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Advanced Molecular > Membrane Trafficking > Flashcards

Membrane Trafficking Flashcards

lecs 12,13 yeast screening and membrane compartmentalisation

1
Q

What is membrane trafficking and why do eukryotic cells need it?

A
  • The movement of membrane-bound vesicles within the cell
  • Allows complexity
  • Enzymes can modify specific subsets of proteins in certain environments
    -Retrieval of proteins back to ‘resident’ compartment
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2
Q

What is the exocytic pathway?

A
  • AKA secretory/biosynthetic
  • Protein synthesis takes place on ribosomes on the rough ER
  • Transported into ER membrane and folded in the lumen
  • Packed and bud off into vesicles at trans golgi network
  • Trafficked to function within an organelle where they fuse with the plasma membrane via SNAREs
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3
Q

What is the endocytic pathway?

A
  • AKA recycling or degradative
  • Material coming from outside of the cell entering a vesicle
  • Can be sent to lysosome for degradation or Golgi apparatus for recycling
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4
Q

Where are proteins modified?

A

Moving through the ER and golgi

  • Processes such as glycosylation and addition of oligosaccharides
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5
Q

Are mitochondria part of the membrane trafficking pathway?

A

NO

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6
Q

What is glycosylation?

A
  • When protein enters ER it can be modified
  • Addition of sugars
    -Transferred as a large single unit in the ER
  • Sugars can be modified and add complexity
  • Each sugar added is a similar size to an amino acid
  • If it is added as transport into ER happens it will effect the folding
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7
Q

What is the purpose of glycosylation?

A
  • To assist folding
  • As a ligand
  • Outside the cell for interactions with extracellular matrix with proteins/sugars on other cells
  • Interactions with pathogenic molecules
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8
Q

What genetic organisms are used to identify trafficking pathways?

A
  • Yeast
    -Drosophila
  • Slime mould
  • Zebrafish
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9
Q

What makes a model suitable for studies on membrane trafficking?

A
  • Simplicity (single-celled organism)
  • A model system that is able to perform the function that your interested in
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10
Q

What are the pros in using yeast as a model organism?

A
  • Can grow as haploid and diploid so recessive mutations can be studied
  • Entire genome is known and annotated
  • Cheap and easy to grow in large quantities
  • Limited gene diversity
  • Fundamental pathways are conserved
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11
Q

What are the cons in using yeast as a model organism?

A
  • Limited cell-cell contact
  • Very small
  • Has a cell wall which can preclude some types of study
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12
Q

What is the Sec Yeast screening used for?

A

Identify genes that were mutated and cause a defect in the capacity for cells to secrete proteins to the cell surface

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13
Q

What is the End yeast screening used for?

A

Identify genes involved in endocytosis

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14
Q

What is the VPS yeast screening used for

A

In yeast the lysosome is called the vacuole
- Identified genes that were destined for the vacuole/lysosome

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15
Q

Who did the key experiment regarding the Sec Pathway?

A

Novick and Schekman (1980)
- Secretory pathway in yeast

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16
Q

What did Novick and Schekman predict?

A

That if proteins couldn’t be secreted the cell would increase in density as the vesicles carrying the proteins accumulate

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17
Q

What was N + S’s experimental analysis?

A
  • Cells were analysed for their ability to secret enzymes (invertase and acid phosphatase) at permissive and restrictive temps
  • Denatured enzymes= cannot secrete
  • Secretory mutants were the strains that couldn’t export the enzymes but carried on synthesising proteins in restrictive conditions
  • Also looked through electron microscopy at subcellular structures
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18
Q

What genes were identified through Sec screening?

A

At least 23 genes were identified by grouping mutants with similar phenotypes where the producing products are required to ensure transport of proteins from ER to PM

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19
Q

What is alpha factor?

A
  • Secreted by cells as a pheromone
  • Pre-protein, glycosylated in the ER
  • Size can be determined by western plotting
  • In later stages of the pathway the signal sequence is cleaved off and the remaining protein is cut into smaller peptides and secreted
  • example of a protein which enters the ER, is glycosylated and is cleaved
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20
Q

Why weren’t all of the genes/proteins involved in the exocytic pathway identified by N + S ?

A
  • Set up only with temperature sensitive mutants
  • Only considered secretion to the plasma membrane so defect in transport to endosome or vacuole would not be identified
  • Any redundantly functioning genes would not be identified
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21
Q

What is gene redundancy?

A

A biochemical process being encoded by two or more genes

22
Q

What compartment decides whether the protein is going to the lysosome or the plasma membrane?

A

The Trans Golgi Network (TGN)

23
Q

Why is endocytosis important?

A
  • Downregulation of signals
  • Remodelling cell surface lipids and protein composition
  • Homeostasis
  • Nutrient and neurotransitter recycling
  • Retrieval of molecules that form secretory vesicle
23
Q

What is endocytosis?

A

Process through which the plasma membrane invaginates the cell resulting in the production of a vesicle that is then able to fuse with endosomes

24
Q

What are the stages in the endocytic pathway? (END screening)

A
  • PM to endocytic vesicle
  • Endocytic vesicle to early endosome
  • Early endosome to late endosome or recycle to PM
  • Late endosome to Golgi vacuole (degradtion)
25
Q

How is endocytosis studied in yeast?

A
  • Mutants that cannot internalise fluid phase marker dye on a pheromone alpha-factor (Flouresence-based assays)
  • actin cytoskeleton was found to be important
  • Knockout genes and flourescent microscopy (labelling endoctyic proteins to track under microscope)
26
Q

What is autophagy?

A

destruction of damaged or redundant cellular components occurring in vacuoles within the cell.

27
Q

How are resident enzymes of the lysosome transported there?

A
  • Through the secretory pathway
  • At the TGN it is decided whether the enzymes go to the PM or the lysosomes
  • Yeast identified this sorting pathway
28
Q

What does the VPS pathway look at?

A
  • What would stop enzymes reaching the lysosome and being able to degrade material
  • The resident enzymes of the lysosome are instead in the PM or intracellular space
29
Q

What is an example of an enzyme and the research around it that is normally transported to the lysosome?

A

Carboxypeptidase Y
- Labs look for mutagenized cells which generate CPY and those which do were investigated using microscopes and biochem techniques
- 60 vacuolar protein sorting (vps) genes were identified

30
Q

What is shown in a biochemical analysis of CPY?

A
  • CPY is glycosylated and proteolytically cleave at different stages
    -This helps to follow its process
31
Q

How are vacuolar mutants divided?

A

Depending on the stage at which they block the route to the vacuole

32
Q

How is CPY recognised and moved to appropriate compartment?

A
  • CPY synthesised and transported through ER to Golgi
  • In late Golgi CPY recognised by Vps10 receptor
  • Adaptor proteins Gga1 and Gga2 and clathrin that recognises cytoplasmic tail of Vps10 when bound to CPY
  • Packaged into vesicles taken to late endosome
  • CPY dissociates from Vps10 and enters vacuole
  • Vps10 is recognised and retrieved by signalling to late Golgi to be reused
33
Q

What is a nuclear pore?

A
  • Formed at junction of the inner and outer membrane of nuclear envelope
  • Material allowed in and out (gated transport)
  • Made up of 30 different nucleoporins (proteins)
  • During cell division the nucleoporins get distributed as the nuclear envelope breaks down
34
Q

What do the nuclear pore complexes do?

A
  • Moves substances across nuclear envelope
  • Histone molecules are shuttled in as they are needed for DNA synthesis and made in the cytosplasm
  • For translation to occur, ribosomes need to enter the nucleus from the cytoplasm and then leave
35
Q

How dosubstances get in and out of nuclear pores?

A
  • Diffusion: larger molcules are slower until they weigh 60,000 then diffusion is blocked and they need a ‘permit’ to be moved in by active transport
  • Energy and signal is needed for active transport to open up the pore to a wider diameter
36
Q

What is the experimental evidence supporting active transport?

A
  • mRNA transport into the nucleus inhibited on cooling to 4 degrees (???????)
  • ATP hydrolysis is required in vitro
  • In absence of ATP the protein binds to the pore complex but remains outside
  • Add ATP and the protein starts to appear inside the nucleus
37
Q

What does co-translational translocation and post-translational translocation mean?

A

Co= It is made at the same time it is transported
Post = It is made and then transported

38
Q

Translocation into the ER requires a signal, describe the signal hypothesis (co-translational, soluble protein)

A
  • (E.g.) mRNA translated at ribosome and has a signal on its N-terminus
  • Recognised by ER membrane protein called Sec61
  • Signal sequence stays in Sec61 and the polypeptide threads through as it is translated
  • Once translated, signal peptidase cleaves off signal sequence from protein and the peptide is in the lumen
39
Q

What makes co-translational, co-translational like what gives it the name?

A

The ribosome on the outside of the membrane is v. closely associated with Sec61, there’s no leakage of ions when the nuclear pore opens for the sequence to bind so, the translation and transportation occur at the same time as it feeds through

40
Q

How are membrane proteins (insoluble) inserted into the ER?

A
  • Membrane proteins have transmembrane domains which allow anchoring
  • Signal sequence at N-terminus recognised by Sec61
  • Protein is threaded through but the hydrophobic transmembrane domain is recognised as a stop transfer sequence
  • Signal peptidase cleaves signal peptide
  • C-terminus in cytoplasm and N-terminus anchored in ER by the transmembrane domain
41
Q

Why would it be that the N-terminus remains in the cytoplasm during translocation rather than the C-terminus?

A

Because the signal peptide is contained within the peptide so it is recognised by Sec 61 then the N-terminus (which is above) remains in the cytoplasm and the C-terminus feeds through

42
Q

What helps the soluble proteins to fold within the ER lumen?

A

Chaperone proteins, disulphide bonds

43
Q

How do antibodies (IgG) get folded into their structure in the ER

A
  • Two heavy chains and light chains in a ‘Y’ shape
  • BiP is an ER chaperone that remains associated with a subunit of an antibody until its fully assembled allowing the antibody to be packaged and transported away
44
Q

Give an example of a protein folding defect that has lead to disease

A

The cystic fibrosis delta 508 mutation
- Leads to a misfolded protein that gets stuck in the ER, this leads to the ‘unfolded protein response’ (UPR) because the ER becomes full
- Protein synthesis is then basically shut down

45
Q

How do proteins get into the mitochondrial matrix?

A
  • Example of post translational translocation
  • N-terminal signal sequence is recognised by the TOM complex on the outer mitochondrial membrane
  • Protein translocates through TOM and then through the intermembrane space and then through TIM23 which is on the inner mitochondrial membrane
  • Signal is cleaved
  • TOM and TIM23 allow the soluble molecule to pass through hydrophobic membrane
46
Q

What is different about the signalling sequence for a protein that enters the mitochondria?

A

Receptor is recognised by the structure of amino acids rather than the order
- E.g. Cytochrome oxidase signal sequence in a amphipathic alpha helix

47
Q

How are proteins translocated into the outer membrane (e.g. porin) in mitochondria?

A
  • Protein goes through TOM complex
  • Translocated into intermembrane space
  • Chaperone proteins bind
  • Binds to SAM complex on the outer membrane
  • This is the weird one from last year like why cant it just bind straight to the SAM complex
48
Q

How are proteins translocated into bacterial outer membrane?

A
  • Proteins translocated through Sec proteins
  • The same process used in the SAM complex for mitochondria happens
49
Q

How are proteins translocated into chloroplasts?

A
  • Same mechanism but TOC and TIC instead of TOM and TIM23

Advanced Molecular (13 decks)

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