BIO230 > Lecture 5 > Flashcards

Lecture 5 Flashcards

(55 cards)

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1
Q

three types of protein movements

A
  • within cell from different compartments
  • out of cell
  • into cell
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2
Q

why is protein sorting important?

A
  • protein synthesis is initiated on ribosomes in the cytosol
  • proteins must be sorted to the correct location
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3
Q

state two types of protein sorting processes

A
  1. post-translational process
  2. co-translational process
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4
Q

post-translational process

A
  • proteins fully synthesised in the cytosol before sorting
  • unfolded: mitochondria, plastids (i.e. you stay unfolded til you get there)
  • folded: nucleus, peroxisomes
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5
Q

co-translational process

A
  • endoplasmic reticulum (ER)
  • proteins with ER signal sequence associate with ER during protein synthesis
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6
Q

draw a flow chart of protein sorting mechanisms

A
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7
Q

distinguish between transmembrane transport and vesicular transport

A

transmembrane transport: requires protein translocators
vesicular transport: vesicles move protein between compartments

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8
Q

define gated transport

A

proteins moving between cytosol and nucleus mediated by nuclear pore complexes

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9
Q

nuclear pore complex

A
  • involved in selective transport of macromolecules (proteins, RNA)
  • free diffusion of small molecules (<5000 daltons)
  • made up of nucleoporins (proteins)
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10
Q

describe the two types of cargo transport through the nuclear pore complex

A
  1. nuclear import: from cytosol to nucleus
  2. nuclear export: from nucleus to cytosol
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11
Q

nuclear import receptor

A
  • binds to nuclear localisation signal
  • binds to nucleoporins in NPC
  • transport into nucleus
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12
Q

nuclear localisation signal (NLS)

A
  • rich in Lys and Arg
  • cargo proteins to be imported into the nucleus have an NLS
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13
Q

nuclear export receptor (NER)

A
  • binds to nuclear export signal
  • binds to nucleoporins in NPC
  • transport into cytosol
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14
Q

similarity between nuclear export and import receptors

A

NLS and NES are structurally related to each other

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15
Q

nuclear export signal (NES)

A
  • rich in Leu
  • cargo proteins to be exported have an NES
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16
Q

examples of cargo proteins that have an NES

A

newly assembled ribosomal subunits, RNA, proteins with regulated nuclear import and export

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17
Q

what is the function of Ran GTPase?

A

required for nuclear import and export

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18
Q

what two states does Ran GTPase cycle between?

A
  • GDP-bound
  • GTP-bound
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19
Q

what is Ran GTPase regulated by?

A
  • Ran-GAP (GTPase-Activating-Protein): stimulates GTP hydrolysis by Ran
  • Ran-GEF (Guanine nucleotide Exchange Factor): promotes exchange of GDP for GTP by Ran
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20
Q

location of Ran-GAP

A

cytosol

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21
Q

location of Ran-GEF

A

nucleus

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22
Q

Ran-GTP concentration gradient

A
  • high [Ran-GTP] in nucleus; low [Ran-GTP] in cytosol
  • this is critical for the direction of transport
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23
Q

describe the movements of Ran-GTP and Ran-GDP

Study These Flashcards
A
  1. Ran-GTP: to cytosol with nuclear import/export receptors
  2. Ran-GDP: to nucleus with NTF2 (nuclear transport factor 2)
24
Q

process of nuclear import of cargo proteins

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A
  1. nuclear import receptor binds cargo in the cytosol
  2. the receptor and cargo move to the nucleus
  3. Ran-GTP binding causes cargo release
  4. empty import receptor and Ran-GTP move to the cytosol
  5. Ran Binding Protein and Ran-GAP promote GTP hydrolysis and the release of the import receptor
25
process of nuclear export of cargo proteins
1. nuclear export receptor binds Ran-GTP and cargo in the nucleus 2. receptor and cargo and Ran-GTP move to the cytosol 3. Ran Binding Protein and Ran-GAP promote GTP hydrolysis, release of cargo, and release of export receptor 4. empty export receptor returns to the nucleus
26
give an example of how nuclear import and nuclear export can be regulated
NFAT (nuclear factor of activated T cells): high [intracellular Ca2+] > nuclear import - calcineurin (protein phosphatase) removes phosphate groups from NFAT, blocking the NES and exposing the NIS - NFAT enters the cells and activates transcription of genes for proteins needed to fight infections low [intracellular Ca2+] > nuclear export - ATP and active protein kinase phosphorylate NFAT to expose its nuclear export signal
27
experiment to demonstrate the work of NFAT
1. express NFAT-GFP fusion protein in T cells 2. add calcium ionophore (a substance that helps transport a particular ion across the membrane) 3. monitor fluorescence
28
transmembrane transport
- transport into ER, mitochondria, plastids, peroxisomes - requires protein translocators
29
protein translocators
- transport of protein across membrane - protein usually unfolded
30
why do mitochondria and chloroplasts require proteins?
they have their own genomes and ribosomes but most proteins are nuclear encoded - translated in cytosol - imported into organelle (post-translational, proteins unfolded)
31
how do proteins imported into the mitochondria and chloroplasts remain unfolded?
proteins remain unfolded in cytosol by association with hsp70 chaperones
32
draw a diagram of the mitochondria and chloroplasts
33
importing proteins to the mitochondrial matrix
- protein translocators: TOM and TIM23 complexes - precursor protein has a mitochondrial signal sequence (peptide) consisting of an N-terminal amphipathic alpha-helix - signal sequence binds receptor and moves through TOM and TIM23 complexes into the matrix space, where it is then cleaved - proteins can also be further sorted
34
importing proteins into the chloroplast
- protein translocators: TOC and TIC - precursor protein has a chloroplast signal sequence consisting of an N-terminal amphipathic alpha-helix, a signal sequence cleaved in the chloroplast - if targeting to thylakoid, it will have a hydrophobic thylakoid signal sequence which is unmasked when chloroplast signal seq cleaved
35
are mitochondrial and chloroplast signal sequences the same? why/why not?
chloroplast and mitochondrial signal sequences are different for correct targeting in plants
36
sorting proteins to the peroxisome
- precursor protein contains a peroxisomal targeting signal consisting of 3 amino acids at the C-terminus (SKL) - protein folded - transported across membrane by large translator complex
37
how are proteins sorted to the ER? how can they be further sorted?
- sorted by transmembrane transport - can be further sorted by vesicular transport to other compartments or to the surface
38
topological transport
- orientation stays consistent throughout all of vesicular transport - for example, if its sorted into the ER lumen it will remain in the lumen of organelles
39
two main functions of the ER
- synthesis and modifications of proteins - synthesis of lipids
40
give two types of proteins sorted to the ER
- soluble proteins, transmembrane proteins - proteins destined for Golgi, secretion, lysosomes
41
how can proteins to be sorted to the ER be distinguished from other proteins?
they have an ER signal sequence - this consists of hydrophobic amino acids at the N-terminus
42
describe the process of protein sorting to the ER
1. mRNA and ribosomes bind 2. translation starts: the ER signal sequence emerges first 3. SRP binds to this signal sequence and the ribosome 4. ribosomes are directed to the ER membrane 5. co-translational translocation
43
SRP cycle
- signal recognition particle (SRP, a promiscuous protein) and SRP receptor have GTPase domains that bind GTP - SRP + ribosome = low affinity - SRP + ribosome + ER signal sequence = high affinity > binds SRP receptor 1. ribosome forms a tight seal with the translocator, preventing diffusion of ions and small molecules 2. the binding of SRP to SRP receptor causes GTP hydrolysis, leading to the release of the SRP + SRP receptor complex
44
what is a translocon?
a protein translocator channel (gated channel)
45
protein sorting of soluble proteins to the ER
1. the ER signal sequence (N-terminal start-transfer sequence) binds to the translocator 2. a signal peptidase cleaves the ER signal sequence 3. the ER signal sequence laterally diffuses into the lipid bilayer (as the translocator is gated in a 2nd direction) 4. translocated protein is released into the ER
46
for single pass and multi-pass transmembrane proteins, the N and C terminal ends are sorted out during
translocation
47
protein sorting of transmembrane proteins (Single-Pass 1) to the ER
- ER signal sequence: NH2 start-transfer - TM domain is a stop-transfer signal which laterally diffuses into the lipid bilayer - protein synthesis continues in the cytosol (COOH in cytosol)
48
protein sorting of transmembrane proteins (Single-Pass 2, 3) to the ER
- TM domain is an internal start-transfer sequence and is not cleaved, it laterally diffuses into the lipid bilayer - orientation is determined by amino acids flanking the internal start-transfer sequence
49
how is orientation of transmembrane proteins determined?
- protein translocator is (-) facing the cytosol; more positive amino acids face the cytosol - protein translocator is (+) facing the ER lumen; more negative amino acids face the ER lumen
50
Rhodopsin
1st TM = start-transfer (- to + AAs) 2nd TM = start-transfer 3rd TM = stop-transfer 4th TM = start-transfer
51
what do ER targeting sequences (N terminal, internal) and stop transfer sequences consist of? how are they predicted?
they consist of specific hydrophobic sequences and are predicted by stretches of hydrophobic amino acids
52
three types of membrane proteins
- integral (including transmembrane) - lipid anchored - peripheral
53
describe the chemical composition of most transmembrane proteins
most transmembrane proteins are glycosylated on extracellular face
54
what does GPI anchored proteins stand for?
glycosylphosphatidylinositol anchored proteins
55
formation of GPI anchored proteins
- target protein has C-terminal hydrophobic domain (signal for GPI-anchor) - GPI anchor is preformed in the membrane - ER transaminase enzyme transfers protein to GPI anchor - GPI-anchored protein ends up on ER luminal side and can go to cell exterior surface
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