Endomembrane System Part 3 Flashcards

1
Q

How do the membranes form?

A

Membranes do not form de novo - all membranes arise from pre-existing membranes

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2
Q

Where are membrane proteins and lipids synthesized?

A

Most are synthesized at the ER

Glycolipids synthesized in the Golgi and unique chloroplast and mitochondrial proteins & lipids

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3
Q

Where do ER membrane proteins and lipids traffic?

A

Nascent ER membrane proteins & lipids can traffic to other membranes in the cell
For example, move to other ER subdomains (via lateral diffusion through the bilayer) OR to other ‘downstream’ organelles of the endomembrane system (via transport vesicles)
Results in each organelle possessing a unique complement of membrane proteins & lipids

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4
Q

Where are ER membrane proteins and lipids distributed?

A

Nascent ER membrane proteins and lipids are distributed and/or oriented in the lipid bilayer in an asymmetric manner

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5
Q

What are the three types of membrane proteins and what they do?

A

Integral Membrane Proteins: Different regions of protein located on either the cytoplasmic or exoplasmic (i.e., ER lumen) face of the ER membrane
Peripheral Membrane Proteins: Located on either the cytoplasmic or luminal side of the ER membrane
Membrane phospholipids: Distributed unequally between the cytoplasmic and exoplasmic leaflets of the ER membrane bilayer

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6
Q

Why does membrane topology need to be the right way?

A

Needs to be set up the right way to ensure the correct protein function

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7
Q

How is protein and lipid asymmetry established?

A

It is established at the ER and maintained throughout the rest of the endomembrane system, the orientation remains the same
Cytoplasmic and endoplasmic faces of cellular membranes are conserved in the endomembrane system
ER luminal protein or region(s) of ER membrane-spanning protein facing the lumen are located in the lumens of all other endomembrane compartments in which it resides or at the plasma membrane, on the exterior of the cell

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8
Q

How are newly synthesized proteins in the ER processed?

A

The final steps in the co-translation translocation pathway involve ‘processing’ of the newly synthesized protein in the ER lumen
1) Signal sequence cleavage: removal of the N-terminal signal sequence by signal peptidase
2) Initial stages of glycosylation: covalent addition of unique carbohydrate side chains to specific amino acids of the nascent protein (required for proper folding, protein-protein binding, etc) –> Add sugar groups to help determine if folded directly
3) Protein folding and assembly: nascent protein is folded into the proper 3D conformation and oligomeric assembly by molecular chaperones (reticuloplasmins)
4) Quality Control: Misfolded and/or improperly assembled proteins are recognized and degraded
ER serves as ideal processing and quality control site for nascent proteins since it represents the first compartment in the endomembrane system (biosynthetic & secretory pathway)

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9
Q

Explain glycosylation

A
Most proteins (soluble and membrane) synthesized in the ER are glycoproteins (have sugar groups added)
Glycoproteins are proteins linked to one or more sugar chain (oligosaccharides) attached to specific amino acids within the nascent polypeptide
Sugar groups aid in the protein's proper folding AND serve as binding sites for other macromolecules that interact with the protein.
Glycosylation ensures proper folding of a protein
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10
Q

What is N-linked glycosylation?

A

It is the most common type of glycosylation
The addition of specific short chains of sugar monomers (linked together in a specific order to form an oligosaccharide) to the terminal amino group of asparagine (N)

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11
Q

What does N-linked glycosylation consist of?

A

Two stages:
i) Core glycosylation
ii) Core modification
For some glycoproteins that are transported to other post ER compartments, the core modification stage continues in the Golgi

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12
Q

What is Core glycosylation?

A

The first stage of N-linked glycosylation
Various ER membrane-bound glycosyltransferases synthesize the core oligosaccharide –> highly branched oligosaccharide chain consisting of 14 sugar residues, including a 3-glucose long terminal branch (important for quality control)
The process begins with the addition of the first sugar to dolichol phosphate - membrane lipid serving as an ‘anchor’ and ‘carrier; for the new, growing core oligosaccharide

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13
Q

What does Tunicamycin do?

A

Blocks the first step of N-linked glycosylation (inhibits glycosyl-transferase action), preventing the subsequent proper folding of nascent ER proteins

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14
Q

What are the steps of core glycosylation?

A
Glycosyltransferases continue to add (one at a time) sugar units at specific positions on the growing core oligosaccharide 
Synthesis of core oligosaccharide begins on the cytoplasmic face of the ER membrane and ends on the ER luminal (exoplasmic) face --> core oligosaccharide precursor is 'flipped' across the ER membrane during its synthesis 
Final sugars (mannose & glucose units) added via 'flipping' carrier dolichol phosphates
The final step involves the transfer of core oligosaccharide from dolichol lipid carrier to nascent soluble or integral membrane protein while still being synthesized via the Sec61 co-translocational translocation pathway 
--> empty dolichol carrier recycled (& flipped) for another round of core oligosaccharide synthesis 
--> Core only transferred to the luminal-facing portions of nascent ER proteins with a specific amino acid sequence motif: N-x-S/T (core attached to asparagine residue)
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15
Q

What is core modification?

A

It is the second stage of N-linked glycosylation

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16
Q

What are the core modification steps?

A

After transfer to the nascent protein, the 14 sugar oligosaccharide(s) is gradually trimmed and modified
Two (of the three) terminal glucose units are removed (‘trimmed’) by ER lumen glucosidases
Subsequent removal (and re-addition) of the last glucose unit is important for the process that ensures proper protein folding/assembly (i.e, quality control)
During N linked glycosylation and modification, the nascent protein is rapidly folded into the proper 3D conformation which is mediated by several ER lumen and membrane proteins
Core oligosaccharide(s) added to nascent protein during N-linked glycosylation also contribute to proper protein folding/assembly and stability and participate in protein quality control
The addition of sugar and all listed above ensure proper folding

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17
Q

What is reticuloplasmins?

A

ER molecular chaperones, including BiP, calreticulin, and calnexin
Bind transiently (reversibly) to nascent ER proteins to prevent misfolding or aggregration
Involved with core-modification
BiP = Binding Immunoglobin Protein

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18
Q

What is protein disulfide isomerase (PDI)?

A

The enzyme catalyzes the formation of intra/intermolecular disulfide bonds
The disulfide bonds between cysteine residues on the same or different (nascent) polypeptides promote proper folding and assembly by stabilizing their proper 3D conformation
Involved with core-modification

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19
Q

What happens with ER protein quality control?

A

Reticuloplasmins (and PDI) bind to nascent glycoprotein (with one remaining glucose unit) while still being synthesized via the Sec61 co-translational translocation pathway –> this helps to mediate protein’s proper folding, oligomeric assembly, stability, etc
ER lumen glucosidase removes (‘trims’) the last glucose unit from the core oligosaccharide during the latter step in the N-linked glycosylation process
Protein is released from calnexin and calreticulin (reticuloplasmins)

20
Q

What happens in ER protein quality control if the protein is properly folded/assembled?

A

One mannose unit is removed by the ER lumen mannosidase enzyme (step 4)
Then functions in ER (subdomain) as an ER-resident protein OR
Transported (via vesicles) from the ER to the Golgi (where N-linked glycosylation continues) and then resides in the Golgi moves onto another compartment(s) in the endomembrane system (a lysosome, pm)

21
Q

What happens in ER protein quality control if the protein is released from calnexin and calreticulin is misfolded/misassembled?

A

Recognized by the UGGT monitoring enzyme –glucosyltransferase that acts as a ‘confirmation-sensing protein’
–> Recognizes hydrophobic residues that are usually ‘masked’ (buried) inside a correctly folded protein
UGGT adds back a single glucose to the trimmed oligosaccharide core
Misfolded/misassembled protein binds (again) to calnexin and calreticulin
–> Mediates again and with BiP proper protein folding, oligomeric assembly, etc…

22
Q

How does ER protein degradation occur?

A

The entire process of ER protein quality continues(repeated) until the protein is properly folded/assembled
Eventually, any misfolded/misassembled (abnormal) proteins are degraded (within 60 mins after synthesis in the ER)

23
Q

What are the three steps in the ER protein degradation process?

A

Recognition
Dislocation
Degradation

24
Q

What is recognition in the ER degradation process?

A

Mannose units in core oligosaccharide in misfolded/unfolded protein distinguished from properly folded/assembled proteins by mannosidases
–> Enzymes remove (>1) mannose units from the core oligosaccharides
‘Trimmed’ oligosaccharides on proteins destined for degradation recognized by ER membrane protein OS-9

25
Q

What is the dislocation in the ER degradation process?

A

Misfolded/misassembled proteins recognized by OS-9 as destined for degradation are transported out of ER lumen into the cytosol
Protein transport across the ER membrane involves ER membrane protein complex ERAD
ER- Associated Degradation pathway (not well understood) -
–> involves AAA ATPase p97- ER membrane protein utilizes ATP hydrolysis to ‘pull’ misfolded/misassembled proteins into the cytosol

26
Q

What is Cystic Fibrosis?

A

Most patients possess mutant transporter protein (CFTR) that is not properly glycosylated and, thus, degraded by OS-9/ERAD; rather than being properly targeted to the plasma membrane

27
Q

What is the degradation in the ER degradation process?

A

In the cytoplasm, oligosaccharide chains removed, and misfolded/misassembled protein is poly-ubiquitinated – protein is linked to a chain of repeating (poly) ubiquitin units

28
Q

What is ubiquitin units?

A

Mono-UB

Poly- UB

29
Q

What is ubiquitin (UB)?

A

Small (76 amino acids) protein involved in diverse cellular functions

30
Q

What is Mono UB?

A

Serves as a ‘signal’ for targeting membrane proteins intro intraluminal vesicles of late endosomes/multivesicular bodies

31
Q

What is Poly UB?

A

Serves as a ‘signal’ for ER protein degradation and for most other cellular proteins destined for normal turnover
Poly UB is dislocated out of ER

32
Q

What is Poly UB degraded by?

A

Proteasome

33
Q

What is the proteasome?

A

Barrel-shaped, multi-subunit protein-degrading machine located in the cytoplasm (and nucleus)
Garbage can –> where degraded proteins go

34
Q

What are the steps in the proteasome degradation process?

A

UB- protein binds to the ‘cap’ or ‘lid’ of the proteasome
Poly-UB chain removed (recycled)
Protein ‘threaded’ into the proteasome and degraded (via proteolysis)
Free amino acids are reused for new protein synthesis

35
Q

What happens when under certain conditions, misfolded/misassembled proteins accumulate in the ER to high levels?

A

Overload the OS-9/ERAD protein degradation pathway
In various diseases (CF, Alzheimer’s, etc.) misfolded proteins accumulate in the ER and form toxic aggregates leads to cell death
Results in ER stress that signals various Unfolded Protein Response (UPR) pathways

36
Q

What are the various pathways involved when misfolded proteins accumulate in the ER to high levels?

A

Ire1
PERK
ATF6( sense when ER stress occurs, by detecting when too many misfolded proteins are in the ER)

37
Q

What are the various pathways that are mediated by when misfolded proteins accumulate in the ER to high levels?

A

Each pathway are mediated by a unique protein sensor which is an ER integral membrane-spanning proteins

38
Q

What are the PERK and ATF6 UPR pathways?

A

Both membranes bound PERK and ATF6 possess ER luminal-facing ‘stress-sensing domains’
Bind to molecular chaperones (BiP) in the ER lumen
In non-stress conditions, PERK and ATF^ sensors are inactive due to their binding to BiP
In the ER-stress conditions (increase in misfolded/misassembled proteins) UPR pathways are activated

39
Q

What are the steps of the PERK mediated UPR pathway?

A

1) BiP released from PERK to aid in folding of accumulating (misfolded/misassembled) ER proteins
2) PERK dimerizes and becomes active
3) Cytoplasmic-facing kinase domains of ‘activated’ PERK dimer phosphorylate (inhibit) eIF2alpha
- –> Cytosolic protein translation factor required for initiation of protein synthesis – participates in ribosome- mRNA binding
4) Decrease in cellular protein synthesis (including at RER), should not try to make proteins, since having enough proteins, and want to make the misfolded protein, active
- -> Molecular chaperones (BiP) available to focus on pre-existing (misfolded/misassembled) proteins in the ER
5) ER stress is alleviated or (if not) cell death occurs

40
Q

What are the steps of the ATF6 mediated UPR pathway?

A

1) In ER stress conditions, BiP released from ATF6
–> BiP needed for folding accumulating (misfolded/misassembled) ER proteins
2) Active’ ATF6 moves from ER to Golgi (by transport vesicles) (active when in Golgi)
3) At the Golgi, the cytoplasmic-facing, transcription factor domain of ATF^ is cleaved off by a Golgi-associated protease: TF is now available
–> ATF6 transcription domain targets the nucleus via an exposed NLS (not exposed in full-length ATF6)
—-> More BiP, calnexin, etc.…
—> Here, up-regulating proteins involved in preventing/fixing protein misfolded
4) In the nucleus, the ATF6 transcription factor domain upregulates a number of genes encoding key proteins in ER quality control including:
Reticuloplasmins- assist in protein folding in the ER (BiP)
ER export components: assist in moving (via transport vesicles) properly-folded proteins out of the ER to Golgi and/or other compartments in the endomembrane system
ERAD components – assist in degrading any remaining misfolded/misassembled proteins in ER
5) ER stress is alleviated or (if not) cell death occurs

41
Q

What is the fate of a newly-synthesized protein in the ER?

A

Properly folded/assembled and glycosylated soluble and membrane proteins in the RER are either:
—-> Retained in the ER (remains localized in the RER)
OR
—-> Move (diffuses) laterally through the RER lumen or membrane bilayer to another ER subdomain where it functions (SER, nuclear envelope)

42
Q

When and how does exit from the ER happen?

A

Exit from the ER (within minutes after synthesis

Move to ER subdomain involved in the formation of transport vesicles destined for the Golgi – ER exit sites (ERES)

43
Q

What is the ER Exit Site?

A

Distinct subdomain of the ER, usually located next to the cis face of a Golgi complex

44
Q

What are the ERES enriched with?

A

ERES enriched with the molecular machinery responsible for the formation (‘budding’) of membrane-bound transport vesicles destined for the Golgi

45
Q

What is ERES machinery responsible for?

A

ERES machinery also responsible for proper packing of vesicles with correct luminal and membrane ‘cargo’ proteins (& lipids) destined for the Golgi

46
Q

What happens to resident ER proteins at ERES?

A

Resident ER proteins (BiP) prevented (but not always) from entering Golgi-destined transport vesicles

47
Q

What is protein trafficking throughout the endomembrane system like?

A

Throughout endomembrane system (ER to Golgi) is not non-selective bulk flow
—-> Most proteins in biosynthetic and secretory pathways specifically transported between compartments of the endomembrane system via unique targeting signals and receptors –> specific processes to move proteins