaved off

Answer 9

• recognized by translocons (complementary receptors)

- signal peptidases removes the signal sequence

Question 10

how are microsomes purified

Answer 10

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]

Question 11

leader peptide

Answer 11

the last peptide of the of the ER signal sequence

Question 12

what happens when microsomes are added after protein synthesis with proteins that have an ER signal sequence

Answer 12

they are not transported into the microsome and the

Question 13

explain the 1970 experiment with protein synthesis (insert s7) where microsomes were add after

Answer 13

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

Question 14

explain the 1970 experiment with protein synthesis (insert s8) where microsomes were add during protein synthesis

Answer 14

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

Question 15

in protein synthesis of ER proteins, how are the ribosomes targeted to the ER?

Answer 15

with the help of a signal recognition particle (SRP)

Question 16

for ER proteins, how is the signal sequence inserted into the channel

Answer 16

ER signal sequence inserted as a hairpin loop into the channel through the translocation channel

Question 17

what state is the ER protein in when it traverses the ER membrane

Answer 17

in the unfolded state

Question 18

SRP structure

Answer 18

elongated particle made by six different polypeptides bound to small RNA molecule

Question 19

what is the function of the RNA molecule in SRP

Answer 19

acts as a scaffold to organize the SRP proteins

Question 20

how do the ends of the SRP interact with the ribosome

Answer 20

• one end blocks the amino acid+tRNA entry door of the ribosome (stops protein synthesis)
• one end interacts with emerging signal sequence

Question 21

how does the SRP guide the ribosome to the ER?

Answer 21

• 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

Question 22

why is the specific sequence of the SRP so important

Answer 22

revents protein misfolding/aggregation/activation in the cytosol