Module 18 - Molecular Traffic in Cell Flashcards
Describe where nuclear protein synthesis and folding occur.
They occur in cytosolic ribosomes. Nuclear proteins complete synthesis by these ribosomes and pass through the pores in fully folded state.
Describe the mechanism by which both small and large molecules pass between cytosol and the interior of nucleus
Small molecules (<5kD-40kD) pass through the nuclear pore complex by diffusion, while large molecules (>40kD) enters through active transport.
In general terms describe the structure of the nuclear pore
- made up of 30+ different protein types
- octagonal arrangement
- central aqueous pore: molecular sieve (polypeptide chainswith Phe and Gly repeats)
- 39 nm in diameter
Describe how the nuclear localisation signal/patch targets specific proteins to the nucleus (importins)
Proteins that is transported to the nucleus have a continous stretch of AA 15-60 residues long often at the N terminal, (signal sequence) or in regions which when folded forms a signal patch. These are recognized by complementary nuclear import receptors (importins), which interact with Nuclear Pore Complex proteins to transfer cargo in/out of nucleus.
The NLS are commonly rich in Lysine (PKKKRKV) and mutation in sequence may lead to cytoplasmic retention.
Explain the mechanism of binding and dissociation of importins to NPC and how it leads to the transport of cargo into nucleus.
- Cargo protein, along with NLS, binds with importins
- Importin-cargo shuttles into the nucleus via the NPC (F-G repeats)
- Ran GTP binds to the importins to discharge cargo.
- Importin-Ran shuttles out via the NPC
- Ran-GTP is hydrolused to Ran-GDP in the cytosol
Note: Movement of Importin-cargo and Importing-Ran is achieved through antiporter means.
How does the mechanism of exportins compare to the importins?
Exportins export cargo proteins out of the nucleus with a reverse mechanism compared to importins. In this case, the binding of Ran-GTP allows for the binding of the cargo protein and the exportin, allowing it to be exported outside the nucleus.
Explain how compartmentalisation of Ran-GTP and Ran-GDP maintained betwee the cytosol and the nucleus.
High levels of Ran-GDP is maintained in the cytosol by Ran-GAP. While high levels of Ran-GTP is maintained in the nucleus by Ran-GEF.
Any Ran-GTP in the cytosol will be dephosphorylated by Ran-GAP, while any Ran-GDP in nucleus will be replaced with a GTP bye GEFs (Guanine Exchange Factors)
Describe where mitochondrial protein synthesis and folding occur
Mitochondrial protein synthesis occurs in the cytosolic ribosome, however unlike nuclear proteins binding of chaperones prevent protein from folding in the cytosol.
Mithochondrial proteins translocated as fully synthesized but unfloded polypeptide chains.
Describe the the role of the signal sequence in the mechanism of protein import into the mitochondrial matrix.
Mitochondrial translocation depends on amphiphilic signal sequences. These signal sequences form an α-helix, with non-polar as well as polar and hydrophilic regions. The hydrophobic region matches the groove in the receptor of the protein. Its binding with the TOM and TIM complexes allow for the translocation of the mitochondrial protein
Describe the mechanism of protein import into the mitochondria.
Signal sequence in mitochondrial precursor protein binds to TOM receptor complex in the outer membrane. This leads to the insertion of the protein into the intermembrane space, while TOM aligns itself with the TIM complex. The signal sequence then binds with the TIM complex and it gets translocated through the inner membrane into the matrix. Signal peptidase enzymes cleave the signal sequence allowing the mature mitochondrial protein to fold.
The translocations through TIM and TOM complexes are aided by chaperone proteins that binds to the peptide. It induces ATP hydrolysis that provides energy for the translocation mechanism.
Describe the role of TIM and TOM complexes and chaperones in transferring proteins from the cytosol to the mitochondrial matrix
The TIM and TOM complexes act as receptors for cargo protein as well as a translocation channel for them to pass through. TOM complex is responsible for transport through outer membrane. TIM is responsible for the inner membrane.
Chaperone proteins (Hsp70) is responsible for providing the energy required, through ATP hydrolysis, to translocate protein through TOM (cytoplasmic Hsp70) and TIM (mitochondrial Hsp70) complexes.
Describe the sites of synthesis and folding of proteins that pass into the ER lumen.
Synthesis of ER-targeted proteins begins on free cytosolic ribosomes, but is not completed after ribosome binds to ER through a process called co-translational translocation. This differs form nuclear or mitochondrial proteins where translocation happens after translation (post-translational translocation). Follding would occur in the ER.
How does the co-translational translocation mechanism occur?
During translation, the binding of SRP protein with the signal sequence in the polypeptide causes a pause in the translation process. The SRP-bound ribosome attaches to the SRP receptor in the ER membrane, which causes the SRP to be displaced. This allows translation to continue alongside translocation through the protein translocator (Sec61)
Describe the anatomy of Sec61 translocator.
In a closed mechanism, it has a plug preventing translocation through the bilayer. However, when it binds to the signal sequence of the peptide it opens in a hinge-like motion, displacing the plug.
Describe the role of the ER lumen as a common way-station for a number of destinations in the cell
Many proteins pass through the ER lumen en route to other destinations in the cell. ER’s role includes the folding and transport of various proteins, specifically carrying them to the Golgi apparatus. The ER is also responsible for marking these proteins that it transports with a signal sequence. Other proteins are headed outside the ER, so they are packed into transport vesicles and moved out of the cell via the cytoskeleton.
Describe the mechanism of translocation of soluble proteins into the ER lumen and of single-pass integral membrane proteins into the ER membrane. Explain the role of peptide signal sequences in these processes.
After the SRP protein dissociated from the ribosome, the signal sequence of the peptide chain is recognised by the Sec61 translocator in the ER membrane. This activates the translocator allowing the growing nascent polypeptide chain to pass through into the ER lumen. Then, a signal peptidase enzyme cleaves the signal sequence allowing the mature soluble protein to fold up in the lumen.
In the case of a single-pass integral protein being produced, insertion of the transmembrane protein involves the same binding of the signal sequence (Start Transfer) to the Sec61. However, an additional hydrophobic “Stop Transfer” sequence causes polypeptide chain to stop in the translocator, as the remaining -COOH terminal growth continues in the cytosol.
When is the orientation of transmembrane proteins determined?
Not all TM proteins have the same orientation. Most TM proteins have the N-terminal located in the lumen. However, in cases where the N-terminal is located in the cytosol, it only involves a slight modification where the signal sequence is not located in the N-terminal but rather within the sequence.
Hence, the orientation of TM proteins is determined by the location of the peptide sequence (Start and Stop transfer sequences) during co-translational translocation.
Explain the significance, site and mechanism of initial protein glycosylation
Protein glycosylation occurs in rough ER as proteins enter the lumen. Precursor oligosaccharide is attached initially to Dolichol, which lies adjacent to the Sec61 translocator. Then, the transfer of precursor oligosaccharide (glucose, mannose, N-acetylglucosamine) to N atom on Asn is aided by the oligosaccharyltransferase enzyme associated with the translocator.
Explain how protein folding is regulated.
Oligosaccharides are used as tags to mark the state of protein folding. In the initial unfolded state, glucose trimming occurs, removing glucose from the precursor oligosaccharide until only the terminal glucose is present. This glucose the binds to calnexin, where its arm helps the folding of the protein. Glucosidase enzyme then cleaves the last glucose.
If the folding is correct, it is transported out of the ER through vesicular transport. If it is not correct, the terminal glucose is added back by glucosyl transferase and UDP-glucose and the process repeats.
How are misfolded proteins handled in the ER?
Misfolded proteins are recognised by chaperones and other factors and somehow leaves the ER where it is digested by proteosomes after ubiquitination.
Describe which compartments are involved in the biosynthetic-secretory and endocytic pathways and the directions of traffic between those compartments
ER, Golgi, Endosomes, Lysosomes, and secretory vesicles are involved in these pathways. Bio-synthetic secretory pathways involve the movement of materials from inside the cell to extracellular space and vice versa for the endocytic pathway.
Describe the functional roles and significance of protein coats around vesicles
Clathrin, COPI, COPII are examples of protein coats. They are all involved in the formation of the vesicles itself (not its destination) by bending it to give vesicles its shape.
Clathrin: Involved in endocytosis
COP I: Used for recycling from Golgi back to ER
COP II: Used for ER to GolgI
Describe the mechanism by which specific cargo molecules are selected as well as vesicle formation/budding.
Cargo receptors, located in the Golgi membrane, binds with specific cargo proteins in the Golgi lumen. This induces the binding of adaptins to the receptor. Adaptor proteins (adaptins) binds both clathrin triskelions and cargo receptor together. As more clathrins bind, it interlocks and bends the membrane. Dynamin proteins then bend the membrane even further until the vesicle dissociates from the membrane. The clathrins, as well as the adaptins, then dissociate, leaving the naked transport vesicle along with its cargo
What are the Rab proteins? Describe the molecular basis for targeting of vesicles to specific compartments.
Rab proteins are small GTPases that guide vesicle targeting. Rab-GTP is initially bound to the cargo vesicle (by GEFs). It then recognises the Rab effector (tethering protein) in the target membrane. It docks and induces complementary sets of vesicle snare (v-snares) and target snares (t-snares) to form a complex. This then leads to hydrolysis of Rab-GDP to Rab-GTP, which drives the fusion of the vesicle to the target.
