Lecture 7

Question 1

Where do most protiens begin synthesis?

Answer 1

Nearly all proteins, except a few inside mitochondria and plastids, begin their synthesis on ribosomes in the cytosol.

Question 2

Large and small ribosome subiunits?

Answer 2

Large subunit: Contains the active site of the ribosome - the site that creates the new peptide bonds when
proteins are synthesized. They then fold as they are made.

Small subunit: finds a messenger RNA strand and ensures that each codon pairs with the anticodon

Question 3

how does the ribosome work for protein synthesis?

Answer 3

The subunits find an mRNA molecule and clamp it, then they scan the mRNA and make a protein based on the mRNA sequence they are translating.

Question 4

what happens after proteins are synthesized from ribosomes?

Answer 4

New proteins must go from a ribosome in the cytosol to the organelle where it functions. It does so by using sorting signals in its amino acid sequence.
- Any protein that is secreted will be made in the cytoplasm and will get incorporated into the ER and then go to the Golgi and throughout the secretory pathways from there.

Question 5

Types of movement in the secretory pathway?

Answer 5

• Protein translocation (through membrane)
• Gated transport
• Vesicular transport (in vesicles)
• Engulfment

Question 6

What are the main steps of the secretary pathway?

Answer 6

1. Translates from mRNA in the
   ribosomes in the cytoplasm
2. Enters the ER lumen
3. Goes from the ER to the Golgi in a
   vesicle
4. Transits the Golgi
5. Leaves the Golgi in a vesicle
6. The vesicle fuses the cell membrane
7. It is outside

Question 7

Electron microscopy in the secretory pathway?

Answer 7

Electron microscopy give snapshots of the secretory pathway:

We can observe changes from RER -> Golgi -> vesicles -> membrane

Question 8

How can we screen for defective yeast?

Answer 8

Genetic screen for secretion defective yeast:
- Mutated yeast cells used to determine which protein was necessary for transportation in a cell
- Proteins move from mother cell to budding cell by vesicles
- In the mutated one, vesicles cannot dock to the membrane, it just builds up and makes holes.

Question 9

Mutant categories?

Answer 9

Mutants fall into distinct categories depending on where the mutated protein was required:
1. Fail ER import
2. Fail to produce ER vesicles
3. Vesicles don’t fuse to Golgi
4. Fail to leave the golgi, Golgi
vesicles do not form
5. Vesicles don’t fuse with cell
membrane

Question 10

Yeast proteins for trafficking?

Answer 10

The main proteins involved in protein trafficking were discovered in yeast, including the Sec proteins.
- Sec proteins are serotonin effector cells
- They are required to grab vesicles after the Golgi and fuse them to the membrane.
- Sec proteins unload from the ER

Question 11

How is the ER organized?

Answer 11

The ER is organized into a netlike labyrinth of branching tubules and
flattened sacs that extends throughout the cytosol. The ER has a single internal space, called the ER lumen. This means that you can get anywhere in the ER lumen from a different place.

Question 12

rough vs smooth ER?

Answer 12

The rough ER has ribosomes bound to the membrane surface. The
smooth ER lack ribosomes and is dedicated to other functions such as the biosynthesis and metabolism of lipids.

Question 13

What are peptides?

Answer 13

A signal peptide is a short peptide (usually 16-30 amino acids long)
present at the N-terminus of newly synthesized proteins that are destined toward the secretory pathway.
- Ribosome subunits associate and move from the 5 to 3 end, synthesizing the new peptide.

Question 14

Cotranslational import?

Answer 14

Cotranslational import into the ER is the first step in protein secretion. As the protein is being translated, it will be imported into the ER.

Question 15

How does cotranslational import work?

Answer 15

The ribosomes attach to the mRNA strand and begin synthesizing the peptide into the ER.
- There is a cleaved signal sequence that breaks off the signal peptidase and allows there to be a newly synthesized protein in the ER.
- If the signal is not present, it will be made ion the cytosol. When the signal is present, it is made in the ER lumen.
- This process has t be efficient because if it isnt then it will not fir through the membrane.
- the result is a mature protein inside the ER

Question 16

How is the ER signal sequence guided to the ER membrane?

Answer 16

The ER signal sequence is guided to the ER membrane by at least two
components: a signal-recognition particle (SRP), which binds to the signal sequence, and an SRP receptor in the ER membrane.
- Prevents ribosome from finishing translation right away
- This docks at the ER lumen, allowing protein synthesis to occur in lumen

Question 17

What happens when a signal sequence binds?

Answer 17

When a signal sequence binds, SRP exposes a binding site for an SRP
receptor, which is a transmembrane protein complex in the rough
ER membrane.

Question 18

Membrane bound vs Free ribosomes protein synthesis?

Answer 18

Membrane-bound ER ribosomes make proteins that are co-translocated across the ER membrane. Free ribosomes, unattached to any membrane,
synthesize all other proteins

Question 19

where does the polypeptide chain pass through?

Answer 19

The Polypeptide Chain Passes Through a Signal Sequence–gated
Aqueous Channel in the Translocator (or Translocon).
- The signal sequence is incorporated into the memrbane
- Proteins are free to go into the membrane of the ER after being synthesized

Question 20

Polypeptide chain in multiples proteins?

Answer 20

In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer.
- As they are synthesized, hydrophobic regions of the protein go into the memrbane and stay there!!

Question 21

What is involved in the ER protein folding?

Answer 21

Protein folding in the ER involve: Chaperone proteins, Disulfide bonds, and glycosylation

Question 22

ER molecular chaperones?

Answer 22

Prevent protein misfolding and aggregation (BiP)

Question 23

Disulfide bonds?

Answer 23

Folds properly and stably this way. Uses PDI: protein disulfide-isomerase.

Question 24

BiP and PDI?

Answer 24

Together, with one preventing misfolding/aggregation and the other promoting stability and proper folding, they combat any misfolding.
- Proteins have more time to be folded improperly, so these two items are very important for preventing aggregation or misfolds

Question 25

N-linked glycoslyation?

Answer 25

OST performs N-linked glycoslyation in the ER protein folding. OST = oligosaccharyltransferase

Question 26

What happens to polypeptide chains in the ER?

Answer 26

Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER

Question 27

Disulfide bonds in the ER?

Answer 27

Disulfide bonds are post-translational modifications that occur in the ER (not required in the cytoplasm). - During protein folding, disulfide bonds form the mature tertiary structure. - PDI (protein disulfide isomerase) forms disulfide bonds.

Question 28

Types of diuslfide bonds?

Answer 28

1. consecutive intra-chain disulfide bonds 2. non-consecutive intra-chain disulfide bonds They help protiens fold into their specific proper shapes!

Question 29

How are proteins synthesized in the rough ER?

Answer 29

Proteins Synthesized in the Rough ER Are Glycosylated (sugars added) by the Addition of a Common N-Linked Oligosaccharide. - N of asparagine is covalently attached to sugars/carbs

Question 30

What are oligosaccharides used for?

Answer 30

Oligosaccharides Are Used as Tags to Mark the State of Protein Folding. - if folded, a glucosidase removes the last glucose. - if not folded properly, a glucosyl transferase adds a glucose.

Question 31

What is calnexin and what does it do?

Answer 31

Calnexin binds to monoglucosylated on incompletely folded proteins and retain them in the ER. ● Calnexin is a chaperone ● Recruits an oxidoreductase (ERp57) to add more disulfide bonds ● If folding is good, GlsII removes the final glucose residue.

Question 32

ER chaperones are used for?

Answer 32

ER Chaperones prevent protein misfolding and aggregation. e.g, Hsp70/BiP

Question 33

BiP?

Answer 33

● Protects peptides from interacting with other misfolded proteins ● Create a folding environment ● give folding a second chance

Question 34

What is the main function of chaperones?

Answer 34

chaperones unfold misfolded proteins to give them a 2nd chance of folding properly

Question 35

Misfolded proteins are recognized how?

Answer 35

Misfolded proteins are recognized because they have exposed hydrophobic residues ● Hydrophobic residues should not be outside