L4

Question 1

Give 2 types of signal sequence in membrane traffiking?

Answer 1

signals can mediate protein sorting

linear - signal sequence at end terminus

patched - several signal sequences along protein that require correct folding of protein for sequences to come together to form signal patch.

Question 2

what is the signal sequence for return to the ER (and ER retention)?

Answer 2

KDEL (HDEL in yeast)
K- lysine
D - aspartic acid
E - glutamic acid
L - leucine

(4 amino acids)

Question 3

give a function of the smooth ER?

Answer 3

SER makes fatty acyl components and lipids.

highly abundant in lipocytes

Question 4

why is RER easy to isolate?

Answer 4

• RER has a characteristic morphology
• 50% of cellular membrane - can be isolated when homogenised
• rough and smooth microsomes form vesicular structures

Question 5

what are differences in densities between smooth and rough microsomes?

Answer 5

• smooth microsomes have low density - float at low sucrose concentrations.
• rough microsomes have high density - float at higher sucrose concentrations.

(can separate smooth and rough microsomes)

Question 6

How was it determined that KDEL is both necessary and sufficient for ER retention?

Answer 6

• proteins containing KDEL sequence and proteins without KDEL were compared and see which were retained at ER. (can be isolated)
• KDEL sequence added to protein which is usually secreted (resides in cytoplasm)+ see if retained by ER (was retained). (transfection approach)

Question 7

what recognises the ER signal sequence?

Answer 7

signal recognition complex (RNA and 6-7peptides)

- signal sequence binding pocket - can recognise different ER signal sequences - lined by methionine)

Question 8

what does binding of signal recognition complex/particle do?

Answer 8

couples translation to translocation
- brings nascent polypeptide with ER signal sequence to membrane (binds to receptor in ER membrane) as it is being translated to be translocated

(binding of SRP to signal sequence causes pause in translation)

Question 9

what is sec61?

Answer 9

membrane protein translocator

• facilitates transfer of nascent chain across ER membrane into ER lumen
• must undergo conformational change to open and allow nascent protein to be fed through pore into ER lumen

Question 10

when is pore in ER membrane open?

Answer 10

when ribosome sits on top, the sec61 pore opens- translocation begins - protein fed through pore
- ribosome bought over to pore/sec61 by SRP (binds to signal sequence on peptide) binding to SRP receptor in ER membrane
(so translations and translocation are coupled?)

Question 11

how is the signal sequence degraded?

Answer 11

ER signal sequence is cleaved by signal peptidase and then released into ER and degraded.

Question 12

what is the ER lumen rich in?

Answer 12

rich in chaperones and glycosylating enzymes

- chaperone essential for function of secreted and membrane proteins - correct folding

Question 13

why is quality control by the ER important?

Answer 13

• ensures correct protein folding
• prevents transport of aberrent proteins
• retains precursor proteins in environment where they can mature
• favours correct folding by increasing subunit conc
• reduces toxicity risk by inhibiting aggregation and degrading terminally misfolded proteins
• maintains secretory pathway homeostasis
• developmental regulation of protein secretion
• stores proteins for regulated secretion (e.g. seed formation in plants)

Question 14

what happen is protein is correctly folding in ER?

Answer 14

packaged into ER exit sites and undergoes secretion from ER in COPII coated vesicles
(via ERGIC53 and VPL)

Question 15

what happens when protein is incorrectly foled in ER?

Answer 15

• glucose is removed (by glucosidases) and another glucose is transferred to unfolded protein (by UGGT)
• gets another chance to fold correctly (undegoes CNX cycle again)- interacts with chaperones
• knows incorrectly folded if hydrophobic aa’s exposed
• targetted to ERAD pathway after a few rounds of incorrect folding (mannose trimming by manosidase acts as timer)

Question 16

what is ERAD?

Answer 16

endoplasmic reticulum associated degradation

- regulated mechanism to remove incorrectly folded proteins (reverse translocation)

Question 17

what is UPR?

Answer 17

unfolded protein response

• ER stress induced signalling pathway
• aims to reduce abundance of unfolded proteins in ER

Question 18

what are the sensors/signalling arms in UPR?

Answer 18

IRE1
AFT6
PERK
(sense abundance/ misfolded proteins?)

Question 19

what does acitivation of UPR signalling arms do?

Answer 19

• causes upregulation of genes which increase oxidative protein folding in ER
• prevent protein entry into ER (prevent translocation)
• upregulate ERAD
• mRNA degradation of secretory proteins
• decrease protein translation

(aims to reduce abundance of unfolded proteins in ER)

Question 20

How does magnitude of ER stress determine cellular outcome?

Answer 20

low ER stress- cells survive
High ER stress- pro-apoptotic (if UPR does not reduce unfolded protein levels ER stress remains high)

(cancer cells make use of this)

Question 21

How can you determine components required for protein translocation into the ER lumen biochemically?

Answer 21

use labelled protein containing ER signal sequence to see if is transported/traslocated into nucleus by:

• seeing if labelled protein cofractionates with organelle/ER during centrifugation
• see if signal sequence removed by specific protease in ER (will see 2 bands on gel if is present in ER)(one on own, one in ER)
• protein protected from digestion if proteases added to medium (protected in organelle/ER) but not if detergent used first (disrupts ER membrane)

Question 22

give genetic approaches to study protein translocation?

Answer 22

enzyme (HDH) which reside in cytoplasm produces histadine from histodinol in yeast
-histadine is required for yeast to make proteins (otherwise yeast dies)
- ER signal sequence added to HDH and can see if yeast cells can survive without a histodine environment (cells which produce own histodine don’t need histodine environment)
(cells with ER sequence - HDH targetted to ER - yeast dies as histodine not produced)
(mutant translocation apparatus - see if HDH in cytoplasm/ER or not- if not all transported-cells can survive)