Targeting proteins to Organelles Flashcards
Mechanism of Protein Targeting
Specific amino acid sequences dictate delivery of protein to target
ER retention signals
Keep resident ER proteins such as BiP from entering the secretory pathway
- C-terminal KDEL sequence binds KDEL receptors to get packaged into COP1 vesicles from Golgi to ER
- Similar C-terminal signals: KKXX, KXKXX and RKR
- Some proteins can only be secreted when they’ve successfully assembled:
- SOR1 and Kir6.2 proteins express the RKR retention signal if expressed alone (when they successfully form Potassium-ATP channel, the RKR signals are masked allowing successful ER transit)
Different signals target different organelles
Mitochondria - Amphipathic, 20-50 residues
Nucleus - Cluster of basic residues: …ppKKRKv…
Peroxisomes - ‘SKL’ at C-terminus
Mitochondrial Targeting
Protein’s signal:
- 20-50 amino acids long
- Rich in hydrophobic and positively charged amino acids (arginine and lysine) whilst lacking negatively charged
- Amphipathic (hydrophobic charge one side of helix, hydrophilic on other side)
Mitochondrial Protein Import
Proteins imported into matrix as precursors and fold into final conformation within.
- Import requires outer membrane receptors and translocons in both membranes
Mitochondrial Outer Membrane Import receptor
TOM 20 and 22 (Translocation of the Outer Membrane)
Mitochondrial Import proteins
HSP70 and HSP90 use ATP to keep proteins unfolded so they can be taken up
TOM70 can serve as importing receptor through binding to HSP90
Mitochondrial Import Channels
Import receptors transfer protein to an import channel
TOM40 = a general import pore wide enough to accommodate an unfolded polypeptide chain
- It’s a passive pore: driving force from matrix
Mitochondrial Inner Membrane Translocons
Used for proteins destined for the Matrix: TIM23 and TIM17 (Translocon of the Inner Membrane)
- translocation occurs at ‘contact sites’ where the inner and outer membranes are in close proximity
Mechanisms of Mitochondrial Import (stages)
1) TOM20 recognises matrix targeting sequence
2) Protein translocated to the TOM40 pore where it diffuses through
3) Protein interacts with TIM23 and TIM17 to enter matrix
4) Upon entering, N-terminal mitochondrial targeting sequence is removed by a protease
5) Protein interacts with HSP70 via TIM44 (complex can interact with TIM23/17)
6) HSP70 is cleaved and protein can fold into its final conformation.
Sourcing energy for mitochondrial targeting
1) ATP hydrolysis by HSP70
2) ATP driven release of HSP70 from translocating peptide to ‘trap’ it in the matrix
3) H ions electrochemical gradient (proton motive force) allowing for transfer
Targeting proteins to inner mitochondrial membrane
Pathway A
Same machinery as matrix targeted protein
- targeting sequence recognised by TOM20/22 - transfered through TOM40 & TIM23/17
- BUT, hydrophobic Stop Transfer sequence prevents translocation through TIM23/17 so protein is laterally inserted into inner membrane
Targeting proteins to inner mitochondrial membrane
Pathway B
Matrix target sequence + Internal Hydrophobic domain recognised by Oxa1 Protein
- TOM20/22 recognition, translocation via TOM40
- Hydrophobic domains interact with Oxa1 to insert into membrane
Targeting proteins to inner mitochondrial membrane
Pathway C
Multi-pass protein with >6 TMDs lacking usual N-terminal matrix targeting sequence
- TOM70/22 recognise internal sequences, TIM22/54 translocate the protein
- Transfer through TIM9/10 act as chaperones to prevent protein folding/aggregation in the intermembrane space
- TIM22/54 insert hydrophobic regions into inner membrane
Targeting proteins to the mitochondrial intermembrane space
Pathway A
Protein carries 2 signals:
1) N-terminal matrix sequence cleaved by protease
2) Hydrophobic Stop Transfer sequence
- Membrane sequence laterally diffuses from TIM22/17 and gets cleaved
Targeting proteins to the mitochondrial intermembrane space
Pathway B
No N-terminal matrix sequence present on protein
- general import pore TOM40 delivers protein to intermembrane space
- No involvement of TIMs
- Protein trapped due to disulphide bonds between Mia40 and Erv1
Targeting proteins to the mitochondrial outer membrane
Outer membrane proteins (e.g. TOM40) have a Beta barrel conformation
- Proteins interact with TOM40 and the Sorting & Assembly Complex (SAM)
- SAM complex (similar to bacterial BamA protein) can integrate proteins into membrane
Peroxisomes
Small single-membrane organelle with no associated ribosomes (proteins synthesised from cytosolic ribosomes)
- Oxidise substrates, convert toxic Hydrogen peroxide into water using catalase
- most abundant in liver
Targeting the peroxisomes
Using the C-terminal Peroxisomal Targeting Sequence 1 (PTS1)
- Sequence = Ser-Lys-Leu (SKL)
- First discovered in luciferase (resident protein in peroxisome)
Importing into peroxisome matrix
E.g. Importing catalase:
1) PTS1 binds to Pex5 receptor
2) Pex5 binds to Pex14 receptor in the peroxisomal membrane
3) Protein released from Pex14 into into the interior of the peroxisome through the Pex2/10/12 complex
- translocated in folded stated whilst PTS remains intact
Different mechanisms for incorporating proteins into the peroxisome membrane or matrix discovered by mutation
Zellweger Syndrome = mutation in Pex5 transporter
- Proteins can’t be imported into matrix
- Still have full complement of peroxisome membrane proteins (thus mechanism is different)
Importing into peroxisomal membrane
Occurs during peroxisome biogenesis at the ER
- Pex3 and Pex16 inserted into ER membrane by translational translocation
- Pex3/16 recruit Pex19 at specialised region of ER that can bud off to form an empty peroxisome (preperoxisome)
Mechanisms of import/export in the Nucleus
Depends on the Nuclear Pore Complex (NPC)
- Macromolecules such as RNA and proteins must associate with transport factors to pass
Nuclear Import
Proteins for IMPORT carry a Nuclear Localisation Signal (NLS) that bind IMPORTINS
- NLS = 7 residues, rich in basic amino acids near C-terminus
- proteins incorporate in a folded state
Ran proteins regulating importins
Small Ras-related GTPases regulate importin ability to transport cargo:
- alpha subunit of importing recognises NLS
- beta subunit binds to NPC to bring cargo to pore before dissociating to allow cargo through
- Hydrolysis of RanGTP to RanGDP triggers release of cargo by importin alpha subunit to allow transport through pore into nucleus
Nuclear Export
Proteins for EXPORT carry a Nuclear Export Signal (NES) that binds EXPORTINS
- NES = Leucine-rich sequence of 4 hydrophobic residues (e.g. LXXXLXXLXL)
- First discovered HIV1 Rev protein and cAMP PK1
- Exportin 1 recognises NES on HIV1’s Rev
