Lecture 10 - Endomembranes ER import Flashcards

(93 cards)

1
Q

Where do all proteins begin synthesis?

A

ribosomes in the cytosol

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2
Q

Where do proteins need to end up?

A
  • in the cytoplasm
  • in an organelle
  • in the plasma membrane (or ER/Golgi)
  • outside the cell (secreted
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3
Q

What are the 2 protein sorting pathways?

A
  • Secretory pathway (Co-translational import)
  • Non-secretory pathways (post-translational import)
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4
Q

What are the 2 distinct locations where polypeptides are synthesized?

A
  1. 1/3 of proteins synthesized at the Rough ER
    - secreted proteins
    - transmembrane proteins
    - soluble proteins residing in ER, golgi, lysosomes, endosomes, vesicles, vacuoles (endomembrane system)
  2. remaining proteins are synthesized on ‘free’ cytosolic ribosomes
    - proteins destined to remain in cytosol
    - peripheral proteins of the cytosolic surface of membranes
    - proteins transported to nucleus
    - proteins incorporated into peroxisomes, chloroplasts, and mitochondria
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5
Q

Explain Proteins in Co-translational import

A
  • proteins carrying an ER signal sequence that direct the ribosome-polypeptide complex to the RER
  • translation is completed on RER
  • proteins processed and sorted in the ER and Golgi
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6
Q

Explain Proteins in Post-translational import

A
  • proteins lacking ER signal sequence
  • therefore, complete synthesis on ribosomes that are free in the cytoplasm
  • proteins released into cytoplasm and ones with an organelle-specific sorting signal are imported into the organelle
  • cytoplasmic proteins do not have sorting signals and remain in the cytoplasm
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7
Q

What are the 4 important points to consider with respect to protein targeting?

A
  1. Sorting Signal
    - tells proteins where to go as they are being synthesized
    - information is coded in primary sequence
  2. Receptor for the Signal Seq.
  3. Nature of the Translocation Channel
  4. Source of Energy
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8
Q

give rough overview of the endomembrane system and what each does

A
  • Rough and Smooth ER and the Golgi are site for protein (and lipid) synthesis, processing, and sorting
  • Endosomes carry and sort materials brought into the cell
  • Lysosomes digest ingested material and unneeded cellular components
  • Transition, Transport, and Secretory Vesicles move mol. btwn the compartment and plasma membrane
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9
Q

Explain structure of ER

A
  • has tubular membranes and flattened sacs (cisternae)
  • internal space is called lumen
  • luminal space of the rough and smooth ER is contiguous (ex. they are not separate organelles and don’t need vesicle transport to move cargo btwn them)
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10
Q

What is Co-Translational Import (ER-docked ribosomes) used for?

A
  • used by ribosomes synthesizing polypeptides destined for export from the cell and intracellular compartments (ex. lysosomes, endosomes, golgi)
  • these ribosomes are attached to ER early in translation, and polypeptides are transferred across ER membrane as synthesis takes place
  • an ER signal seq. directs polypeptide/ribosome to ER membrane
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11
Q

how are some polypeptides deposited into the ER lumen?

A
  • co-translational translocation of soluble proteins deposits the polypeptide into the ER lumen by a ribosome that is attached to the ER membrane
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12
Q

Who got the 1999 Nobel Prize in Medicine and for what?

A
  • Gunter Blobel
  • found that proteins have intrinsic signals that govern their transport and localization in the cell
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13
Q

Explain the Signal Hypothesis

A
  • address codes
  • proposes that intrinsic mol. signals determine the localizations of some polypeptides
  • if this signal is deleted, targeting of protein is lost
  • if signal is transplanted (to another protein), targeting is passed on to recipient protein
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14
Q

How long is the ER signal seq. and what does it direct it to?

A
  • 15-30 aa long
  • direct the complex (mRNA/polypeptide/ribosome) to the RER surface
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15
Q

ER signal seq. are usually very different. What is the little thing that they have in common

A
  • seq at the N-terminus usually has a core of 6-12 hydrophobic aa
  • a hydrophilic, often positive region, proceeds the hydrophobic core
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16
Q

What is the signal sequence recognized by?

A

the Signal Recognition Particle (SRP)

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17
Q

What stops translation/protein synthesis?

A
  • stops when the ER signal seq. has been formed
  • the SRP binds to this and blocks further translation
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18
Q

What happens when SRP binds to the ribosome?

A
  • directs ribosome to a structure in the ER membrane called the translocon
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19
Q

What is the SRP comprised of?

A
  • 6 proteins and 300 nucleotide RNA
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20
Q

What are some examples of SRPs?

A

Note: all ‘P’ can be ‘SRP’ instead. (ex. P54 = SPR54)

  • P54: binds ER signal seq.
  • P68/P72: required for protein translocation
  • P9/P14: Interact with ribosomes
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21
Q

Where does the complex bind the SRP receptor?

A
  • binds the receptor, which is an integral membrane protein, on the cytoplasmic surface of the ER membrane, in close proximity to a translocon
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22
Q

What happens once the SRP is released?

A
  • the ER signal seq is inserted into the translocon (channel protein in membrane)
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23
Q

What happens if SRP is not released?

A
  • binding halts translation and blocks the nascent polypeptide from entering the translocon
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24
Q

What happens once the ER signal seq. enters the translocon?

A
  • contact with the interior of the translocon displaces the plug, opening the channel to the ER lumen
  • as the polypeptide elongates, it passed into the ER lumen
  • a signal peptidase cleaves the seq, which is quickly degraded (final is 15-20 aa shorter)
  • chaperones bind the nascent peptide to facilitate folding
  • ribosome detaches from ER membrane, subunits dissociate and release mRNA and repeat!
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25
What is the translocon also called?
sec 61 complex
26
How does the SRP get released?
- when the SRP and receptor hydrolyze GTP
27
What do G proteins act as?
- G proteins (GTPases) act as "molecular switches" - they "switch" btwn 2 conformations (the GTP- and GDP-bound state) - in different conformations, they have different affinities for ligans
28
Explain how the GTPase goes from active to inactive and vice versa
Active (bound to GTP) ----GTPase-accelerating protein (GAP)----> Inactive (bound to GDP) ----Guanine exchange factor (GEF) and GTP (which replaces GDP)------> Active - Guanine exchange factor: proteins that kicks out GDP for GTP to bind
29
What do G-proteins play roles in?
- cell signaling (G-protein coupled receptors) - cell division - protein synthesis - cargo selection - vesicle fusion
30
How is the SRP released?
- SRP and SRP receptor are both G proteins - in GTP-bound form, they have high affinity for each other - hydrolysis of the GTP will change their conformation and reduce affinity, releasing it
31
What is the group of polypeptides synthesized on the RER destined to become?
- destined to become integral membrane proteins
32
Explain transmembrane proteins
- have hydrophobic a-helical transmembrane segments - can either be single or multipass
33
explain single and multi pass
Single-Pass (one transmembrane segment) - Type I: C-terminus is in the cytosol and N-terminus is in ER lumen - Type II: N-terminus in cytosol and C-terminus in ER lumen Multi-Pass (2 or more transmembrane segments) - both C and N terminus in cytosol (loop out and back inside) - this orientation is determined during synthesis, with the asymmetry maintained during membrane budding and fusion events - asymmetry reflects function - oligosaccharide chains are always present on the non-cytosolic side
34
explain the translocation of proteins into the ER lumen and membrane (don't be scared. it's a long answer but not bad)
1. polypeptide comes into inactive protein translocator 2. N-terminal signal seq stays in membrane as the rest goes through 3. Signal peptidase disconnects the signal seq and chain 4. protein translocator becomes inactive again, N-terminal still in membrane, mature soluble protein is now in ER lumen If the protein has the N-terminal signal seq and an internal stop transfer anchor sequence, it forms a hydrophobic a-helix that remains in the membrane: 1. Polypeptide comes into inactive protein translocator 2. N-terminal signal seq stays in membrane and the chain goes through till the stop-transfer sequence 3. signal peptidase disconnects n-terminal signal seq from the chain 4. protein translocator disconnects/becomes inactive, N-terminal signal seq. is in membrane and the chain has the C-terminus still in the cytosol side and the N-terminal in the ER lumen (stop-transfer seq in membrane) this protein is called mature single-pass transmembrane protein in ER membrane
35
How can hydrophobic transmembrane domains integrate into the ER membrane?
They can "dissolve" into the lipid bilayer through a seam along one side of the translocon.
36
What determines the orientation of hydrophobic transmembrane domains?
The charge of residues flanking the hydrophobic domain.
37
What happens to proteins that lack a cleavable N-terminal ER signal sequence? Explain process
- proteins use internal signal anchor seq. instead Type I: 1. ribosome attach, peptide enters through translocon, N-terminus in lumen, hydrophobic signal anchor seq. in translocon 2. N-terminus is negative so it stays in positive lumen 3. ribosome detaches, n-terminus in lumen and c-terminus in cytosol still Type II: 1. ribosome attach, peptide enters 2. N-terminus goes into lumen but back out to cytosol at the hydrophobic signal anchor seq because the N-terminus is now positive 3. Positive N-terminus is back in cytosol and the C-terminus goes all the way through translocon into the lumen 4. ribosome detaches, N in cytosol and C in lumen
38
What is the dual function of an internal signal anchor sequence?
- ER signal seq and membrane anchor
39
What contributes to the charge asymmetry across the ER membrane?
- abundance of negatively charged phosphatidylserine (PS) on the cytosolic side
40
What feature do both the translocon and the ER membrane share that influences protein insertion?
both have charge asymmetry, favouring orientation with pos. charges on the cytosolic side
41
What determines the orientation of a multi-pass integral membrane protein?
- the charge and orientation of the first transmembrane domain
42
How is the orientation of each subsequent transmembrane domain determined?
- must have the opposite charge/orientation of the previous domain
43
What allows multiple transmembrane domains to be inserted into the membrane?
ribosome must be recruited to the translocon for each new domain
44
What happens to a transmembrane domain after insertion?
- dissolves into membrane, becoming embedded in lipid bilayer
45
Briefly explain multi-pass
- multiple internal start- and stop-transfer seq. 1. N-terminus enters translocon into lumen, back out to cytosol as it is the pos. side 2. chain keeps entering till the next transmembrane domain 3. ribosome detaches, leaving N- and C- terminus in cytosol, with a loop in lumen 4. another ribosome attaches to the translocon to do the same thing with the next domain what happens next depends on the domains' charge orientation
46
What are the other functions of the RER? (another long memorizing one ughhhhhruiwhruihweihwlifhailfhweirhwerhwekhrwehfiweuheiwuhiwuehbrwebrwebfjebfjweb)
site of: - protein modification and maturation 1. glycosylation (in ER and golgi) = glycoproteins 2. folding of chains and subunit assembly 3. disulfide bond formation 4. GPI anchors - recognition and removal of misfolded proteins 1. in ER-associated degradation (ERAD) proteins that are incorrectly folded, modified or assembled are exported for degradation from lumen to cytosol 2. degradation occurs in cytosolic proteasomes - lipid synthesis 1. membrane biogenesis 2. establishing lipid membrane asymmetry
47
Where does N-linked glycosylation occur?
- RER
48
How are proteins synthesized in the RER glycosylated?
- glycosylated on the amide nitrogen of an Asparagine residue by the addition of a pre-formed (14 unit) oligosaccharide = N-linked glycosylation)
49
What is the composition of the 14-unit oligosaccharide used in N-linked glycosylation?
2 N-acetylglucosamine (GlcNAc), 9 mannose residues and 3 glucose units
50
What is the significance of the "N" in N-linked glycosylation?
It refers to the nitrogen atom in the side chain of asparagine where the sugar is attached.
51
What molecule carries the precursor oligosaccharide for N-linked glycosylation?
Dolichol phosphate, a lipid embedded in the ER membrane.
52
How is the oligosaccharide transferred to the protein?
It is transferred from dolichol phosphate to an asparagine residue on the growing polypeptide.
53
When does N-linked glycosylation typically occur?
During translation, as the polypeptide is being synthesized — this is called co-translational glycosylation.
54
What does dolichol phosphate structurally resemble?
A lipid — it has a long hydrophobic chain (~14 isoprene/carbon units) that anchors it in the ER membrane.
55
What is the role of dolichol phosphate in glycosylation?
It acts as a carrier for the pre-assembled oligosaccharide
56
What happens to the N-linked oligosaccharide in the ER lumen during initial processing?
Three glucose and one mannose residues are removed.
57
Why are the three glucose residues initially added to the oligosaccharide?
act as a quality control signal to indicate that a mature oligosaccharide has been formed
58
What role does the re-addition of one glucose residue play in the ER?
involved in protein folding
59
Where does further modification of N-linked glycosylation occur after the ER?
Golgi apparatus
60
Where are O-linked sugars added, and to which amino acids?
In the Golgi, added to serine and threonine hydroxyl groups
61
What are the three main functions of carbohydrate (glycan) groups on proteins?
a) Act as macromolecule binding sites b) Aid in protein folding c) Increase protein stability
62
What happens to polypeptides as they enter the ER lumen?
begin folding into their final 3D shape
63
What are Calnexin and Calreticulin?
lectin chaperones that bind to misfolded or incompletely folded glycoproteins containing one terminal glucose residue
64
When can a protein dissociate from Calnexin/Calreticulin and leave the ER?
After the third (last) glucose is removed from the oligosaccharide
65
What does PDI (Protein Disulfide Isomerase) do in the ER?
catalyzes the formation and rearrangement of disulfide bonds, helping stabilize protein structure
66
What condition must a glycoprotein meet before exiting the ER?
must be properly folded and have no remaining terminal glucose (indicating readiness for export)
67
What type of chaperone is BiP, and where is it found?
BiP is a member of the Hsp70 chaperone family, found in the ER lumen
68
What regions of the polypeptide does BiP bind to and what does it do?
- binds to hydrophobic regions of polypeptide chains - prevents aggregation of polypeptides and interactions between hydrophobic regions of different proteins
69
How does BiP release the polypeptide chain?
ATP hydrolysis causes BiP to release the chain, allowing a brief opportunity for folding
70
What determines whether a polypeptide interacts with BiP again?
If it folds correctly, hydrophobic regions are buried and BiP does not rebind if it misfolds, BiP rebinds the exposed hydrophobic areas
71
How does PDI form a disulfide bond in a protein?
It transfers its own disulfide bond to the protein, changing from oxidized to reduced
72
How is PDI re-oxidized after transferring its disulfide bond?
By another enzyme called Ero1.
73
Where in eukaryotic cells are disulfide bonds formed?
Only in the ER lumen
74
When do disulfide bonds form in the protein synthesis process?
As proteins are being synthesized, between sequentially adjacent cysteines in the polypeptide chain
75
Where does GPI anchor attachment occur?
ER
76
What happens to the protein before GPI anchor attachment?
synthesized and inserted into the ER membrane as a transmembrane protein
77
What enzyme mediates GPI anchor attachment? and how does it do it?
transamidase - cleaves the precursor protein and transfers the new C-terminal carboxyl group to the terminal amino group of the pre-formed GPI anchor
78
What is the result of GPI anchor attachment?
protein becomes anchored to the outer leaflet of the plasma membrane via the GPI anchor, no longer spanning the membrane
79
What triggers the Unfolded Protein Response (UPR)?
When proteins are made faster than they can be folded, processed, or transported, leading to accumulation of misfolded proteins in the ER
80
How do ER sensor proteins detect misfolded proteins?
The chaperone BiP is recruited away from the sensors to bind misfolded proteins, leaving sensors free to activate
81
What happens when UPR is activated?
- Phosphorylation of translation factor eIF2α → reduces overall protein synthesis - Upregulation of genes for: A. ER chaperones (e.g., BiP) B. Transport proteins to export misfolded proteins C. Protein degradation systems (like ERAD)
82
Name the three major sensor pathways in the UPR.
1. PERK 2. ATF6 3. IRE1
83
explain PERK
PERK phosphorylates eIF2α (phosphorylate translation factor), reducing translation to limit new protein entry into the ER
84
explain ATF6
when it is activated, it is trafficked to the Golgi, where it is cleaved to become a transcription factor that activates UPR genes
85
explain IRE1
IRE1 splices XBP1 mRNA, producing a transcription factor that enhances expression of chaperones, ERAD, and lipid synthesis genes
86
What happens to only properly folded proteins in the endomembrane system?
allowed to progress through the system for further processing and transport
87
Which cellular machinery degrades misfolded proteins after ER export?
The proteasome in the cytoplasm
88
What is the ER the primary source of?
- primary source of membrane lipids like phospholipids and cholesterol - most enzymes required are here exceptions: some unique lipids made in mitochondria, chloroplasts, and peroxisomes
89
Where are fatty acids for phospholipids synthesized, and where are they incorporated?
synthesized in the cytoplasm and incorporated into the cytosolic side of the ER membrane
90
How are phospholipids transferred to the lumenal side of the ER membrane?
By enzymes called phospholipid translocators (flippases) - without this, it will be a unilayer
91
How does membrane asymmetry arise in the ER membrane?
Different phospholipid translocators selectively transfer specific phospholipids to the lumenal side, creating distinct lipid compositions on each leaflet
92
Why do different organelle membranes have distinct lipid compositions?
each organelle has different enzymes and lipid transport mechanisms that shape their unique lipid profiles
93
Explain the movement of lipids
1. most organelles have enzymes that modify lipids (converted one type into another) 2. some phospholipids are selectively included in budding vesicles 3. ER can form contact sites with other organelles (ex. mitochondira, chloro., peroxi.) - Lipid Transfer Proteins exchange lipids btwn compartments - allows for movement to membranes outside of endomembrane system