Lecture 2: Protein folding

Question 1

Three basic types of protein transport mechanisms in eukaryotes

Answer 1

1. transmembrane transport (co-translational and post-translations translocation)
2. gated transport
3. vesicular transport

Question 2

Endoplasmic Reticulum

Answer 2

• the membrane of the ER is a network of tubes and sacs that is continuous with the nuclear envelope

–> interior is lumen

• Two distinct regions:
  1. rough ER (ribosomes on surface)
  2. smooth ER (lacks ribosomes)

Question 3

Rough ER and Co-translational Translocation

Answer 3

• has bound ribosomes, synthesizing proteins into its lumen

–> each growing polypeptide chain conatinas a signal peptide directing it to the ER

–> SRP: this sequence binds to a signal recognition particle that then binds to the receptor of the ER membrane

–> in the RER lumen, proteins are folded and eventually further distributed via vesicular transport

Question 4

Where does co-translational translocation occur?

Bacteria, Archaea, Eukaryotes?

Answer 4

• cytosol –> ER lumen

Question 5

Where does post-translational translocation occur?

Eukaryotes? Bacteria?

Answer 5

Eukaryotes: Cytosol –> ER lumen (pulled into compartment of function)

Prokaryotes: cytosol –>extracellular space (pushed into compartment of function)

Question 6

Structure of mitochondria

Answer 6

• smooth outer membranes
• inner membranes folded into cristae

two compartments of inner membrane:

• intermembrane space
• mitochondrial matrix

Question 7

Protein transport complexes of outer mitochondrial membrane

Answer 7

• TOM complex
• SAM complex

Question 8

Inner membrane mitochondrial protein transport complexes

Answer 8

• TIM 23
• TIM 22
• OXA

Question 9

Translocation from cytosol into matrix space

Answer 9

TOM brings it in to intermembrane space

TIM 23 brings it in to matrix

Question 10

Translocation from cytosol into outer membrane

Answer 10

TOM and SAM

TOM brings protein into intermembrane space then SAM needles it back into outer mebrane

Question 11

Translocation from cytosol into inner membrane (without entering matrix)

Answer 11

TOM brings it in

TIM 22 needles it into inner mmebrane

Question 12

Translocation from cytosol into inner membrane via matrix

Answer 12

TOM, TIM 23, OXA

Tom brings it in to inner membrane space

TIM 23 pulls it to matrix

OXA inserts it into matrix

Question 13

Gated transport between nucleus and cytosol

Answer 13

• nucleus contains most of the DNA in a eukaryptic cell and is surrounded by a double membrane (nuclear enevelope) with tiny channels (nuclear pores)
• mRNAs and ribosomes are synthesized int he nucleus and exported into the cytoplasm, while materials needed in the nucleus are imported into the nucleus

–> transport of proteins and other large molecules into and out of the nucleus occurs through the nuclear pore complex

–> recognizes a “suitcase” carrying proteins and lets it in (nuclear localization signal)

Question 14

Vesicular Transport from the ER via the golgi complex to the final destination

Answer 14

• as proteins are synthesized into the ER lumen, they fold into 3-dimensional strucutres and may be modified further
• protein transport is mediated by transport vesicles that bud off the ER and carry their cargo to the golgi complex, where the vesicles fuse and deposit the cargo

–> additional post translational modification steps may occur int he golgi complex

Question 15

What is the key to ensuring that a cargo protein reaches its target destination?

Answer 15

• ability of membrane attached receprots to recognize certain features of the cargo protein (such as protein modifications)

Question 16

Summary of vesicular transport from the ER via the Golgi complex to the final destination

Answer 16

1. proteins are tagged
2. proteins are sorted
3. vesicles bud leaving lumen of Golgi (receptor type proteins on both sides of membrane)
4. external proteins of vesicle interact with receptors on lysosome
5. Fusion and Delivery

Question 17

Protein Folding: general process

Answer 17

• driven by hydrophobic forces
• initially, regions of secindary structure or other marginally stable native-like mictorstructures (foldons) may form
• followed by folding into super secondary structures via foldon interactions
• folding intermediates are rapidly brought to molten globule states (based on hydrophobic collapse) abd then to a single native comformation (due to thermodynamic constraints)

Question 18

Protein Folding model

Answer 18

• process can be pictured as a funnel of free energies
• rim at the rop represents many unfolded states
• polypeptides fall down the “wall” of the funnel to ever fewer possibilities and lower energies as they fold
• kind of “out of the blue” as they suddenly result in a functional protein
• lowest energy state is usually most functional

Question 19

Proteins that facilitate protein folding

Answer 19

1. molecular chaperones: heat shock proteins, function in preventing or reversing improper intramolecular or intermolecular protein aggregation
2. protein disulfide isomerases: thioredoxins, form or break disulfide bridges of cysteine
3. peptidyl propyl cis-trans isomerases: catalyze interconversions of X-pro peptide bonds between their cis and trans conformations. Whether or not proline in cis or trans

Question 20

Protein aggregates that result from Intramolecular contacts

Answer 20

• folding intermediates
• native states
• partially folded states

Question 21

Protein aggregates that result from intermolecular contacts

Answer 21

• amorphous aggregates
• oligomers
• amyloid fibrils

Question 22

what do most human misfolding diseases result from?

Answer 22

• formation of highly ordered protein aggregates
• -> amyloid fibrils or amyloid plaques

Question 23

Examples of amyloid related human disease

Answer 23

• alzheimers –> amyloid b or Ab peptide
• Parkinsons –> a synuclein
• spongiformencephalopathies –> prion protein
• amylotrophic lateral sclerosis–> superoxide dismutase I
• Huntington’s –> huntingtin with polyQ tracts
• Cataract –> y-crystallins
• Type II Diabetes –> Islet amyloid polypeptide (IAPP)
• Injection localized amyloidosis –> Insulin

Question 24

Process of cotrasnalational translocation into ER

Answer 24

• translocon initially closed
• GTP on rna polymerase binds receptor on translocon
• translocon opens, RNA has affinity for translocon
• RNA injected
• signal peptidase cleaves signal sequence

Question 25

TOM

Answer 25

• brings proteins from cytosol into inner membrane space

Question 26

SAM

Answer 26

• needles proteins into outer membrane from inter membrane space

Question 27

TIM 23

Answer 27

pulls proteins from inner membrane space into matrix

Question 28

TIM 22

Answer 28

• needles proteins into inner membrane from intermembrane space

Question 29

OXA

Answer 29

• needles proteins from matrix into inner membrane

Question 30

Golgi apparatus

Answer 30

• cis face recieves from ER
• vesicles bud from trans

Question 31

Molecular chaperones

Answer 31

• unfold misfolded proteins
• help protein get to its native state

Question 32

Protein disulfide isomerases (PDI)

Answer 32

• interact with cysteine to facilitate formation of disulfide bridges

Question 33

Peptidyl propyl cis-trans isomerases (PPI)

Answer 33

• interconversion of cis-trans isomers of pro peptide bonds