The endomembrane system and ER Flashcards
which organelles make up the endo-membrane system
- ER
- Golgi
- Endosomes
- lysosome/vacuoles
- secretory granules
- plasma membrane
- nucleus
Which organelles are excluded from the endomembrane system
- peroxisomes
- chloroplasts
- mitochondria
what is the purpose of the endo-membrane system
- “indirect communication” through exchanging (trafficking) materials between each organelle via small, membrane-bound transport vesicles
general vesicle transport: step 1
‘cargo’ containing vesicle buds off the donor membrane compartment
general vesicle transport step 2
nascent vesicle is transported through the cytosol to the recipient membrane compartment
- involves molecular motors and the cytoskeleton
general vesicle transport: step 2
nascent vesicle is transported through the cytosol to the recipient membrane compartment
- involves molecular motors and the cytoskeleton
general vesicle transport: step 3
the vesicle fuses with the proper recipient membrane and cargo proteins are incorporated into the recipient
general vesicle transport step 4
the entire process can occur on the reverse direction
- could be recycling of any ‘escaped’ proteins from the original donor
3 main proteins in the ER
- soluble
- transmembrane
- resident protein of donor compartment
Biosynthetic trafficking pathway
ER to gogli to endosomes to lysosomes OR plama memb
constitutive secretory pathway
ER to Golgi to PM and/or released from cell
- cargo is ER-derived materials (that you make all the time)
- secretory transport vesicle membrane components are incorporated into the PM and vesicle lumen cargo is released
regulated secretion pathway
ER to Golgi to secretory granule to PM
- occurs in specialized cells (only occurs when your body tells you to
- ER-derived materials stored in secretory granules are release in response to a cellular signal
- materials released via exocytosis
endocytic trafficking pathway
- opposite direction of secretory pathways
PM to endosomes to lysosomes - incorporates materials from the PM and/or extracellular space destined for degradation
endoplasmic reticulum structure
lumen: aqueous space inside ER tubules and cisternae
tubules and cisternae: ER integral membrane proteins that regulate ER curvature - in constant flux
rough vs smooth ER
rough: mostly cisternae with bound ribosomes, involved in protein and membrane phospholipid synthesis
smooth: mostly curved tubules lacking ribosomes, involved in Ca2+ storage and hormone synthesis
ER subdomains
- smooth ER
- rough ER
- Outer nuclear membrane
- MAM and PAM
- ER exit sites
fate of proteins synthesized on free ribosomes
- remains in the cytosol
or - targets to the proper intracellular destination
fate of proteins synthesized on RER ribosomes
- remains in the RER or localizes to another ER subdomain
or - targets from the ER to another post-ER compartment in the endomembrane system
co-translational translocation of a soluble protein into the RER lumen
- insertion of a protein made on a free ribosome in the cytosol into the RER lumen
what is an SRP
signal recognition particle
- a ribonucleoparticle that binds to the ribosome and stops protein translation
- the SRP and its receptor are both G-proteins
- SRP targets the entire ribosome + polypeptide complex to the surface of the ER where it binds to its receptor
what is an SRP receptor
- hetero-dimeric ER integral membrane protein complex
- serves as a docking site for the incoming SRP
what is a translocon
a structure in the ER membrane that feeds the nascent protein into the membrane
- polar molecule with +ve AAs on its lumen side and -ve AAs on the cytoplasmic side
what causes displacement and return of the plug
displacement: when the signal sequence interacts with the interior of the translocon
return: release of the ribosome
what is the main difference in translocation of a soluble vs. integral membrane protein across the ER membrane
- soluble enters the lumen (goes all the way through translocon)
- integral membrane have TMDs
what makes Type 1 integral membrane proteins unique from the others
has a signal sequence
- has a stop-transfer anchor sequence like a soluble protein, no SAS
what makes Type 2 integral membrane proteins unique from the others
- has a signal anchor sequence
- +ve residues before the SAS
what makes Type 3 integral membrane proteins unique from the others
- has a signal anchor sequence
- +ve residues after the SAS
Insertion of tail-anchored proteins
- their hydrophobic alpha-helix is on the C-terminus
- upon emergence from the ribosome, the alpha-helix is bound by Get3-ATP
- when translation is complete, Get3-ATP binds with Get1/2-ATP, which hydrolyzes it to transfer the a-helix to a membrane-spanning channel
what proteins and lipids are not synthesized at the ER
- glycolipids synthesized at the Golgi
- chloroplast and mitochondrial lipids
different types of membrane proteins
integral membrane: different regions of the protein facing the cytosol and/or ER lumen
peripheral membrane: located on either the cytosolic OR lumen side of the membrane
membrane phospholipids: distributed unequally between cytosolic and lumen leaflets
what steps are involved in the processing of a new protein in the ER lumen
- signal sequence cleavage: removal of the N-terminal signal sequence
- initial glycosylation: addition of a sugar side chain
- protein folding and assembly: fold into proper 3D confirmation by reticuloplasmins
- quality control: misfolded or improperly assembled proteins and recognized and fixed or degraded
what is glycosylation
-addition of oligosaccharide chains to proteins
- the order of monomers in oligosaccharides impact the function of the glycosidicprotein
what are functions of oligosaccharides on glycoproteins
- assists in binding with other molecules
- assists in protein folding
- important for intracellular trafficking - targets proteins
what is N-liked glycosylation
- addition of specific short-chain sugars to the terminal amino group of an asparagine
consists of 2 stages
1. core glycosylation
2. core modification
what does the core oligosaccharide consist of
14 monomers total…
2 NAc
9 Mannose
3 glucose
process of core glycosylation
- various ER membrane-bound glycosyltransferases synthesize the core oligosaccharide
- the core oligosaccharide is a branched chain consisting of 2 NAc, 9 mannose and 3 glucose
- starting to build it on the dol phosphate, each enzyme adds a specific sugar to a specific position on the growing core oligosaccharide
- in the final step, glycosyltransferase links the core sugar to a specific N residue on the protein
- it is only transferred to luminal-facing N residue on the sequence N-x-S/T-
process of core modification
- after transfer to the nascent protein, the 14 sugar core oligosaccharide is gradually trimmed
- the first 2 terminal glucose units are removed by glucosidase 1 and 2
- the nascent protein is also folded by reticuloplasmins
- during folding, glycoprotein is also subjected to ER quality control
- mature protein is released and either retained in the ER or sent to the Golgi
what is calnexin
a membrane-bound reticuloplasmin that mediates a glycoprotein’s final folding steps
- after glucosidase II removes the terminal (last) glucose unit from the core oligosaccharide the protein is released from calnexin
what happens if the protein is misfoled when released from calnexin
- the protein is recognized by GT (glucotransferase) monitoring enzyme - recognizes hydrophobic residues that are usually masked in correctly folded proteins
- GT monitoring enzyme adds back a single glucose to the terminal end of the core
- misfolded protein binds again to calnexin to redo the final step
- entire process continues until the protein is properly folded OR it is degraded via the ERAD pathway
how does the protein enter the ERAD pathway
if the protein is misfolded it is translocated out of the ER and into the cytosol via the translocon = retrotranslocation
ER-associated degradation
- in the cytosol, oligosaccharide chains are removed and misfolded protein is ubiquitinated
- Ub-protein binds to the lid of the proteasome
- the Ub chain is removed, the protein is threaded into the proteasome where it is degraded and its amino products can be reused
what is a proteasome
a complex barrel-shaped subunit protein-degrading machine located in the cytosol (and nucleus)
mono vs poly ubiquitin
mono-Ub: serves as a signal for membrane protein import into endosomal vesicles
poly-Ub: serves as a signal for ER protein degradation and most other cellular proteins destined for normal turnover via degradation by the proteasome
unfolded protein response (UPR) pathways
- occur when misfolded proteins accumulate in the ER in levels too high for the ERAD pathway
- signalled by ER stress
- mediated by protein sensors (PERK or ATF6)
- posses ER lumen-facing stress-sensing domains that bind molecular chaperons (BiP)
- BiP binding makes the sensors inactive in non-stress conditions
PERK mediated UPR pathway
- when misfolded proteins accumulate, BiP is released from PERK
- PERK sensors dimerize and become active
- cytosolic-facing kinase domains of the activated PERK dimer phosphorylate and inhibit the TF elF2a
- protein synthesis in the cell decreases so that chaperons can focus on pre-existing misfolded proteins in the ER
- ER stress is alleviated or cell death occurs
ATF6-mediated UPR pathway
- when misfolded proteins accumulate BiP is released from ATF6
- active ATF6 moves from the ER to the Golgi
- at the Golgi, the cytosolic-facing TF domain of ATF6 is cleaved off and targets to the nucleus
- ATF6 TF domain up-regulates a number of genes encoding proteins for quality control of proteins
- ER stress is alleviated or cell death occurs
