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BIOL 2020 > Endomembrane system > Flashcards

Endomembrane system Flashcards

1
Q

Endocytic pathway

A

Cell surface -> Endosomes -> lysosomes (usually)

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2
Q

Autoradiography (pulse-chase)

A

Incubate tissue in radioactive amino acids (PULSE), wait (CHASE). Photographic emulsion to cover tissues, radioactive amino acids react to turn those spots black to figure out where the protein is located.

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3
Q

Typical pulse-chase experiment

A

3 minute pulse - no chase (in ER).
3 min pulse - 20 min chase (in golgi).
3 min pulse - 120 min chase (in vesicles).

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4
Q

Subcellular fractionation

A

Homogenise cells then centrifuge to separate into cells, nuclei, and mitochondria. Centrifuge the supernatant again to pellet the microsomes.

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5
Q

Microsomes

A

Vesicles formed from the ER and golgi after homogenisation.

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6
Q

Centrifugation of the microsomes results in:

A

Smooth layer on top of the rough.

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7
Q

Sec mutants process

A

Expose yeast to low levels of mutagens, then pick out the mutants but only those that are heat sensitive.

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8
Q

Five classes of sec mutants based on the pathway steps.

A
  1. New proteins cant get in the ER.
  2. New proteins accumulate in the ER.
  3. New proteins accumulate in vesicles between the ER and golgi.
  4. New proteins accumulate in the golgi.
  5. New proteins accumulate in secretory vesicles.
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9
Q

Typical sec mutants experiment

A

Grow normal and mutant yeast cells at room temp, increase the heat, the perform a pulse-chase, followed by autoradiography to analyse the protein locations.

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10
Q

Smooth ER functions

A

Make steroid hormones, detoxification using enzymes to oxidise hydrophobic compounds, store calcium.

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11
Q

Rough ER functions

A

Visible ribosomes that are important in synthesising proteins in the secretory pathway

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12
Q

Free ribosomes

A

proteins that remain in the cytosol or stay on cytoplasmic leaf as peripheral proteins. Go to nucleus or mitochondria.

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13
Q

Membrane-bound ribosomes

A

Proteins to secrete or that are integral membrane proteins. Some may be involved in the endomembrane system.

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14
Q

Free ribosomes pathway

A

Complete the polypeptide in the cytosol.

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15
Q

Membrane-bound ribosomes pathway

A

Attached to the ER and feed protein into the ER lumen to be completed and then secreted.

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16
Q

Signal hypothesis (Blobel)

A
  1. All ribosomes are the same.
  2. The amino acid signal on a new protein directs the growing protein and ribosome into the ER.
  3. The protein will be fedd into the ER lumen as it is translated.
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17
Q

Cotranslational import steps to make a secretory protein

A

SRP temporarily stops translation.
SRP binds receptor for docking.
Nascent polypeptide goes into pore.
Signal peptidase cleaves the signal sequence.
Polypeptide is deposited into the ER lumen for chaperones to help.

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18
Q

SRP

A

Signal recognition particles - 6 polypeptides plus a small bit of RNA

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19
Q

Components of the cotranslational import translocon

A

SRP receptor
Pore
signal peptidase

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20
Q

Evidence Blobel was correct

A

Subcellular fractionation in the presence of microsomes shows signal peptidase activity.
Addition of the signal sequence directs proteins to the functional ER (microsomes).

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21
Q

Transmembrane protein type I

A

Amino in lumen, ER signal sequence and stop-transfer sequence.
ST halts translocation (movement across membrane), signal removed, protein released laterally.

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22
Q

Transmembrane proteins type II

A

Amino in cytosol, internal start-transfer seq.
Internal transfer creates a cytosolic loop until the C terminus moves through the translocon and the protein is released laterally.

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23
Q

Multi-pass transmembrane proteins

A

Multiple internal start- and stop-transfer sequences.
Start-transfer begins transfer, continues til stop-transfer, portion released laterally and next start-transfer repeats the process.

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24
Q

N-linked oligosaccharides production

A

Core oligosaccharide made in ER, carbohydrate chain grows on dolichol phosphate in the ER membrane.
Glycosyltransferases add monosaccharides to cytoplasmic side of DP.
Flippase flips dolichol and the sugar chain to face lumen of ER, more sugars added.
Oligosaccharide protein transferase take the sugar chain and add it to the Aspargine.

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25
Glycosyl transferases
Add monosaccharides to the dolichol on the cytoplasmic side
26
Dolichol phosphate
Lipid upon which the carbohydrate chain will grow using glycosyltransferase.
27
Oligosaccharide protein transferase
Takes the core oligosaccharide sugar chain off the dolichol and adds it to the asparagine of a protein.
28
Flippase
Flips the dolichol and attached sugar chain from the cytoplasmic side the lumen of the RER.
29
Quality control process of core oligosaccharides
2-3 glucoses are removed and the protein will bind Calnexin to fold. Glucosidase II will then remove the thrid glucose. If not correctly folded, a gylcosyltransferase UGGT adds a glucose back on so that calmnexin rebinds to try again. If irreparable, the protein gets fed back out for reverse translocation to be destroyed by a proteasome.
30
Calnexin
A chaperone involved in the folding of N-glycosylated proteins.
31
Glucosidase II
Removes the third glucose.
32
Glycosyltransferase UGGT
Adds glucose back on if a N-glycosylated protein has been incorrectly folded.
33
Proteasome
Barrel-shaped, capped, protein degrading machines driven by ATPases, and digest proteins that have been ubiquitin tagged.
34
Ubiquitination uses three enzymes
(8.5 kDa peptide ubiquitin) E1/E2 - ubiquitin carriers. E3 - ubiquitin ligase, recognises misfolded proteins and transfers ubiquitins from E1/E2 to the protein.
35
Difference between proteasomes and lysosomes
P - barrel-shaped, degrade ubiquitin tagged proteins, ATPase activity. L - specialised organelle, acid hydrolases, M6P tag.
36
BiP bound to sensors in the ER membrane.
If there are too many misfolded proteins, it gets recruited and the sensors become active.
37
Active BiP- absent sensors functions (2)
1. DImerise and phosphorylate to elFα to bind the small ribosome subunit and reduce protein synthesis. 2. Undergo proteolytic cleavage and cytosolic portion acts as a transcription factor to make more stress-alleviating proteins.
38
More modifications to N-linked glycosylation. happens in the _____, as the ______ protein progresses through different _________.
Golgi; cargo; compartments.
39
Cisternae
Stable compartments moving in the cis-trans direction, cargo gets shipped from compartment to compartment.
40
Cisternae maturation model
Cis - fusion of vesicles in the ERGIC. “Mature” as the mave from cis to trans face. Cargo stays in the same compartment. Golgi-resident enyzmes are shipped backwards by retrograde transport to return to “home” compartment.
41
Evidence of cisternae maturation model (3)
1. Cargo exclusively found in cisternae, not vesicles - maturation not transport. 2. Golgi-resident proteins are found in both cisternae and vesicles as the move in the retrograde direction. 3. Mutations to ER vesicle formation cause the golgi to disappear.
42
Three types of coat proteins
Involved in selecting what components get included, and form on the cytosolic face. 1. COPII 2. COPI Clathrin
43
COPII-coated vesicles function
Anterograde from the ER to the golgi Interact with ER export signals involving GTPase activity.
44
COPI-coated vesicles
Retrograde from the Golgi to the ER, and from the trans to cis face of the Golgi
45
Clathrin-coated vesicles
From the trans golgi out, and from the plasma membrane to endosomes.
46
COPII-coat formation (ER->Golgi)
1. Sar1 (GTPase) binds GTP (active). 2. Hydrophobic tail swings out of Sar1 and enters the bilayer, curving the membrane. 3. Sar1-GTP recruits two COPII adaptor polypeptides (sec23/24), a heterodimer that binds the tails of transmembrane cargo receptors. 4. Two more COPII outercoat polypeptides join (sec13/31) to form an outer coat.
47
COPII adaptor polypeptides
Sec23 and Sec24 - form a heterodimer.
48
COPII outercoat polypeptides
Sec13 and Sec31
49
Sec31 mutants
Affected in the formation of vesicles as they leave the endoplasmic reticulum, causing cargo proteins to accumulate in the ER.
50
COPI-coat formation
Same mechanism as COPII but GTPase is Arf1 instead of Sar1, and different coat proteins (7) form a coatamer in a triskelion shape.
51
Retrieval sequence for retrograde transport
lys-asp-glu-leu (lets all go lie (down)) or KDEL
52
Clathrin coat formation
3 heavy chains, 3 light chains, in a triskelion structure forming a lattice. Also uses Arf1 (GTPase) bending an αhelix into the membrane. Adaptor proteins vary - GGAs (trans golgi), AP2 (endocytosis).
53
GGAs
recruit clathrin from the trans golgi
54
AP2
recruits clathrin from the plasma membrane - endocytosis.
55
Targeting acid hydrolyases to lysosomes occurs through the ________ using the tag ___.
Secretory pathway; MP6 (mannose-6-phosphate)
56
Mannose-6-phosphate binds
Tag used on lysosomal enzymes that binds a M6P-receptor on the trans face of the golgi to be packaged in clathrin coated vesicles
57
GGA adaptor protein in lysosomal vesicles
Binds M6P receptor, Arf1-GTP, and clathrin.
58
Role of dynamin in clathrin coats
Polymerise around the clathrin lattice, pinching and bring the membrane in close proximity before GTP hydrolysis cause vesicle fission.
59
Nonhydrolysable GTPs in Dynamin functioning (GTPγS)
Excess polymerisation of dynamin in a ring around the membrane but no fission so it just sticks out.
60
How can a monomeric G protein be broken? (2 ways)
1. nonhydrolysable GTPγS 2. Sec mutant for Sar1
61
Rabs, function and mechanism
Small, GTP-binding proteins that specify vesicle destination. Tethering and docking: Associate with membranes via a lipid anchor, recruit tethering proteins to loosely attach the vesicle.
62
SNARES, function and mechanism
Membranes that mediate vesicle fusion via α-helix domains in a coiled-coil (Docking). - t-SNAREs on the target membrane - v-SNAREs on the transport vesicle
63
Tethering process
Rabs bring in tethering proteins to loosely hold vesicle in place.
64
Docking process
SNAREs make a coiled-coil to pull the vesicle into close proximity with the target membrane, and then promote fusion via interaction of t and v snares.
65
Dissociation during membrane fusion process
NSF uses ATP to untwist the SNAREs so that parts may be reused.
66
Receptor-mediated endocytosis in chicken oocyte yolk proteins.
Yolk particles accumulate in a coated pit with inner surface clathrin, deepening of pit forces curvature to trap additional particles. Vesicle formation and budding with intact clathrin coat.
67
Lateral diffusion to coated pits
Domains in the cytoplasmic rich in particular receptor types form pits via clathrin coating indentations.
68
AP2 in receptor-mediated endocytosis
Adaptor protein complex of four polypeptides that connects the membrane receptors and clathrin coat
69
Phagocytosis
Engulfment of relatively large particles
70
Autophagy
Destruction of organelles by isolation in a double-membraned vesicle, followed by fusion with a lysosome
71
How proteins get into the mitochondria
Hsp70/90 deliver proteins to the mitochondria in an unfolded state so that they go through the TOM complex.
72
TOM functions (2) from the outer mitochondrial membrane
Send to the inner mitochondrial membrane TIM22. If destined for the matrix it will send it to TIM23, which feeds it through.
73
TIM22
In inner mitochondrial membrane for embedded proteins.
74
TIM23
Protein moves through and transit sequence is cleaved by mitochondrial transit peptidase. It then binds to chaperone Hsp60

BIOL 2020 (8 decks)

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