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BIO230 > Lecture 9 > Flashcards

Lecture 9 Flashcards

1
Q

review: animal cells

what is:
- extracellular matrix
- lysosomes

A

extracellular matrix: specialized materials outside of the cell – very specific

lysosomes: function in the degradation in cellular components that are no longer necessary

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2
Q

what are the typical organelles that are in plant and animal cells vs. just plant cells

A

all cells:
* nucleus
* Endoplasmic reticulum
* Golgi apparatus
* Peroxisome
* Mitochondria
* Plasma membrane

plant specific:
- vacuole (2 kinds, storage and degradation)
- chloroplasts
- cell wall

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3
Q

what is protein sorting?

A
  • movement of proteins:
    ➢within cell from different compartments
    ➢out of cell
    ➢into cell
  • protein synthesis is initiated on ribosomes in cytosol
  • proteins must be sorted to the correct location
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4
Q

what are the two types of protein sorting

A
  1. Post-Translational Process
    * proteins fully synthesized in the cytosol before sorting but after translation
    * stays unfolded if it goes to the – then gets folded: mitochondria, plastids
    * protein is folded first before it reaches: nucleus, peroxisomes

ex. going to the nucleus: protein will be made first and will be folded before it gets there

  1. Co-Translational Process (next class)
    * Endoplasmic Reticulum (ER)
    * proteins with ER Signal Sequence
    –> associated with ER during protein synthesis
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5
Q

what are the three transport mechanisms of protein sorting
- where does it move to

A

gated transport:
- proteins move between nucleus and cytosol

transmembrane
transport:
* requires protein translocators
- only goes one way from cytosol
- goes to plastids, mitochondria, peroxisomes, ER

vesicular transport
* vesicles move protein between compartments
- can go bidirectionally
- goes to: perixosomes, ER, golgi, endosome, lysosome, early endosome, plasma membrane, cell exterior

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6
Q

what is gated transport? and what complex does it use

A
  • proteins moving between cytosol and nucleus
  • Uses the nuclear Pore Complex (NPC)
    –> selective transport of macromolecules
    –> free diffusion of small molecules (<5,000 Daltons)
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7
Q

what is nuclear import and export in relation to NPC

what are NPC made up of?

A
  • Transport of cargo through Nuclear Pore Complex (NPC)
  • this transport can occur in both directions

1) Nuclear Import
* from cytosol to nucleus
2) Nuclear Export
* from nucleus to cytosol

NPC is made up of nucleoporins

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8
Q

Nuclear import:

the proteins that want to go in the nucleus (cargo proteins) has what attached to it?

what does the nuclear import receptor bind to and what does it do?

A
  • Cargo proteins have a Nuclear Localization Signal (NLS)

Nuclear Import Receptor:
* binds to NLS (rich in Lys and Arg)
* binds to nucleoporins in NPC
* cargo protein with the NLS binds to the receptor to transport complex in the nucleus

  • different nucleus import receptors to bind to NLSs - NLS are specifc to receptor
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9
Q

Nuclear export:

the cargo proteins that wants to leave the nucleus has what?

what does the nuclear export receptor bind to and what does it do?

A

Cargo proteins have a Nuclear Export Signal (NES)
* newly assembled ribosomal subunits, RNA, proteins with
regulated nuclear import & export

Nuclear Export Receptor
* structurally related to nuclear import receptor
* binds to NES (rich in leucine)
* binds to nucleoporins in NPC
* transport into cytosol

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10
Q

Ran GTPase = Ran = Ran protein

  • what can Ran cycle being bound to?
  • what are the two ways Ran GTPase is regulated by? (GAP and GEF)
A

Ran GTPase protein enzyme cycles between being:
* GDP-bound on cytosolic side
* GTP-bound on nucleic side
- Ran GTPase is required for nuclear import and export

Ran GTPase is regulated by:
* Ran-GAP (GTPase-Activating Protein) in the cytosol
➢stimulates GTP hydrolysis by Ran (cuts Ran-GTP to Ran GDP in cytosol)

Ran-GEF (Guanine nucleotide Exchange Factor) in the nucleus
➢promotes exchange of GDP for GTP by Ran in the nucleus

note: high [Ran-GTP] in nucleus; low
[Ran-GTP] in cytosol

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10
Q

where does Ran-GTP and Ran-GDP form and move to? how are they transported?

A
  1. Ran-GTP: forms in the nucleus, moves to cytosol
    * with nuclear import/
    export receptors
  2. Ran-GDP: formed in cytosol. doesnt really move to cytosol
    * transported by NTF2
    (Nuclear Transport Factor 2)
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11
Q

what is the pathway of nuclear import in the nucleus and cytosol?

look at the diagram on slide 16 in notes

A
  1. Nuclear import receptor
    ➢binds cargo in cytosol
  2. Receptor + cargo move to nucleus by binding to nucleoporins on Nuclear Porin complex (NPC)
  3. Ran-GTP exchanges binding spots with cargo on the receptor
    ➢ causes cargo released in nucleus
  4. Import receptor + Ran-GTP move to cytosol
  5. Ran protein and Ran-GAP promote:
    ➢GTP hydrolysis to Ran-GDP + P
    ➢release of import receptor back in cytosol to be reused
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12
Q

what is the pathway of nuclear export in the nucleus and cytosol?

look at the diagram on slide 17 in notes

A
  1. Nuclear export receptor
    ➢binds Ran-GTP + cargo in nucleus
  2. Receptor + cargo + Ran-GTP move to cytosol
  3. Ran Protein and Ran-GAP promote:
    ➢GTP hydrolysis to GDP + P
    ➢release of cargo to cytosol
    ➢release of export receptor for reuse
  4. Empty export receptor returns to nucleus
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13
Q

what does NFAT stand for and what is its general movement in the nucleus

A

NFAT = Nucleated factor of activated t cells

  • if your body recognizes a bacteria it wants to attack it to mitigate the bacteria spread asap. You need NFAT to transcribe the gene that activates the t-cells to attack the bacteria.
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14
Q

how is NFAT used as an example for nuclear import so that it can fight bacterial cells

A

nuclear import: cytosol to nucleus
1. resting t-cell is chilling but somehow there is an increase in [Ca2+] in the resting t cell
2. activates protein called calcineurin (protein phosphatase)
3. calcineurin removes the phosphate groups on NFAT which creates a nuclear import signal
4. calcineurin blocks the nuclear export signal that already defaultly exists on NFAT
5. thus the nuclear import signal is free and thus stimulated the movement in the nucleus
ACTIVATION OF GENE TRANSCRIPTION

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15
Q

how is NFAT used as an example for nuclear export

A

nuclear export: nucleus to cytosol
steps:
1. resting t-cell is chilling but somehow there is a low [Ca2+] in the resting t cell
2. ATP + active protein kinase phosphorylates the NFAT protein and the nuclear export signal moves the NFAT in the cytosol

16
Q

summarize NFAT role and how it moves based on concentration of Ca2+

A
  • high [intracellular Ca2+] → nuclear import
  • low [intracellular Ca2+] → nuclear export
17
Q

2nd way of protein sorting: Transmembrane transport

which organelles use transmembrane transport for protein sorting? what does transmembrane transport require to be conducted?

A
  • ER, mitochondria, plastids, peroxisomes
  • can only move one way (import)
  • Protein translocators
    ➢transport of protein across membrane
    ➢protein usually unfolded
18
Q

where are mitochondria and chloroplasts translated? do proteins enter the mitochondria and chloroplast as folded already or unfolded?

A

Mitochondria and chloroplasts have their own genome and ribosomes BUT most proteins are nuclear-encoded
➢translated in cytosol

  • proteins enter unfolded and then get folded inside
  • they remain unfolded in the cytosol by hsp70 chaperons which only help folding at the right time (ie when they enter)
19
Q

importing proteins in the mitochondrial matrix:

look at diagram slide 22

A
  • The protein translocators that assist in protein sorting in mitochondria are: TOM (translocase outer mitochondrial membrane) and TIM23 (translocase inner mitochondrial membrane) complexes
  • Precursor unfolded protein has a mitochondrial signal sequence (peptide) which composes of a specific N-terminal amphipathic alpha-helix
  • signal sequence binds to the receptor → moves through TOM and TIM23 complexes into the mitochondrial matrix space
  • the signal sequence is then cleaved and protein folds once inside
  • Proteins can also be further sorted
20
Q

Importing Proteins to the Chloroplast

LOOK AT DIAGRAM 23

A
  • Protein translocators in chloroplast membranes (TOC, TIC complex)
  • Precursor protein has a chloroplast signal sequence which is also made of a specific N-terminal amphipathic alpha-helix
  • signal sequence binds to receptor and moves through TIC, TOC complexes into chloroplast stroma/matrix
    ➢Signal sequence cleaved in chloroplast
    ➢Different from mitochondrial signal sequence for correct targeting in plants
  • If the precursor protein targets the thylakoid…(which is like the cistra in mitochondria) then
    ➢ the hydrophobic thylakoid signal sequence is unmasked when chloroplast signal seq. is cleaved, thus transported in the thylakoid
  • protein becomes folded
21
Q

Importing Proteins to the Peroxisome

reference diagram slide 24

A

Precursor Protein that is being sorted has a peroxisomal targeting signal of 3 amino acids at C-terminus (SKL)

  • the protein folded before entering
  • attaches to a receptor and transports to a docking protein on peroxisomal membrane, then transports the protein in the matrix through the large translocator on the peroxisomal membrane leaving the receptor in the cytosol
  • thus successful sorting of peroxisomal matrix protein
22
Q

review the mechanism, signal and if the protein is folded before transport for the following protein sorting locations:
- nucleus
- mitochondria
- peroxisomes
- chloroplasts

A

Nucleic transport:
1. destination: nucleus
mechanism: gated
signal: NLS rich in Lys and Arg
protein folded before transport: yes

transmembrane transport:
2. destination: mitochondria
mechanism: transmembrane
signal: amphipathic a-helix
protein folded before transport: no

  1. destination: peroxisomes
    mechanism: transmembrane
    signal: 3 amino acid sequence (SKL) at C-terminus
    protein folded before transport: yes
  2. destination: chloroplasts
    mechanism: transmembrane
    signal: N- terminal amphipathic a-helix
    specific to chloroplasts
    protein folded before transport: no

BIO230 (15 decks)

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