Protein Sorting Flashcards
endoplasmic reticulum
continuous with the outer membrane of the nuclear envelope
consists of vast networks of tubules which extend throughout cytoplasm
organelles arent completely discrete
RER- responsible for protein synthesis
SER- responsible for lipid synthesis eg phospholipids
SER- functions
synthesis of lipids eg phospholipids +cholesterol- forming cell membranes
synthesis of steroid hormones eg testosterone + cortisol
storage and release of Ca2+ from sarco retic
detoxification eg of alcohol- lots of SER in liver
RER
site of protein synthesis and processing for mebrane bound proteins destined for ER, golgi, plasma membrane, lysosomes, endosomes
transmembrane and to be secreted from the cell
protein synthesis in ER
in cytosol
small and subunit bought together in ribosome
section of mrna- coding for specific protein- is bound to ribosome
protein is transcribed and leaves ribosome
protein synthesis in RER
same as above
but protein binds to ER and transferring the protein into the cell from the outside
co translational translocation
when proteins are imported into the ER during protein synthesis
one end of the protein attaches to ribosome, other end inserts itself to ER, protein will remain in primary sequence
once a protein has been translated and entered ER it will be modified- fold into 3D conformation (ionic,H, disuplhide bonds + van der waals) and glycosylation
process of co translational translocation
- recognition
signal recognition particle (SRP) will recognise signal on N terminal of growing polypeptide
binds to signal sequence and ribosome - (pauses synthesis- bring about translational pause domain)
SRP consists of RNA and 6 proteins
2.targeting
signal sequence must be recognised by ER and embedded within ER membrane
SRP receptor on RER membrane
brings it close to protein translocator, protein moves through translocator and enter ER
3.release
once ribosome has translated, mRNA is released back to free ribosome pool
SRP released from receptor and is recycled
mrna may still remain bound to ER as it can be translated to multiple ribosomes
exocytosis- proteins
if a protein is to be released fro cell, it will be fully transported into ER lumen
if a protein is destined to be a membrane protein, it will get embedded into the ER membrane
glycosylation
addition of a sugar to a protein
very specific to proteins that enter the ER- quality control, recognition, protection
precursor oligosaccharide composed of 14 sugars is added to proteins via N terminus of asparagine side chain (N linked glycosylation)
occurs during protein synthesis as proteins enter ER lumen
glcyoslation importance- quality control
quality control- recognises correctly folded proteins
1. if protein is not folded correctly 3 glucose and 1 mannose are cleaved from N linked oligosaccharide
2. still not correct, glucosyl transferase enzymes add single glucose back (proteostasis) (signal to calnexin)
3. calnexin binds to unfolded protein to prevent aggregation
4. removal of terminal glucose releases protein from calnexin
5. glucosyl transferase determines if protein is correctly folded, if not a single glucose is added back
if protein remains misfolded, it will be released from ER for targeted degradation within cell
unfolded protein response
misfolded protein accumalate in the cell, causes ER stress and trigger UPR
-inhibits protein synthesis
-degrades misfolded proteins
-increases transcription of chaperone proteins incl BiP, calnexin
apoptosis (cell death) may occur if problem persists
pulse chase experiment
used to determine how things can move around a cell
1.addition of radiolabelled amino acids to cell
2-newly formed proteins are radiolabelled (pulse)
washed away radiolabelled amino acids (chase)
3. cells returned to non radioactive medium (chase)
7 min- into golgi
120 min- into vesicles which leave the cell
membrane fusion
for vesicular cargo to reach target organelle, membranes must fuse (using SNARE proteins)
V-SNAREs (vesicle) and T- SNARE(target)- specific to one another
wrap around each other to form stable trans-SNARE complex
draw vesicle closer and closer until it can fuse
releases cargo inside membrane
during fusion, a Rab-GAP (GTPase activating protein) causes Rab to hydrolyse GTP to GDP
Rab-GDP is then released from the vesicle and bound by Rab-GTP dissociation inhibitor (GDI) to keep Rab inactive (recycled)
ER to golgi
COPII coated vesicles bud off from ER carrying cargo (protein)
mutiple vesicles can fuse (SNAREs must match) forming vesicle tubular clusters
(pathway from golgi back to ER= retrieval pathway)
golgi apparatus
stacks of flattened membrane enclosed compartments called cisternae
cis and trans face
cis golgi network receives things from ER
trans GN secretes onwards from ER
unclear how things move through golgi- by vesicles/ network is fluid
function of golgi
further processing of oligosaccharide chains
-ensures correct folding of proteins
-prevents unwanted aggregation
-acts as a signal for sorting and targeting correct pathway
lysosome
function to digest unwanted material
contain many enzymes to break down macromolecules acid hydrolases
active at 4.5-5pH - very acidic + enzymes only active at this pH
degradation occurs inside lysosome
pH maintained via vacuolar ATPase
tagged with mannose 6 phosphate
endosomes
types of lysosomes
transporters of material that has been digested by the cell (endocytosis)
fusion of late endosomes with endosomes of lysosomes generate endolysosomes- become more acidic
once digestion of endocytosed material is complete, they form lysosomes
mannose 6 phosphate tag
acid hydrolases (in lysosomes) tagged with mannose 6 phosphate
mp6 directs proteins to lysosomal network
mannose binds with hydrolase precursor (want to get into lysosome)
P=GLcNAc added which adds on phosphate tag (M6P signal) + moves through golgi network
binds to M6P receptor on edge of golgi
clathrin coated endosome containing hydrolase
transported to early endosome (will become lysosyme)
fuses with membrane
vesicle is recycled/ retrieved
endocytosis
transport into cell from plasma membrane
after endocytic vesicle fuses with early endosome, ingested material can be either
degraded- moves towards cell and membrane composition alters, no longer recycles- starts degradation
recycles- vesicles containing unwanted material for recycling to plasma membrane bud off and fuse with recycling endosome
exocytosis
two types of pathway
-consitutive secretory pathway- all of the cargo is released at once
-regulated secretory pathway- cargo is maintained in vesicle until its needed
relies on signal such as hormone or neurotransmitter
eg digestive cells (enzymes after eating), endocrine cells (insulin)
vesicles
clatharin- from plasma membrane (endocytosis) + between golgi and endosomes
cop1 coated- from golgi
cop2 coated- budding from ER
ensuring specificity- membrane fusion
Rab GTP tells vesicles which membranes are correct to bind to
GTP bound- active
GDP bound- inactive
GEF found on vesicle membrane activates Rab- activates Rab-GDP in cytosol to form Rab- GTP
Rab GTP binds to Rab effector protein on target membrane- tethering of vesicle to target membrane
