Intracellular Trafficking + Sorting of Proteins (Chapter 49) Flashcards
What is Signal Hypothesis?
Proteins synthesised on membrane bound ribosomes contain a signal sequence on their N-terminal.
What are Preproteins?
These are proteins which possess signal peptide that must be removed.
What cystolic proteins are formed?
- Mitochondrial
- Nuclear
- Perioxsomal
- Cystolic
What RER proteins are formed?
- GA Membrane
- Plasma Membrane
- ER Membrane
- Lysosomal enzymes
What is the exocytic pathway?
RER - > GA - > PM
What is “Constitutive pathway” and “Regulated Pathway”?
Constitutive is when we have constant formation of vesicles passing through the secretory pathway.
Regulated when the formation of vesicles can be turned off/on.
Roles of the GA?
- Processing of Oligosacharides
- Especially N-linked Glycoproteins
- also have enzymes involved in O-Glycolysation.
- Protein sorting as well (Trans part only involved in this)
1) What do Chaperones do?
2) What do they show?
3) Which region of protein do they bind to?
1) They stabilise unfolded or partially folded proteins.
2) Chaperones exhibit ATPase Activity.
3) Chaperones bind to Hydrophobic regions of Proteins - preventing their aggregation.
How do Chaperones work?
ADP-Chaperones have a high affinity to UNFOLDED Proteins.
They bind to it.
Then ADP is replaced for ATP, and with each cycle more folded protein is released.
1) What are Chaperonins?
2) What do Chaperonins do?
1) These are a second major class of Chaperones.
2) They form barrel like structures which separates the unfolded protein allowing them the environments for correct folding.
Give examples of Chaperonins in Bacteria and in Humans.
Bacteria - GroEL
Human - Hsp60
What is the uptake signal?
A signal found on cystolic proteins which targets them to their appropriate organelle.
Examples of Uptake Signals?
N-terminal Sequence --- ER KDEL/HDEL --- Membrane of ER Amino Leader --- Mitochondria NLS --- Nucleus PLS --- Perioxosome Mannose-6-phosphate --- Lysosome
What is Translocation?
The movement of a molecule through 2 membranes.
What do Proteins targeted to Mitochondria posses?
What’s the most common type of AA found in this sequence?
An Amino Leader sequence.
It has many:
-Hydrophobic AA and Positive AA.
What does TOM20/22 do?
Acts as a receptor on Mitochondria.
What is condition for protein to pass through the pore in Mitochondria?
What ensures this?
The pore is called TOM40.
To pass through it must be unfolded. This is ensured by chaperones such as Hsp70.
Which Protein which enters the mitochondrial matrix does not have a pre-sequence?
Cytochrome C.
Diameter of the Nuclear Pore?
9nm.
watch this be the multiple choice question that will make you pass… just wait.
Under which weight are molecules able to simply able to diffuse through the nuclear pore complex?
Below 40kDa.
What is involved in Nuclear Import?
- Ran
- GAP
- Importins
Explain the process of Nuclear Import.
1) Importin binds to cargo. This complex then associated with Ran-GDP. It enters the nucleus.
2) GEF converts GDP into GTP, which causes the release of the cargo.
3) Ran-GTP-Importin Complex leaves the NPC.
4) GAP then converts GTP into GDP allowing the release of Importin.
What are Karyopherins?
These are the family of proteins of the importins and exportins.
1) How is mRNA exported out of the Nucleus?
2) Does the process use Ran?
1)mRNA is exported as an RNP (Ribonucleoprotein).
This binds to mRNP Exporter (which is a heterodimer).
2) NO RAN USED. But ATP AND RNA HELICASE IS.
Properties of Signal Sequence directed to the ER?
- N-terminal located
- Methione is usually the amino-terminal Amino Acid
- Possess hydrophobic AA clusters.
Two methods of translocation of proteins to the ER?
- Cotranslational Pathway
- Post translation pathway.
1) What is important for the protein to be in the co-translational pathway?
2) Explain the steps for proteins which follow the co-translational pathway.
1) The protein must be unfolded.
2) a)Signal peptide recognised by SRP
b) SRP binds to SRP-R
c) Hydrolysis of GTP (bound by SRP and SRP-R) causes the release of SRP and the binding of the ribosome to translocon (Sec61 complex).
d) Signal peptide binds to HYDROPHOBIC AA in translocon which causes opening of the plug.
e) Signal Peptidase removes signal peptide so that Ribosome is released.
1) What is SRP made up of?
2) What does SRP binding to Signal Sequence do?
1) 6 proteins and 1 RNA Molecule.
2) It arrests elongation.
How does the Signal Peptide open the Translocon Plug?
Binds to Hydrophobic region in the translocon.
What does Post-Translational Translocation for ER Destined proteins require?
- Sec 61
- Sec62/63
- Hsp70
- BIP
Steps involved in Post-Translation translocation for ER destined proteins.
1) When protein enters, it binds to sec61 - causing release of Cystolic chaperones.
2) BIP binds to protein, and ATP-bound BIP interacts with Sec62/63.
3) Hydrolysis of ATP on BIP causes pulling of protein in.
What does ATP-Bound BIP interact with?
It interacts with Sec62/63.
When hydrolysed it pulls in the Protein.
What is the purposes of BIP?
1) Ensure proper folding.
2) Binds to abnormal protein preventing them from leaving ER.
Name the different routes for proteins targetted to be inserted into the membrane of ER.
1) Cotranslational Insertion
2) Post translational Insertion
3) GA retention
4) Retrograde insertion.
Explain Type I Cotranslation Insertion.
Example for when it is used.
Used for LDL receptors.
The N-terminal is inside the lumen.
They have a stop signal which is hydrophobic.
Proteins exit via lateral gate
what does stop signal sequence consist off?
consist of hydrophobic AA.
Explain Type II Cotranslational Insertion.
Differene between Type I and Type II
What is it used for.
Used for Asiaglycoproteins.
There is no cleaving occuring.
And C-terminal is position in th ER.
Type III Co-translational Insertion used for?
Used for Cytochrome P450.
N-terminal inside the lumen.
Type IV Cotranslation insertion/
Used for Glucose.
Transverses more than once.
What are topogenic Sequences?
These are sequences that determine a structure of a protein in a membrane.
Example of Post-translational Insertion?
Cytochrome B5.
GA retention:
- What sequence do these proteins have?
- what route do they take?
They have carboxyl located KDEL and HDEL Sequences.
They travel to GA in a COPII Vesicle.
Then interact with KDEL-Receptor.
Then travel back to ER Via COPI vesicle.
What chaperones and Enzymes do we find in the ER?
Chaperones:
- Calnexin - CA2+ binding protein, it is membrane bound
- Calreitculin - Ca2+ binding protein. NOT membrane bound.
Enzymes: -Protein Disulfide Isomerase (PDI): Rearranges formation of Sulfide bonds. -Protein Proyl Isomerase (PPI) Catalyses trans and cis isomer of proline.
What is ERAD?
Endoplasmic Reticulum Associated Degradation.
Useful in removing unfolding proteins.
What is ER Stress and UPR?
ER stress:
Occurs when there is an accumulation of unfolded proteins.
UPR:
This is Unfolded Protein Response. This recognises ER stress and initiates mechanism to fix this.
What are the effects of activation of ER Stress Sensors?
1) Stops the translation of the fucked protein.
2) Increases transcription of chaperones.
3) Increases protein synthesis of the degrading proteins.
What is the ERAD Pathway,
what provides the energy for this?
Firstly misfolded proteins move back out of the ER.
Energy supplied by p97 an AAA-ATPASE
Possible pathways for ERAD?
Can be:
- Sec61
- Derlin1
- HRD1
- Doa10
- ERAD E3 ligase.
Which proteins escort Polyuniquinated proteins?
Polyuniquinated Binding proteins.
Where is Ubiquination involved in?
- Cell cycle regulation
- DNA repair
- Inflammation
- Immune response
What COPI Involved in?
- intra GA transport
- Retrograde transport
What is COPII involved in?
Anterograde transport
What is Clathrin involved in?
Post GA transport i.e// to PM and Trans Golgi Network.
Key Steps involved in Vesicle Formation
Ben Takes Dick Man
- Budding
- Tethering
- Docking
- Membrane Fusion.
What is ARF involved in forming?
ARF is a GTPase
COPI And Clathrin Coated Vesicles.
What is NSF?
THis is a ATPase.
What is Sar1?
A GTPase, involved in forming
COPII.
What is Sec12p?
This is GEF, interconverts SarGDP into SarGTP.
Alpha-SNAP
This is an NSF attachment protein.
