Cells and immunity
Mike Fry
Intracellular Tracking; Protein targeting, vesicle trafficking, Secretion
Protein Synthesis; regular vesicles trafficked out of the cell or to late endosome to a lysosome
Inward Traffic; bring stuff in from membrane via early – late endosome [amino acids,
nutrients, water] end in lysosome
Retrograde Traffic; bring stuff in, go back to ER [receptor activated, internalized to
deactivate and return to plasma membranes] like the nervous system = recycled.
Advantages of Compartmentalization;
- Reaction confined to particular region of a cell
- Reactions don’t interfere with one another
- Greater efficiency through more organized reactions and processes
Problems with Compartmentalization;
- Need to deliver all components to correct place [targeting mechanisms]
- Mechanisms to specifically move components around / in / out of the cell
- Maintain organelles, compartments and cell integrity.
Sorting in a polarized cell;
1. Direct Apical Sorting;
- Influenza HA virus glycoprotein and glycophospholipid anchored proteins are
trafficked via vesicles to apical side
2. Basolateral Sorting;
- Apical protein sometimes gets mistaken and taken to basal side] alongside VSV G
Glycoprotein
3. Exocytosis;
- Leaving the cell via vesicles
4. Endocytosis
- Entering the cell via vesicles
5. Transcytosis;
- Helps move apical proteins across the cell to the apical side.
What’s needed to get protein and cellular components to correct compartment?
1. Specific targeting sequences [recognizable]
- Protein recognition and specific protein – protein interactions [sequence on one
matches the other]
- G protein cycles [Active/Inactive]
- Cell signaling
2. Specific recognition mechanism
, Cells and immunity
Mike Fry
3. Specific delivery mechanisms
Targeting Sequences;
- Signal sequence [code in amino acids]
- Transmembrane domains
- Organelle specific uptake sequences
- Nuclear localization sequences
- Protein/lipid binding domains
Targeted Organelle Location Nature of Signal
6-12 hydrophobic amino
Endoplasmic Reticulum N-Terminal acids with 1< basic amino
acids
3-5 nonconsecutive Alg or
Mitochondria N-Terminal Lys with Ser, Thr. No Glu or
Asp [no acids]
Not common. High conc of
Chloroplasts N-Terminal Ser, Thr, small hydrophobic
amino acids, low conc of
Glu, Asp
Peroxisome C-Terminal Not removed Ser-Lys-Leu
Not removed, 5 basic amino
Nucleus Internal acids or 2 smaller basic
residue separated by 10
amino acids.
Nuclear Pore Complex [NPC];
- Basket filaments; gated mechanisms for in/out nucleus
- Transport of RNA and proteins
- mRNA; only needs a small port // Protein needs a larger port
Pyruvate Kinase GFP + SV40 NLS Pyruvate kinase SV40NSL-GFP
[NLS = nuclear localization sequence]
- NLS causes molecule to only go to NPC [nucleolus still clear of protein]
- NLS is rich in basic amino acids
1. Cargo Protein + importin α + Importinβ + GDP RAN molecule
2. Alpha importin conformed to change shape and allows beta importin, cargo and GDP
RAN to attach
3. All 4 complexes are recognized by the NPC, allowed into the nucleus.
4. RCCI [Guanine nucleotide exchange factor] exchanges GDP to GTP, which released
cargo and alpha importin to nucleus.
Mike Fry
Intracellular Tracking; Protein targeting, vesicle trafficking, Secretion
Protein Synthesis; regular vesicles trafficked out of the cell or to late endosome to a lysosome
Inward Traffic; bring stuff in from membrane via early – late endosome [amino acids,
nutrients, water] end in lysosome
Retrograde Traffic; bring stuff in, go back to ER [receptor activated, internalized to
deactivate and return to plasma membranes] like the nervous system = recycled.
Advantages of Compartmentalization;
- Reaction confined to particular region of a cell
- Reactions don’t interfere with one another
- Greater efficiency through more organized reactions and processes
Problems with Compartmentalization;
- Need to deliver all components to correct place [targeting mechanisms]
- Mechanisms to specifically move components around / in / out of the cell
- Maintain organelles, compartments and cell integrity.
Sorting in a polarized cell;
1. Direct Apical Sorting;
- Influenza HA virus glycoprotein and glycophospholipid anchored proteins are
trafficked via vesicles to apical side
2. Basolateral Sorting;
- Apical protein sometimes gets mistaken and taken to basal side] alongside VSV G
Glycoprotein
3. Exocytosis;
- Leaving the cell via vesicles
4. Endocytosis
- Entering the cell via vesicles
5. Transcytosis;
- Helps move apical proteins across the cell to the apical side.
What’s needed to get protein and cellular components to correct compartment?
1. Specific targeting sequences [recognizable]
- Protein recognition and specific protein – protein interactions [sequence on one
matches the other]
- G protein cycles [Active/Inactive]
- Cell signaling
2. Specific recognition mechanism
, Cells and immunity
Mike Fry
3. Specific delivery mechanisms
Targeting Sequences;
- Signal sequence [code in amino acids]
- Transmembrane domains
- Organelle specific uptake sequences
- Nuclear localization sequences
- Protein/lipid binding domains
Targeted Organelle Location Nature of Signal
6-12 hydrophobic amino
Endoplasmic Reticulum N-Terminal acids with 1< basic amino
acids
3-5 nonconsecutive Alg or
Mitochondria N-Terminal Lys with Ser, Thr. No Glu or
Asp [no acids]
Not common. High conc of
Chloroplasts N-Terminal Ser, Thr, small hydrophobic
amino acids, low conc of
Glu, Asp
Peroxisome C-Terminal Not removed Ser-Lys-Leu
Not removed, 5 basic amino
Nucleus Internal acids or 2 smaller basic
residue separated by 10
amino acids.
Nuclear Pore Complex [NPC];
- Basket filaments; gated mechanisms for in/out nucleus
- Transport of RNA and proteins
- mRNA; only needs a small port // Protein needs a larger port
Pyruvate Kinase GFP + SV40 NLS Pyruvate kinase SV40NSL-GFP
[NLS = nuclear localization sequence]
- NLS causes molecule to only go to NPC [nucleolus still clear of protein]
- NLS is rich in basic amino acids
1. Cargo Protein + importin α + Importinβ + GDP RAN molecule
2. Alpha importin conformed to change shape and allows beta importin, cargo and GDP
RAN to attach
3. All 4 complexes are recognized by the NPC, allowed into the nucleus.
4. RCCI [Guanine nucleotide exchange factor] exchanges GDP to GTP, which released
cargo and alpha importin to nucleus.
