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BLGY1115 > Proteins and Transport > Flashcards

Proteins and Transport Flashcards

1
Q

Ribonucleoproteins

A

proteins that contain RNA

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2
Q

What are two types of ribonucleoproteins?

A

Ribosome - more than 60% ribosomal RNA which plays an active catalytic role in ribosomes
Signal Recognition Particle

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3
Q

tRNA

A
  • activates amino acids so they an be attached to a polypeptide chain at a ribosome
  • each tRNA carries one amino acid that corresponds to the anticodon on the tRNA
  • the anticodon binds to the complimentary codon on the mRNA at the ribosome which also corresponds with the amino acid
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4
Q

Amino Acid Activation

A
  • activated by adenylation
  • costs energy
  • a 2 step reaction
  • AminoAcid + ATP + tRNA + H2O -> Amino-acyl-AMP+2Pi
  • Aminoacyl-AMP +2Pi -> Aminoacyl-tRNA + AMP
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5
Q

Aminoacyl tRNA synthetase

A
  • 2 classes of the enzyme
  • transfers activated amino acids to the correct tRNA
  • -needs to be able to recognise amino acids, ATP and tRNA
  • have to make sure the correct tRNA and amino acid are linked
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6
Q

Translation

A
  • protein synthesis always starts with the N terminus and ends with the C terminus
  • amino acids are bonded together by condensation reactions
  • movement of the ribosome along the mRNA requires energy
  • movement by one codon requires 1GTP
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7
Q

Protein Folding

A

-very generally proteins want to fold so that their hydrophobic regions are masked on the inside and their hydrophilic regions are on the outside

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8
Q

Aggregates

A

-Before protein folding or after incorrect protein folding that leaves hydrophobic regions on the outside, multiple proteins join together to mask their hydrophobic regions

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9
Q

The Role of Chaperones in Protein Folding

A
  • chaperones bind to hydrophobic regions to prevent aggregates forming while the protein folds
  • they have a protein binding domain and ATPase domain
  • chaperone initially binds to hydrophobic region weakly
  • ATP binds changing the shape of the chaperone allowing it to bind more strongly to the protein
  • energy from ATP hydrolysis is used to release the protein
  • ligand release, jolts the protein causing it to refold
  • if it is folded correctly it ill continue on the protein pathway, but, more likely, if it hasn’t chaperones will rebind and the process will continue until it does fold correctly
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10
Q

Chaperones

A
  • heat shock proteins

- heat shock 70 is the most important group

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11
Q

What are the two synthesis roots for nuclear encoded proteins?

A
  • cytosol (SRP independent)

- ER surface (SRP dependent)

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12
Q

Soluble ER Synthesised Proteins

A
  • when the signal peptide emerges from the ribosome in the cytosol, a signal recognition particle binds to it
  • it stops/slows down translation and transports the ribosome to the ER
  • the SRP binds to an SRP receptor close to a translocation pore in the ER membrane
  • the nascent peptide chain is threaded through the translocation pore
  • SRP and SRP receptor are recycled
  • transcription continues, the signal peptide is cleaved off in the ER lumen
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13
Q

BiP

A
  • an ER resident heat shock 70 protein
  • binds to nascent proteins as they emerge from the translocation pore
  • masks hydrophobic regions until final tertiary or quaternary structures have formed
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14
Q

ER and Disulphide Bridges

A

-the only proteins with disulphide bridges are formed in the ER as the ER is an oxidising environment which allow disulphide bridges to form

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15
Q

Type I Transmembrane ER Synthesised Proteins

A
  • signal peptide, coding region, transmembrane domain, cytosolic tail
  • synthesis begins in cytosol
  • SRP binds to signal peptide and transports to ER
  • nascent chain threaded through translocation pore, translation continues
  • when the translated transmembrane domain reaches the translocation pore it interacts with the pore and doesn’t pass through
  • this means that the ‘cytosolic tail’ is translated in the cytosol
  • when synthesis is complete there is an N terminal domain in the ER lumen, a transmembrane domain through the membrane and a C terminal domain in the cytosol
  • it can be transported to other organelles in the membrane of a vesicle
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16
Q

Type II Transmembrane ER Synthesised Proteins

A
  • N terminal domain, transmembrane domain, cytosolic tail, no signal peptide
  • synthesis begins in the cytosol
  • the transmembrane domain is recognised by the SRP and the ribosome is transported to the ER membrane
  • transmembrane domain threaded through pore
  • the rest of the protein is synthesised into the ER lumen
  • when synthesis is complete there is an N terminal domain in the cytosol, the transmembrane domain through the membrane and the C terminal domain in the ER lumen
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17
Q

Peripheral Membrane Proteins

A
  • bind to transmembrane proteins
  • can be washed away with a high pH wash
  • association of peripheral membrane proteins with transmembrane domain is post-translational
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18
Q

Tail Anchored Transmembrane Proteins

A
  • a special case of type II membrane spanning protein
  • no cytosolic tail
  • always post translationally translocated as protein leaves the ribosome before the SRP binds to the transmembrane domain
  • final position of the protein, N terminal domain in the cytosol and transmembrane domain in the membrane
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19
Q

Myristolation and Prenylation

A

-association of peripheral membrane proteins with the membrane via a fatty acid chain

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20
Q

Where are the majority of proteins for organelles synthesised?

A

in the cytosol

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21
Q

What are the three transport mechanisms?

A
  • Gated, e.g. transport between the nucleus and the cytoplasm mediated by nuclear pore complexes
  • Transmembrane, e.g. across a membrane of the ER, mitochondria, chloroplast etc.
  • Vesicular, e.g. between organelles of the endomembrane system
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22
Q

What are the two man stream protein targeting groups after transcription?

A
  • synthesis in the cytosol

- SRP arrest, synthesis begins in the cytosol but is finished at the ER

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23
Q

The Secretory Pathway

A
  • ER
  • Golgi
  • Secretion / back to ER / vacuole (lysosome in mammals)
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24
Q

Ligand

Definition

A

protein with a specific sorting signal

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25
Q

Non Ligand

Definition

A

protein without sorting signal or a different sorting signal for a different receptor

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26
Q

Targeting Proteins for Secretion

A

-for soluble proteins the default pathway is secretion so no signal is required

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27
Q

Targeting Proteins for ER Retention

A

-mostly achieved through retrival from the golgi

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28
Q

Targeting Proteins for Vacuolar/Lysosomal Sorting

A

-signal mediated transport from the Golgi to PVC/endosome

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29
Q

Endocytosis and Vacuolar/Lysosomal Sorting

A

Endocytosis can be thought of as vacuolar soring but in reverse
Endocytosis is mediated transport from the plasma membrane to a PVC/endosome

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30
Q

Uses of Molecular Biology to Study Cell Biology

A
  • modifying specific proteins by genetic engineering
  • creating transgenic cells to study modified genes
  • live bio-imaging and subcellular localisation
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31
Q

Uses of Genetics to Study Cell Biology

A
  • isolating mutants to study a process

- using mutants to isolate interesting genes

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32
Q

Sequencing of Various Genes Encoding ER Resident Protiens

A

-It was found that ER resident proteins had a common end amino acid sequence, HDEL or KDEL

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33
Q

Genetic Engineering to Show that KDEL is the ER Retention Signal

A
  • in an experiment KDEL was deleted from BiP. an ER resident protein
  • the truncated BiP was introduces to a mammalian cell
  • BiP was secreted from the cell
  • -showed that KDEL was necessary for ER retention
  • KDEl was fused to a normally secreted protein
  • fusion gene was introduced to mammalian cells
  • the protein accumulated in the ER
  • -showed that KDEL is sufficient for ER retention
  • -also, if proteins need a signal to be retained in the ER and they are secreted when the ER signal is removed then secretion must be default
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34
Q

Defined Protein Concensus Sequences

A
  • easily recognisable, transplantable, context independent

- e.g. KDEL

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35
Q

Signal Patches

A
  • 3D structures
  • generally no sequence motifs
  • not transplantable
  • context dependent
  • several domains of a protein can contribute to form a patch after correct folding
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36
Q

Modification

A
  • composite signal

- e.g. mannose-6-phosphate

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37
Q

N Linked Glycosylation

A
  • occurs during translocation in the ER
  • occurs as soon as the relevant piece of the protein appears in the ER
  • glycans are attached to the primary polypeptide chain
  • glycans usually end up on the surface of the protein when it is in tertiary form
38
Q

Mannose-6-Phosphate Pathway

A
  • transports proteins to lysosomes in mammalian cells
  • mannose-6-phosphate is a glycan that is phosphorylated in the Golgi (a phosphate is added by an enzyme)
  • the glycan binds to mannose-6-phosphate receptors on the inner surface of the Golgi membrane
  • ligand binding induced the formation of clathrin coated vesicles
  • transport to the endosome occurs when the vesicle is fully shaped and ready for fusion
  • at the endosome, ligand dissociation occurs due to lower pH
  • this is though to induce conformational changes that promote association with retromer to initiate vesicle recycling i.e. initiation of vesicle budding for transport back to the Golgi
39
Q

Mannose-6-Phosphate Receptor

A
  • type I membrane spanning protein
  • large luminal domain which binds to phosphorylated mannose residudes in the Golgi
  • selectively incorporated into clathrin vesicles and transported to early endosome where vesicles fuse
  • releases ligands in early endosome and recycle back to the Golgi via retromer coated membrane structure that is tubular not vesicular
40
Q

How does the Golgi know which glycans to phosphorylate when the glycan all look the same?

A
  • proteins with glycan that require phosphorylation will have a signal patch
  • the signal patch is recognised by the enzyme phosphotransferase which phosphorylates the glycans
41
Q

Genetic Engineering Demonstarting that KDEL MEdiated Retreval is From the Golgi

A
  • take a lysosomal protein and add a KDEL
  • if the KDEL signal is recognised in the ER then the KDEL would be the dominant signal and the protein would remain in the ER never reaching the Golgi or lysosome
  • it was found that the protein accumulated in the ER BUT received Golgi specific modification of mannose-6-phosphate
  • meaning that it must have been in the Golgi at some stage and returned to the ER
42
Q

Why does having a signal to return proteins to the ER make more sense than retaining them in the ER permanently?

A
  • if receptors were in the ER you would need as many receptors as there were proteins to retain
  • this would restrict the movement of protiens which need to move freely in the ER lumen to mediate protein folding
  • as they need to be in the ER umen they would end up being vesicularly transported to the Golgi at some stage anyway
43
Q

Biochemical Approach to Characterise Golgi Derived Vesicle Budding

A
  • Reductionist Approach - based on post-translational modification in Golgi as it gives rise to a change in molecular weight
  • when a protein travels from the ER to the Golgi it is modified
  • isolate Golgi from wildtype cell
  • isolate Golgi from mutant cell lacking the modification enzyme in the Golgi
  • mix the two samples together
  • modified proteins can be found in both Golgi
  • -there must be vesicular transport occurring between the two
44
Q

Purification Using Bio Assay

A
  • cytosolic crude extract containing a lot of proteins including those which mediate vesicular budding from the Golgi
  • separate crude extract into fractions by charge/weight/size etc.
  • try the bio assay with each fraction
  • any fractions which mediate vesicular budding must contain the target protein
  • separate those fractions again by a different characteristic and repeat
  • continue until you have a pure for of the target protein
45
Q

Problems With Purification Using Bio Assay

A
  • during each separation you lose some of the material
  • so eventually you will have to top it up with crude mixture
  • very complex
46
Q

Chromatography Name of Size Fractionation

A

gel filtration

47
Q

Chromatography Name of Separation by Charge

A

ion exchange

48
Q

Chromatography Name of Separation by Hydrophobicity

A

hydrophobic interaction

49
Q

Anterograde Transport

A
  • ER -> Golgi

- COPII coated vesicles

50
Q

Retrograde Transport

A
  • Golgi -> ER

- COPI coated vesicles

51
Q

Finding the HDEL Receptor

Normal Sequence of Events in Wildtype Yeast

A
  • yeast cant take in sucrose
  • but can secrete an enzyme, invertase, to break down the yeast and then take in the breakdown products

Secretion of Invertase

  • HDEL binds to receptor
  • ARP recruitment
  • coatamer
  • COPI vesicle
52
Q

Finding the HDEL Receptor

Mutant Yeast

A
  • invertase-HDEL mutants

- could not grow on sucrose as the invertase enzyme was retained instead of secreted

53
Q

Finding the HDEL Receptor

Experiment

A
  • selected from the invertase-HDEL mutants for mutated mutants that had lost the ability to retain the invertase, i.e. that could grow on sucrose
  • DNA extracted from wildtype yeast
  • DNA fragmented
  • each frag,et inserted into a plasmid
  • plasmids mixed with selected mutants
  • trying to reintroduce the inability to grow on sucrose
  • grow on glucose
  • replica plating
  • find coloies that now cant grow on sucrose, they must have plasmids with the DNA fragment containg the gene for the HDEL receptor
  • extract plasmids from these cells
  • sequence gene
54
Q

Finding the HDEL Receptor

Partial Diploids

A

-when the plasmids are introduced the yeast cells become partial diploids as they contain two copies of the genes in the plasmid, the plasmid copy and the copy in their genome

55
Q

Receptor Mediated Transport of Soluble Proteins

Model

A
  • ligand binds to receptor in the lumen
  • conformation of the receptor domain changes
  • occupied receptors in the membrane have an affinity for each other
  • receptor multimerisation brings the cytosolic domains of the receptors closer together
  • this causes a conformational shift on the cytosolic side
  • adaptor molecules are recruited
  • this stabilises the structure
  • the adapters form a scaffold for the protein coat to bind to
  • the coat proteins cause the membrane to curve
  • eventually it curves so much that a vesicle can bud off
56
Q

Uncoating of Vesicles

A
  • for a vesicle to fuse with a target membrane it first has to be uncoated
  • this requires energy
  • a GTPase inside the vesicle induces hydrolysis of ATP by a GTPase activating protein (GAP)
  • the GDP on the GTPase is then replaced by GTP by a GEF to reset the GTPase
  • the protein coat molecules are also reused
57
Q

Recycling Principle

A
  • often involve spontaneous step(s), then energy requiring steps to return to the starting position
  • if vesicle formation occurs through conformational switch triggering polymerisation of cytosolic protein coats, then uncoating will require energy
58
Q

Which GTPase is present in COPII vesicles?

A

Sar1p

59
Q

SNARE Proteins

A
  • mediate the docking process
  • ensure vesicle fusing with the correct target membrane
  • there are always 4 SNAREs
60
Q

SNARE Protein Model of Vesicular Fusion

A
  • 1 vesicle SNARE (vSNARE) binds to a 3 SNARE tSNARE complex in the target membrane
  • the vSNARE and tSNARE complex fit together in such a way that they can screw together to bring the vesicle closer to the target membrane
61
Q

Limits of the SNARE Model

A
  • genes cannot always be individually studied due to pathway complexity
  • often recycling steps depend on each other
62
Q

Endocytosis

A
  • ligands bind to receptors
  • aggregate
  • recruit adapter molecules
  • adapter molecules recruit clathrin protein coat
63
Q

What controls vesicle coat polymerisation and depolymerisation?

A
  • depolymerisation is controlled by molecular switches (GTPases), guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs)
  • coat polymerisation is thought to be spontaneous
64
Q

Transport Within the Golgi

Model I - Vesicular Transport

A
  • COPII vesicles bud from the ER containing proteins
  • they fuse with the cis Golgi
  • the protein is processed in the cis Golgi
  • a new vesicle containing the protein buds from the cis Golgi and fuses with the medial Golgi
  • the protein is processed in the medial Golgi
  • a new vesicle containing the protein buds from the medial Golgi and fuses with the trans Golgi
  • the protein is processed in the trans Golgi
  • a new vesicle containing the protein buds from the trans Golgi and continues along the secretory Golgi
  • in this model both anterograde and retrograde transport within the Golgi is by COPI vesicles
65
Q

Transport Within the Golgi

Model II - Cisternal Maturation

A
  • COPII vesicles containing proteins bud from the ER
  • these vesicles fuse together along with vesicles that bud from the cis Golgi to form a new cis Golgi
  • at the same time vesicles bud from the medial Golgi and fuse with the old cis Golgi, vesicles bud from the trans Golgi and fuse with the medial Golgi and vesicles bud from the trans Golgi to continue along the secretory pathway
  • this continues until the initial vesicles form part of the trans Golgi
  • the proteins then bud from the new trans Golgi to continue along the secretory pathway
66
Q

Why is Golgi shaped the way it is?

A
  • if you keep fusing spherical vesicles together this cannot form a larger sphere
  • this is because surface area of a sphere is proportional to radius squared but volume is proportional to radius cubed
67
Q

The Secretory Pathway Signals

Soluble Proteins - ER Export

A

no signal required, bulk flow

68
Q

The Secretory Pathway Signals

Soluble Proteins - Secretion

A

no signal required, bulk flow

69
Q

The Secretory Pathway Signals

Soluble Proteins - ER Retention

A

KDEL / HDEL

70
Q

The Secretory Pathway Signals

Soluble Proteins - Vacuolar Sorting

A
  • protein signal in yeast and plants

- mannose-6-phosphate in mammals

71
Q

The Secretory Pathway Signals

Membrane Proteins - ER Export

A
  • di-acidic cytosolic sorting signals (DXE)

- interacts with coat proteins

72
Q

The Secretory Pathway Signals

Soluble Proteins - ER Retention

A

-combination of true retention and signal mediated recycling from the Golgi

73
Q

The Secretory Pathway Signals

Soluble Proteins - Vacuolar Sorting

A

-transport from the Golgi via cytosolic signals

74
Q

The Secretory Pathway Signals

Soluble Proteins - Endocytosis

A

-internalisation of the plasma membrane via cytosolic signals

75
Q

Vacuolar Sorting Receptors

Mammals

A

mannose-6-phosphate

sortilin

76
Q

Vacuolar Sorting Receptors

Yeast

A

VPS10p (vacuolar protein sorting 10)

77
Q

Vacuolar Sorting Receptors

Plants

A

VSR (vacuolar sorting protein)

78
Q

Coated Vesicles

Clathrin

A

-clathrin coated vesicles (CCVs) mediate endocytosis and Golgi derived transport to endosomes

79
Q

Coated Vesicles

COPII

A

-COPII coated vesicles mediate ER to Golgi transport

80
Q

Coated Vesicles

COPI

A

COPI coated (coatomer) vesicles mediate Golgi to ER transport

81
Q

Coated Vesicles

Retromer

A

Retromer mediates recycling of receptors in vesicles from the endosome back to the Golgi
It is also a coated carrier but may be tubular rather than vesicular

82
Q

Vesicle

Definition

A
  • a structure formed by a protein coat
  • short lived
  • 50-100nm diameter
  • do not accumulate
83
Q

Examples of Poorly Characterised Vesicles

A

1) mammalian nerve cells store neurotransmitter in structures often described as vesicles but are actually pre-vacuoles called, secretory granules
2) plants have protein storage vacuoles often described as ‘dense vesicles’

84
Q

Learning How Host Cells Work From Pathogens

A

-some bacterial toxins use the retrograde route of the secretory pathway to enter a host cell

85
Q

Cholera Toxin

A
  • follows the secretory pathway but in reverse
  • AB5 toxin encoded by a bacterium
  • has an affinity for surface receptors
  • contains signals for retrograde transport to the Golgi
  • A-chain contains a KDEL signal for transport to the ER from the Golgi
  • from the ER it can enter the nucleus via a nuclear pore
  • in the cytosol it interferes with a signal transduction mechanism to control a range of cell surface channels
86
Q

How do viruses enter host cells by endocytosis?

A
  • glycoproteins on the surface of the virus bind to receptors in the plasma membrane of the host cell
  • this causes a conformational change of the cytosolic domain of the receptor
  • adaptor molecules and protein coat complexes are recruited
  • a vesicle forms with the virus inside it
  • the plasma membrane of the virus fuses with the membrane of the vesicle causing the vesicle to open and releasing the viral genome into the host cell
87
Q

Phagocytosis of Pathogens

A
  • whole pathogens will not fit into vesicles
  • instead they enter the cell by phagocytosis
  • they bind to cell surface receptors to induce phagocytosis
  • then fuse with the lysosome
88
Q

Pathogenesis - Manipulation of Endomembrane Traffic

A
  • early escape after phagocytosis prior to acidification in lysosome
  • modification of early endosome to prevent fusion with lysosome
89
Q

Pathogenesis - Survival in the Endolysosome

A
  • early endosome acidifies and fuses with the lysosome but the pathogen survives
  • pathogen proliferates in the acidic environment
  • it is also constantly supplied with nutrients and perfectly protected from the host immune system
90
Q

Type III Secretion - Inducing Pathogenesis

A
  • a bacterial type III secretion syringe is composed of 2 dozen proteins
  • the syringe is inserted through the plasma membrane
  • the pathogen can use it to secrete molecules into the host cell
  • it is possible for the pathogen to secrete a protein receptor into the host cell
  • the receptor implants into the host cell plasma membrane
  • a bacterial adhesion protein in the membrane of the bacterium can bind to this receptor and induce phagocytosis

BLGY1115 (10 decks)

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