L4 Flashcards
Give 2 types of signal sequence in membrane traffiking?
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.
what is the signal sequence for return to the ER (and ER retention)?
KDEL (HDEL in yeast) K- lysine D - aspartic acid E - glutamic acid L - leucine
(4 amino acids)
give a function of the smooth ER?
SER makes fatty acyl components and lipids.
highly abundant in lipocytes
why is RER easy to isolate?
- RER has a characteristic morphology
- 50% of cellular membrane - can be isolated when homogenised
- rough and smooth microsomes form vesicular structures
what are differences in densities between smooth and rough microsomes?
- 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)
How was it determined that KDEL is both necessary and sufficient for ER retention?
- 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)
what recognises the ER signal sequence?
signal recognition complex (RNA and 6-7peptides)
- signal sequence binding pocket - can recognise different ER signal sequences - lined by methionine)
what does binding of signal recognition complex/particle do?
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)
what is sec61?
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
when is pore in ER membrane open?
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?)
how is the signal sequence degraded?
ER signal sequence is cleaved by signal peptidase and then released into ER and degraded.
what is the ER lumen rich in?
rich in chaperones and glycosylating enzymes
- chaperone essential for function of secreted and membrane proteins - correct folding
why is quality control by the ER important?
- 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)
what happen is protein is correctly folding in ER?
packaged into ER exit sites and undergoes secretion from ER in COPII coated vesicles
(via ERGIC53 and VPL)
what happens when protein is incorrectly foled in ER?
- 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)
