#23: The Secretory Pathway & Protein Quality Control

What 2 types of proteins go through the secretory pathway?

secreted proteins, integral membrane proteins

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

hormones

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

LDL

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

serum blood albumin

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

blood clotting proteins

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

antibodies

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

snot (mucus)

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

connective tissue proteins

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

digestive enzymes

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

spider silk

secreted

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

ion channels

integral membrane

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

nutrient transporters

integral membrane

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

hormone receptors

integral membrane

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

neurotransmitter receptors

integral membrane

Is this an example of a SECRETED protein or an INTEGRAL MEMBRANE protein?

cell adhesion molecules

integral membrane

___% of the genes in your genome encode membrane proteins

30

Secreted and integral membrane proteins are _____________ ___________ into and through _____ and ______ compartments, then transported to ______ destinations.

sequentially transported, ER, Golgi, final

Palade’s Pulse-Chase Experiment

What was his question?

What type of cells did he study to answer this question?

What route do proteins take through a cell?, guinea pig pancreas exocrine cells (they secrete digestive enzymes)

Palade’s Pulse-Chase Experiment

Cellular process?

Pulse?

Chase?

protein secretion via the secretory pathway, radioactive leucine (³H-Leucine), normal leucine (¹H-Leucine)

Palade’s Pulse-Chase Experiment

Data Collection

• Took __________ ______________ (a photograph taken using an electron microscope) of the cells at various time points where the ____________ __________ appear as black dots

• Measured the % of ______________ at each location after the ______ round and 3 rounds of ______.

electron micrographs, radioactive proteins, radioactivity, pulse, chase

Palade’s Pulse-Chase Experiment

Where was the radioactivity located in the electron micrograph taken immediately after the pulse?

Why does this make sense?

on the rough ER, this is where proteins are being newly synthesized with the radioactive leucine amino acids

Palade’s Pulse-Chase Experiment

Where was the radioactivity located in the electron micrograph taken 37 minutes after the pulse?

in vesicles outside of the ER

Palade’s Pulse-Chase Experiment

% Radioactivity Results:

• Pulse (3 mins) = 86.3% of radioactivity located on the ________ _____

• Chase (+7 mins) = 43.0% of radioactivity located in the ___________ _________ of the ______

• Chase (+37 mins) = 48.5% of radioactivity located in the ______________ _________ of the ______

• Chase (+117 mins) = 58.6% of radioactivity located in __________ ________

rough ER, periphery vesicles, Golgi, condensing vacuoles, Golgi, secretory vesicles

Palade’s Pulse-Chase Experiment

What was the conclusion?

proteins go from the rough ER to the Golgi to secretory vesicles

Palade’s Pulse-Chase Experiment

Is this the electron micrograph taken immediately after the pulse or 37 minutes after during the chase?

immediately after the pulse

Palade’s Pulse-Chase Experiment

Is this the electron micrograph taken immediately after the pulse or 37 minutes after during the chase?

37 minutes after

The Big Questions in Membrane Trafficking

• How do compartments keep their ________ with all the flow of ___________ in and out?

• How to maintain the _____ of compartments?

• How to _____ with the ______ membrane and not with the ______ one? (___________)

• How is trafficking directed between ______________?

• How to overcome the ________ ________ of fusion?

identity, membrane, size, fuse, right, wrong, specificity, compartments, energy barrier

What does it mean for the components of the secretory pathway to be “topologically equivalent”?

whatever is on the inside of the vesicle becomes part of the extracellular environment

What is the central organelle of the secretory pathway?

What is its nickname?

Golgi, the soul of the cell

Rothman’s Golgi Trafficking Complementation Assay

What was his question?

How does protein trafficking through the Golgi work?

Rothman’s Golgi Trafficking Complementation Assay

The Problem: Within the Golgi, trafficking occurs _________ different ______ of ___________, specifically from the ____→ _______→ _____Golgi. We want to be able to ________ the Golgi in a lab setting to be able to study the pathway. It turns out that we can isolate the _______ ______, but we can’t actually isolate the _____________ _________.

between, stacks, membranes, cis, medial, trans, isolate, whole Golgi, individual stacks

a test to identify and measure the amount of a specific molecule in a sample

assay

Rothman’s Golgi Trafficking Complementation Assay

• Rothman isolated 2 different Golgi preparations from 2 different cell lines: mutant and wild type. The mutant cell line expressed the ______ _________ _______ in the _____ Golgi but lacked the ________ needed to _____________ the protein in the ______ Golgi (³H-GlcNAc glycosyltransferase). The wild type cell line did express this _________ in the ______ Golgi but he did not _______ the _____ Golgi with the ______ __________ ________. This meant that on their own, neither cell line would produce ______________ ___________.

• Rothman mixed the 2 preparations until he found a situation that worked. Mixing together the ______ Golgi from the mutant cell line and the ______ Golgi from the wild type cell line _______________ the ______________ _______ upon a successful transport → aka ______________ ___________ were produced!

viral protein VSV-G, cis, enzyme, glycosylate, medial, enzyme, medial, infect, cis, viral protein VSV-G, glycosylated proteins, cis, medial, complemented, glycosylation defect, glycosylated proteins

Rothman’s Golgi Trafficking Complementation Assay

He was able to figure out what is needed for proteins to be able to move between the Golgi ______ to get _____________, ultimately realizing that both ____ (i.e. ________) and the ___________ (i.e. other ____________ in the _____) are needed for Golgi trafficking.

stacks, glycosylated, ATP, energy, cytoplasm, molecules, cell

Rothman’s Golgi Trafficking Complementation Assay

Process of Membrane Trafficking: General Scheme

cargo __________ → vesicle __________ → vesicle __________ → vesicle _________ → vesicle ________

selection, binding, transport, docking, fusion

Rothman’s Golgi Trafficking Complementation Assay

What is one of the factors that he discovered that is required for protein movement through the Golgi and assists with vesicle fusion?

SNARE proteins

Rothman’s Golgi Trafficking Complementation Assay

Specificity: different _________ and different ________ ___________ have different ________ proteins that only work in specific combinations

vesicles, target membranes, SNARE

What is the job of the ER? (7 examples)

protein folding, disulfide bond formation, proteolytic cleavage, formation of multimeric complexes, passing things to the Golgi, lipid synthesis, regulation of cholesterol synthesis

Which of these is in the lowest energy state?

6

Protein Folding

Recall the _____________ _______, where proteins fold to achieve the _______ _______ ______ with the hydrophobic parts on the _______ and the hydrophilic parts on the _______

hydrophobic effect, lowest energy state, inside, outside

Protein Folding

Why is protein folding particularly difficult in the ER? (2)

What helps with protein folding in the ER?

denser concentration of proteins, limited space, chaperones

Protein Folding

One of the most abundant chaperones in the ER

BiP

Protein Folding

As the unfolded protein is being threaded through the __________, _____ grabs onto it and helps it bind and unbind the _____________ parts to help them find their ____-_______ states

translocon, BiP, hydrophobic, low-energy

Protein Folding

Which chaperone also helps to pull the unfolded proteins into the translocon to begin with?

BiP

Protein Folding

The chaperone ____ helps form and rearrange _________ _______, trying different combinations until the protein reaches its ________ ________ state

PDI, disulfide bonds, lowest energy

Protein Folding

when proteins clump together to form insoluble structures

aggregation

Protein Folding

Protein aggregation can result from WHAT?

protein misfolding

Protein Folding

Which type of proteins are particularly susceptible to aggregation?

neurons

Protein Folding

Name 3 examples of protein misfolding diseases.

What do they all have in common?

Alzheimer’s, Huntington’s, Parkinson’s, protein aggregation

Protein Folding

What is the trade-off between? (2)

Which disease is an example of the consequences of this trade-off?

accuracy, time, cystic fibrosis

Cystic Fibrosis

The cystic fibrosis gene encodes a _________ _________. The inability to pass _________ _____ through can lead to a _______ __________________ in our _________. It turns out that the most common mutation in this gene is a ____ ________ on the protein far out from the actual ________. People who express the gene with this mutation are able to create a perfectly ________ _________ ________ protein. The problem is that the protein is _____ to _____. This makes our cells think that the protein could ______________, causing our cells to _________ the protein. This is a good mechanism for preventing ________ _____________, but in this case it causes cystic fibrosis.

chloride channel, chloride ions, mucus accumulation, airways, Phe deletion, channel, normal folded channel, slow, fold, aggregate, degrade, protein aggregation

Protein Folding

What is one clue that might hint to a cell that a protein could aggregate?

slow folding

N-Linked Glycosylation as Quality Control

The __________ of the oligosaccharide on proteins in the ___ ______ into the 5-residue pentasaccharide core starts by removing the ___ _________ _________. It turns out that trimming the _____ _________ is a _____________ step that is used to determine whether a protein is ________ or not.

trimming, ER lumen, 3 terminal glucoses, final glucose, reversible, folded

N-Linked Glycosylation as Quality Control

The chaperone __________ helps with the ________ of the protein and removes the _____ _________ residue from the oligosaccharide. The protein without _________ is now assessed to see if it is ___________ ________. If it is, it _______ the ___. If it is not, it __-_______ the cycle. ________ ____________ recognizes unfolded proteins and will __-________ the ________ if the protein is not completely ________. The ___________ protein now goes back through the ________-removal process with __________ to try again.

Calnexin, folding, last glucose, glucose, properly folded, leaves, ER, re-enters, Glucosyl Transferase, re-attach, glucose, folded, misfolded, glucose, Calnexin

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to the amount of misfolded proteins: turn it UP or turn it DOWN?

down

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to the total translation: turn it UP or turn it DOWN?

What could this do?

down, reduce the load of misfolded proteins

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to the synthesis of chaperones: turn it UP or turn it DOWN?

What could this do?

up, help with protein folding

Protein Folding

Name 3 examples of chaperones

BiP, PDI, Calnexin

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to the transcription of mRNA: turn it UP or turn it DOWN?

What could this do?

What could we do to make sure the rest of the cell is unaffected?

down, reduce total protein load, only target mRNAs on the ER

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to the ER volume: turn it UP or turn it DOWN?

What could this do?

What specific process would we turn up to do this?

up, make more room for protein folding, lipid synthesis

ER Stress & the Unfolded Protein Response (UPR)

To reduce stress in the ER, what could we do to overall cell growth: START or STOP?

stop (slow it down)

ER Stress & the Unfolded Protein Response (UPR)

What does the UPR need to be able to do? (2)

sense misfolded and unfolded proteins, turn that into a cellular response

ER Stress & the Unfolded Protein Response (UPR)

What are the 3 branches of signaling the UPR?

IRE1, PERK, ATF6

ER Stress & the Unfolded Protein Response (UPR)

Both ______ and ______ branches upregulate the transcription of XBP1 mRNA but the ______ branch is required to make the XBP1 mRNA useful by translating into a _____________ XBP1 ______________ __________.

PERK, ATF6, IRE1, functional, transcription factor

ER Stress & the Unfolded Protein Response (UPR)

_____ and _____ are both ________ that sense mis/unfolded proteins and go through _____________, _________________, and downstream signaling very much like the _________ ______.

IRE1, PERK, kinases, dimerization, autophosphorylation, insulin RTK

ER Stress & the Unfolded Protein Response (UPR)

______ is a ______________ ________ that in the absence of stress is held in the ____ but in the presence of stress is passed to the ______. When it gets the _____ ______, it travels to the ______ where it is ________ by the same __________ that cleave _______ in __________ synthesis regulation. This cleavage turns it into an _______ ____________ _______ which can now turn on things that help the ____ deal with this stress of protein ___________ like ____________ and ______ ___________.

ATF6, transcription factor, ER, Golgi, stress signal, Golgi, cleaved, proteases, SREBP, cholesterol, active transcription factor, ER, misfolding, chaperones, lipid synthesis

ER Stress & the Unfolded Protein Response (UPR)

PERK Mechanism:

• The presence of _________ proteins acts as a signal for PERK to _________.

• _____________ triggers ___________________ of the ________ domains.

• PERK phosphorylates _____ (a ____________ __________ _______) which _________ shuts down _____________.

• While almost every other protein synthesis __________, phosphorylated _____ upregulates the translation of the protein _____, which is a ____________ ________ that turns on things like ___________ to assist with protein ________.

unfolded, dimerize, dimerization, autophosphorylation, kinase, eIF2, translation initiation factor, globally, translation, decreases, eIF2, ATF4, transcription factor, chaperones, folding

ER Stress & the Unfolded Protein Response (UPR)

Name the 4 mechanisms that are apart of the UPR

IRE1, PERK, ATF6, ERAD (ER-Associated Degradation)

ER Stress & the Unfolded Protein Response (UPR)

IRE1 Mechanism:

• The presence of ________ proteins acts as a signal for IRE1 to form __________, which then _________________.

• Active IRE1 _________ then ________ _____ sitting on the ___________.

• Active IRE1 _________ also act as an _____________:

  • This _________ mechanism is completely different from the ____________. The ______ mRNA has an _______ that is not recognized by the ____________, meaning that in the absence of ______, _______ does not occur and we produce an ___________ ______ transcription factor protein. In the presence of ______, IRE1 acts as an _______________, cuts out specifically that little _______, and through ligation we form a __________ ______ transcription factor protein that now turns on other stuff to help deal with the stress like ____________, ______ proteins, and ______ __________.  

unfolded, oligomers, autophosphorylate, oligomers, degrade mRNA, translocon, oligomers, endonuclease, splicing, spliceosome, XBP1, intron, spliceosome, stress, splicing, unfunctional XBP1, stress, endonuclease, intron, functional XBP1, chaperones, ERAD, lipid synthesis

ER Stress & the Unfolded Protein Response (UPR)

What is a way to degrade terminally misfolded proteins in situations where we could not prevent or fix the misfolding?

What do we have to do to the proteins targeted for termination?

Why?

ER-associated degradation (ERAD), move them out of the ER into the cytoplasm, the ER does not have proteasomes

ER Stress & the Unfolded Protein Response (UPR)

When it gets too stressful, what does the cell do?

apoptosis (aka cell suicide)

The classic experiments of George Palade and his colleagues first demonstrated the sequence of organelles that constitute the secretory pathway. It is:

A. Rough ER/nucleus/secretory vesicles/cell exterior 

B. Rough ER/Golgi/secretory vesicles/cell exterior 

C. Rough ER/cytosol/Golgi/cell exterior 

D. Rough ER/vacuole/Golgi/cell exterior

B

Which organelle receives proteins and lipids from the endoplasmic reticulum, modifies them, and then dispatches them to other destinations in the cell?

A. An endosome 

B. The Golgi apparatus 

C. A peroxisome 

D. The nucleus 

E. A mitochondrion

B

Which ONE of the following proteins does NOT enter the secretory pathway?

A. SRP 

B. signal peptidase 

C. SNARE protein 

D. Sec61 translocon 

E. All of these proteins enter the secretory pathway

A

Which of the following is true about posttranslational modifications? 

A. Carbohydrate side chains are added only after complete synthesis of the polypeptide 

B. Any amino acid can be modified by phosphorylation 

C. All signal sequences must be removed 

D. Disulfide bonds are common in proteins to be exported

D

Proteins are properly folded with the assistance of…

A. chaperones 

B. signal recognition particle (SRP) 

C. Sec61 translocon 

D. ribozymes

A

Which of the following are used to assist in protein folding?

I.  calnexin

II. calreticulin

III. Hsp70

IV. disulfide bonds

A. III, IV 

B. II, IV

C. I, II, III, IV 

D. I, III 

E. I, II 

C

Which of these diseases does not directly involve protein aggregation?

A. Cystic fibrosis 

B. Parkinson’s 

C. Alzheimer’s 

D. Huntington’s

A

Which disease can be caused by too much ER stress?

A. Type II diabetes 

B. Parkinson’s 

C. Huntington’s 

D. Alzheimer’s

A

The unfolded protein response (UPR) operates at which levels of regulation?

I. transcription initiation

II. mRNA decay

III. translational initiation

IV. protein degradation

A. II, IV 

B. I, III 

C. I, II 

D. I, II, III, IV 

E. III, IV

D

You perform a pulse-chase experiment where you label a newly synthesized M6P receptor with a pulse and then follow the location of the M6P receptor at different times afterwards. Which of the following BEST represents the expected sequence? 

A. Cytoplasmic ribosomes, mitochondria

B. ER, Golgi, vesicles, plasma membrane

C. ER, Golgi, vesicles, outside the cell

D. ER, Golgi, vesicles, lysosome

E. ER, Golgi, vesicles, lysosome, Golgi

D (technically A and E are also right. A is the most “general” answer and E includes how the receptors ultimately get recycled back into the Golgi after they bind to the lysosome and lose their binding affinity)

If a slice of pancreatic tissue had been incubated in ³H-leucine continuously for 40 min, where would you expect to find incorporated radioactivity in acinar cells? 

A. ER

B. ER, Golgi vesicles

C. ER, Golgi vesicles, condensing vacuoles

D. Zymogen granules

E. Condensing granules

C

Which of the following is NOT a way to help relieve protein folding stress in the ER?

A. Decrease translation

B. Decrease transcription

C. Increase chaperone synthesis

D. Increase lipid synthesis

E. All of the above help relieve protein folding stress

E

Mutations in PERK cause early onset diabetes. What components of UPR would you expect to be missing in beta cells lacking functional PERK?

A. Downregulation of eIF2 alpha

B. Cleavage of ATF6

C. Splicing of XPB1 mRNA

D. A & B

E. A & C

A

Which of these is NOT a function of IRE1?

A. Directly senses unfolded proteins

B. It is cleaved to produce a transcription factor that goes to the nucleus and upregulates chaperones

C. Promotes degradation of mRNA that is being translated at ribosome docked on Sec61

D. Splices the XBP1 mRNA

B

Which of these might be a possible treatment of cystic fibrosis caused by the most common genetic variant in CFTR, the cystic fibrosis chloride channel?

A. A drug that blocks the kinase activity of PERK

B. A mutation in the residues of IRE1 that are autophosphorylated

C. A drug that blocks ERAD

D. A mutation in ATF6 that prevents its cleavage by proteases

C