Intracellular Trafficking Flashcards
1
Q
What proteins complete their translation in the cytoplasm and how are they segregated to the right organelles?
A
- Free ribosomes in the cytosol are the site of all protein synthesis for proteins that will be in:
- Cytosol
- Cytoskeletal proteins and soluble enzymes
- Nucleus
- Peroxisomes
- Mitochondria
- They are segregated through post-translational segregation
- Specific amino acid sequences in proteins destined for these organelles provide the signals that are used to target proteins destined to these organelles following their synthesis aka post-translational.
2
Q
What proteins complete their translation in the ER and how are they segregated to the right organelles?
A
- Ribosomes in rough ER are the site for protein synthesis for proteins that are destined for secretory pathway such as:
- Membrane proteins and lipids
- ER
- Golgi
- Secretory Vesicles
- Lysosomes
- They are segregated through Co-translational segregation
- A specific amino acid “signal sequence” on the protein indicates where they should be segregated. However, this protein segregation occurs co-translationally such that proteins begin synthesis on free ribosomes in cytosol and then become segregated to ER where they complete their synthesis on ribosomes attached to the ER membrane,.
3
Q
How are nuclear proteins synthesized on the free ribosomes in the cytosol transported into the nucleus?
A
- Nuclear proteins finish synthesis in cytoplasm
- Nuclear protein has nuclear localization sequence (NLS)
- Importin chaperone proteins in cytoplasm recognize and bind to NLS
- The importin/nuclear protein complex winds its way through nuclear pore and get into nucleus
- Ran-GTP binds to its binding site on importins only if importins have nuclear proteins associated.
- Ran-GTP is in high abundance in nucleus because only Ran-GTPs associated GEF is in nucleus.
- This causes nuclear protein to dissociate from Ran-GTP/Importin complex
- Ran-GTP/Importin is transported into cytoplasm
- Ran-GTP is hydrolyzed to Ran-GDP since Ran-GTPs GAP is only located in cytoplasm.
- Ran-GDP can then dissociate from importin
- Ran-GDP goes back through nuclear pore and is then converted to Ran-GTP via the GEF only found in nucleus
4
Q
How are peroxisomal proteins synthesized and transported into perioxisomes?
A
- Proteins are made on free ribosomes in the cytoplasm
- I.e Catalase is a protein that decomposes H2O2 to H2O
- Amino acid signal destines proteins for peroxisome
5
Q
How are secretory proteins localized to the rough ER?
A
- SRP is a cytoplasmic chaperone recognizes a specific and distinct secretory pathway signaling sequence on the N-terminus of proteins (the first part synthesized by ribosome) when it emerges from ribosome
- Binding of SRP to the ribosome/mRNA halts translation of that protein in the cytoplasm
- SRP receptor protein (dimer and GTP binding protein) in the rough ER membrane binds to SRP.
- This docks mRNA/Ribosome complex onto RER membrane.
- Once SRP binds to SRP receptor, there is GTP hydrolysis that causes conformational changes.
- Conformational changes release the SRP from SRP receptor
- Translation starts again with protein synthesis occurring through a channel into the ER membrane.
- Several protein processing steps begin during translocation through ER
- Signaling peptide is removed via a signal peptidase
- Hydroxylationof lysine and proline residues occurs
- Disulfide bonds form
- Chaperones promote proper folding of proteins
- System doesn’t let misfolded proteins to go through pathway so they are shuttled out of ER and sent to degradation in the proteosome
- Glycosylation of proteins occurs early in folding
- Types of changes that occur during glycosylation help assess the extent of folding that has occured
- Sensors look at glycosylation to assess proper folding
6
Q
Glycosylation of proteins in ER
A
- Membrane lipid donor in RER is ajacent to ribosome with growing polypeptide
- Membrane lipid donor co-translationally adds mannose to Asn residues on peptide
- Oligosaccharise is then modified by compartment specific enzymes in the secretory pathway
- The extent of the modifications allow us to deduce how far a protein has progressed
7
Q
Coat proteins and the transport events they are involved in
A
- COPI
- Retrograde and anterograde transport from Golgi
- COPII
- Exit by ER
- Clathrin
- TGN –> late endosome
- late endosome –> TGN
- Cell membrane –> early endosome
8
Q
Entire process of vesicle formation from donot to target membrane
A
- Sar-1 (COPII) or ARF(COPI) are bound to GDP
- GEF in donor membrane exchange GDP for GTP
- Binding of GTP to Sar-1 or ARF leads to conformational change that exposes lipid tail
- Sar-1 or ARF can embed themselves into donor membrane
- COPI or COPII subunits and v-SNAREs bind to Sar-1 and ARF to help deform membrane
- Cargo/Cargo receptors bind to these subunits
- Coat is separated from donor compartment to form vesicle
- Uncoating and recycling of Coat proteins reveals v-SNAREs
- v-SNAREs tether to t-SNAREs on target compartment membrane with high specificity. Rab also contributes to this specificity.
- Coiled-coil is formed between SNAREs leading to fusion of membrane and release of cargo into target compartment.
9
Q
Misdirected proteins from RER
A
- Material leaves ER to go to Goli in COP II vesicles
- Misdirected proteins or proteins meant to stay in ER will be identified through KDEL sequence and will be returned to ER via COP I vesicles
10
Q
Lysosomal proteins modification and delivery
A
- Lysosomal proteins in the ER are modified such that a phosphotransferase only adds a phosphate to the mannose sugar groups in lysosomal proteins creating a mannose-6-phosphate
- These lysosomal proteins meet up with the mannose-6-phosphate receptor on the TGN.
- These lysosomal proteins or enzymes recognized by mannose-6-phosphate receptor on TGN membrane are put in transport vesicles with a clathrin coat.
- The coat is removed and delivers the vesicles to the late endosome.
- The late endosome eventually matures into lysosome
- The acidic pH of the late endosome causes lysosomal enzymes/proteins to dissociate from mannose-6-phosphate receptor
- The receptor is put into transport clathrin coated vesicles that recycle back to the TGN.
11
Q
Zellweger syndrome
A
- There is one specific receptor in the peroxisomal membrane that recognizes peroxisomal signal sequence and mediates transport to internal part of peroxisome.
- Mutation in this peroxisomal membrane receptor results in all peroxisomal enzymes remaining in cytoplasm
- These peroxisomal enzymes are then degraded
- Therefore, it is not only important to have proper signaling sequence but also a receptor for that signaling sequence.
12
Q
I-cell disease
A
- Mutation in phosphotransferase prevents addition of phosphate on mannose in lysosomal proteins
- Lack of Mannose-6-phosphate on lysosomal proteins prevent their recognition by Mannose-6-phosphate receptor in TGN
- Lysosomal proteins are not packaged into clathrin coated vesicles but instead are secreted
- Lysosomes do not have the enzymes necessary to degrade so lysosomes have inclusion bodies resulting in I-cell disease
- I-cell disease could also result in mutation in mannose-6-phosphate receptor in TGN similar to Zellweger syndrome.
13
Q
Pathway of Endocytosis
A
Vesicles formed by plasma membrane with cargo from outside of cell –> early endosome (sorting station)–> late endosome –> lysosome
14
Q
Legionairre’s disease
A
- Legionairre’s disease bacteria is phagocytosed
- Hijacks host transport vesicles and becomes like RER
- It blocks delivery to lysosome and can proliferate n this way
15
Q
Recycling of receptor after endocytosis can follow the following pathways
A
- From early endosome, receptors can be sorted to
- Surface (as in case of cholesterol)
- Lysosome (as in case of phagosomes)
- basolateralmembrane by bypassing tight junctions in epithalial cells
- How neonates get antibodies to go thorugh epithelial cells lining their intestines and into their circulation.
