Unit 3: Endomembrane System and ER Flashcards
What is the Endomembrane System?
- dynamic, coordinated network of all cell’s organells and related structures except peroxisomes, chloroplasts and mitochondria
What does the Endomembrane System do?
- exchange/ traffic lots of materials between organelles
What does the cell use to traffick things in the Endomembrane System?
- small membrane bound vesicles
What organelles does the Endomembrane System consist of?
- Endoplasmic Reticulum
- Golgi
- Endosome
- Lysosomes/Vacuoles
- Secretory granules
- Plasma membrane
How are the organelles of the endomembrane system structurally and functionally different?
- particular set of proteins
- unique set of activities
- compartmentalization and functional diversity
- conserved in eukaryotes
- dynamic structures
Step One Vesicle Transport
- cargo containing vesicle buds of donor membrane
- vesicle COAT proteins selects which donor membrane and lumenal cargo proteins can enter the nascent transport vesicle
- and regulate vesicle formation and budding
Step Two Vesicle Transport
- nascent vesicle tranported through cytosol to recipient membrane compartment
- vesicle RECEPTOR proteins regulate the intracellular trafficking of the vesicle to the proper recipient of the mebrane
- involves molecur motors and cytoskeleton highways
- motor proteins direct vesicle movement within the cell by linking to vesicle surface and to cytoskeleton elements
Step Three Vesicle Transport
- vesicle FUSES with proper recipient membrane compartment
- RECEPTOR proteins also regulate veiscle recipient membrane fusion
- vesicle donor membrane and lumenal cargo proteins incorporated into recipient compartment
Step Four Vesicle Transport
- entire process of budding and fusion is repeated and can occur in the reverse direction
- other receptor proteins regulate RECYCLING of any proteins that escaped to recipient compartment and bring back to donor membrane compartment
Trafficking Pathway: Biosynthetic Pathway
- ER to
- Golgi to
- Endosome to
- lysosome/vacuole OR plasma membrane
Trafficking Pathway: Secretory Pathway: Constitutive Secretion
- ER derived materials continually to Golgi to PM
- may release to extracellular space
- Secretory transport vesicle membrane componenets are incorporated into PM
- and vesicle lumenal cargo released into extracellular space
Trafficking Pathway: Secretory Pathway: Regulated Secretion
- occurs only in SPECIALIZED cells
- ER derived materials from Golgi stored in Secretory GRANULES
- in RESPONSE to cellular SIGNAL
- granules fuse with PM and
- release (exocytosis) lumenal cargo into extracellular space
Neurotransmitter release by nerve cells is what trafficking pathway?
- Regulated secretion of the secretory pathway
Trafficking Pathway: Endocytic pathway
- operates in opposite direction to secretory pathway
- moves INTO the cell
- materials from PM destined for DEGRADATION
- and extracellular space incorporated into the cell (endocytosis)
- transported into the endosomes and lysosomes (vacuoles)
What organelle is the starting point for the secretory and biosynthetic pathways?
Endoplasmic reticulum
Which organelle has the largest surface area? Describe the organelle.
Endoplasmic reticulum
- it is highly complex network of membrane-enclosed
- rod-like tubules
- and sheet-like cisternae
Two shapes of the ER structure?
ER tubules and cisternae
What mediates shape of the ER tubules and cisternae?
- reticulons
What are reticulons?
- unique ER integral membrane proteins
- hair pin V shaped secondary structure
- regulate curvature of the ER membrane (lipid bilayer)
Are ER tubules and cisternae static?
- No, they are highly dynamic and are in constant flux
- undergo constant bending, fusion, fission etc
Two classic subdomains of the endoplasmic reticulum.
- Rough ER and
- Smooth ER
Structure and role of the RER
- mostly cisternae with bound ribosomes
- involved in protein and membrane phospholipid synthesis
Structure and role of the SER
- mostly curved tubules lacking ribosomes
- involved in Ca2+ storage and hormone synthesis
3 examples of ER subdomains other than the 2 classic
- outer nuclear membrane
- mitochondria and plasma membrane- associated membranes (MAM and PAM)
- ER Exit Sites (ERES)
What are the two main sites for protein synthesis?
- free ribosomes in cytosol
2. RER - ER ‘membrane-bound’ ribosomes
What is the fate of the nascent protein in the cytosol (synthesized in free ribosome in cytosol?
- from the free ribosome in the cytosol
- fate is to remain in the cytosol
OR - target (postranslationally) to proper intracellular destination (i.e. mitochondria, chloroplasts, nucleus)
*recall all translation of mRNA begins on a free ribosome in the cyotosol - moves to RER using cotranslational translocation
What is the fate of nascent soluble or membrane protein (synthesized in the RER)?
- to remain in the RER
- or localize laterally in ER or other ER subdomain
- OR
- target (transport vesicles) from ER to another post-ER compartment in endomembrane system (golgi, endosome, lysosome, PM)
Step One: Cotranslational translocation of soluble protein into RER lumen
- Where is the signal sequence located and what is it?
- on N-terminus of nascent polypeptide
- stretch of 8-15 hydrophobic acids
- serve as ER targeting signal
Step One: Cotranslational translocation of soluble protein into RER lumen
- What recognizes the signal sequence? What is it?
SRP - signal recognition particle
- ribonucleotide consisting 6 proteins and 1 small RNA
Step One: Cotranslational translocation of soluble protein into RER lumen
What does the SRP do?
SRP binds to the ribosome and stops protein translation
Step Two: Cotranslational translocation of soluble protein into RER lumen
- What targets the entire complex?
- SRP targets complex (ribosome, stalled polypeptide, mRNA) at surface of ER
Step Two: Cotranslational translocation of soluble protein into RER lumen
What does the SRP bind to
SRP receptor
What is the SRP receptor
hetero-dimeric ER integral membrane protein complex
Step Two: Cotranslational translocation of soluble protein into RER lumen
What is the docking site?
- cytosolic facing domains of SRP receptor (docking site for SRP)
Step Three: Cotranslational translocation of soluble protein into RER lumen
When the SRP released from SRP receptor, what occurs simultaneously?
- ribosome binds the translocon (Sec61) on the cytosolic side
Step Three: Cotranslational translocation of soluble protein into RER lumen
What does SRP release require?
GTP hydrolysis
- conformational change in SRP and SRP receptor
Step Three: Cotranslational translocation of soluble protein into RER lumen
What does ribosome to translocon binding induce?
continuation of protein translation
Step Three: Cotranslational translocation of soluble protein into RER lumen
What is the interaction within the interior of translocon?
- signal sequence interacts with interior
- results in conformational change in translocon subunits
- opening/widening of pore ring and displacement of plug
Step Four: Cotranslational translocation of soluble protein into RER lumen
What happens to the signal sequence as it enters the lumen
- cleaved by signal peptidase
What is the signal peptidase?
ER integral membrane protein (protease) located next to translocon
- catalytic domain faces ER lumen
Step Four: Cotranslational translocation of soluble protein into RER lumen
What happens to the cleaved nascent protein as it enters the lumen?
- glycosylated and begins to be properly folded by reticuloplasmiins
What is a reticuloplasmin?
Chaperones at operates in the ER
- bind to nascent proteins and mediate their proper folding and oligomeric assembly
Step Four: Cotranslational translocation of soluble protein into RER lumen
What happens after protein translation and translocation is terminated?
- ribosome is released from translocon
- ribosome returns to cytosol for another round of protein import
Step Four: Cotranslational translocation of soluble protein into RER lumen
What does the release of the ribosome lead to?
- closing of translocon pore ring
- return of the plug
What are the two mechanisms of maintaining membrane asymmetry?
- lipid composition
- modification and orientation of integral membrane proteins
How are integral membrane proteins modified and oriented?
- lumenal domains of IMP
- – remains in the lumen of endomembrane compartments; glycosylated
- – forms extracellular domain on exoplasic face of PM
- cytoplasmic domain always in cytoplasm
What is the main difference in translocation and translation of soluble protein into the ER membrane and insertion of integral membrane protein into ER lumen?
mechanistic differences resulting in mature protein being inserted/anchored into the ER membrane
Step One: co-translational insertion of an integral membrane protein into RER
- term enters translocon
- 1st/only TMD enters translocon interior
- hydrophobic TMD interacts with hydrophobic pore ring stops anything further translocation of nascent protein through translocon
What is the TMD?
trans membrane domain
- typically alpha helical
- hydrophobic 16-25 a.a.
- serves as stop-transfer sequence (stop transferring protein through bilayer) of translocon
Step Two (Nlumen-Ccytosol): co-translational insertion of an integral membrane protein into RER
- interaction TMD with hydrophobic pore ring blocks translocation and signalss the translocon to open laterally
- TMD segment of protein released laterally into membrane lipid bilayer
Step Three (Nlumen-Ccytosol): co-translational insertion of an integral membrane protein into RER
Synthesis of cytosolic facing C terminus resumes
Step Two (Ncytosol-Clumen): co-translational insertion of an integral membrane protein into RER
- translocon’s interior interacts with protein TMD to stop translocation
- several +ve charged amino acid residues located upstream (N term) of TMD
What is the positive outside rule? (Step 3 of Ncytosol-Clumen)
- nascent membrane protein TMD reoriented (reversed) by translocon so that positively charged residues face cytosol
- reoriented TMD segment is released laterally into membrane bilayer
Why is a reorientation necessary?
Positively charged amino acids determine topology of all membrane proteins
Therefore, translocon +ve on ER side and +ve amino acids are near N terminus, it must be flipped so that it is in the cytosol.
Step Four (Ncytosol-Clumen): co-translational insertion of an integral membrane protein into RER
- synthesis of proteins C terminus resumes
C term is now in the lumen
How are membranes synthesized?
- not de novo
- they come from pre-existing
Where are most membrane proteins and lipids synthesized?
ER
- ER membrane proteins andl ipids can be trafficked to other membranes
How are ER membrane proteins distributed and oriented in the lipid bilayer?
- asymmetric
3 examples of protein asymmetry in the ER membrane?
- integral membrane proteins (regions face cytosol/lumen)
- peripheral membrane proteins
- membrane phospholipids (distributed unequally between leaflets)
Where is protein and lipid assembly established?
ER and is maintained throughout rest of endomembrane system.
What are the final 4 steps to co-translational translocation pathway in the ER lumen?
- signal sequence cleavage (always done) N-term cut
- Initial stages of glycosylation
- Protein folding and assembly
- Quality control
What is the most common glycosylation?
N-linked glycosylation
adding it to the amino group of asparagine (N)
Two stages of N-linked glycosylation.
- core glycosylation (just adding it)
- core modification (modifiying oligosaccharide)
Protein Glycosylation: 1st step - core glycosylation
What synthesizes the core oligosaccharide
ER membrane bound glycosyltransferases
Protein Glycosylation: 1st step - core glycosylation
What is the final step?
glycosyltransferase linking core oligosaccharide to specific N reidue on soluble or integral protein that is still being synthesized
Protein Glycosylation: 1st step - core glycosylation
Oligosaccharide ist transferred to which side of the synthesizing membrane?
only transferred to lumenal facing N residue part of sequence
- N-x-S/T-
Protein Glycosylation: 2nd step: core modification
What happens after the nascent protein has a sugar oligosacch. attahed?
- gradually trimmed and modified
Protein Glycosylation: 2nd step: core modification
What trims the oligosaccharide?
- glucosidase I and II (ER lumenal soluble proteins)
- trims 2 out of 3 glucoses
Protein Glycosylation: 2nd step: core modification
How is the glycoprotein modified?
- proper folding
- ER quality control - proper attachments
What is the name of the reticuloplasmin that mediates the final steps of glycoprotein final folding?
calnexin
- nascent glycoprotein binds to calnexin
For quality control, what removes the last glucose from the core oligosaccharide?
Glucosidase II
-released after from calnexin
What happens if protein released from calnexin is misfolded?
it is recognized by GT monitoring enzyme
What is GT monitoring enzyme and what does it do?
- ER lumenal glucosyltransferase tat recognizes the hydrophobic residues that are masked by attached sugars
- adds back single glucose residue at terminal end of trimmed core
UGGT?
recognizes misfolded protein in ER lumen
What happens to misfolded proteins?
- ERAD pathway for degredation
- retrotranslocation from ER lumen to cytosol
What binds misfolded proteins as a signal molecule for ER protein degredation?
Poly-Ub
for ERprotein degredation and othe proteins for normal turnover
What is the complex for degredation? (organelle?)
proteasome
- proteasome is a complex barrel shaped multisubunit protein located in the cytosol and nucleus
What happens when too many misfolded proteins accumulate in the ER?
- very high levels in ER of misfolded proteins means
ER stress
What does ER stress signal?
unfolded protein response (UPR)
What is the role of ER integral membrane proteins in UPR pathway?
- mediate UPR as protein sensors
- possess ER lumenal stress sensing domains that bind to molecular chaperones in the ER lumen
2 examples of protein sensors in UPR?
PERK
ATF6
In non-stress conditions PERK and ATF6 sensors are ____ due to ____
inactive
due to binding to BiP (molecular chaperones)
What are the similarities between ATF6 and PERK?
- bip is released to activate
What happens in PERK?
- PERK dimierize and active
- cytosolic facng domain phophorylate and inhibit protein translation factor e eIF2a
- therefore, protein synthesis decrease
- slows down so available molecular chaperones can focus on preexisting proteins
- ER stress alleviated
What happens ATF6?
UPR pathway
- active ATF6 moves from ER to Golgi (via transport vesicles)
- at golgi - cytosolic facing
- transcription factor domain of ATF6 is cleaved off and targets nucleus
- ATF6 transcription factordomain upregulates number of genes encoding
What is upregulated in the ATF6 pathway?
- ATF6 transcription factor domain upregulates
- ER molecular chaperones (proper folding)
- ER export components (assis in moving out of ER)
- ERAD components (assist in degrading misfolded proteins)
