Targeting proteins to Organelles

Question 1

Mechanism of Protein Targeting

Answer 1

Specific amino acid sequences dictate delivery of protein to target

Question 2

ER retention signals

Answer 2

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)

Question 3

Different signals target different organelles

Answer 3

Mitochondria - Amphipathic, 20-50 residues

Nucleus - Cluster of basic residues: …ppKKRKv…

Peroxisomes - ‘SKL’ at C-terminus

Question 4

Mitochondrial Targeting

Answer 4

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)

Question 5

Mitochondrial Protein Import

Answer 5

Proteins imported into matrix as precursors and fold into final conformation within.
- Import requires outer membrane receptors and translocons in both membranes

Question 6

Mitochondrial Outer Membrane Import receptor

Answer 6

TOM 20 and 22 (Translocation of the Outer Membrane)

Question 7

Mitochondrial Import proteins

Answer 7

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

Question 8

Mitochondrial Import Channels

Answer 8

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

Question 9

Mitochondrial Inner Membrane Translocons

Answer 9

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

Question 10

Mechanisms of Mitochondrial Import (stages)

Answer 10

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.

Question 11

Sourcing energy for mitochondrial targeting

Answer 11

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

Question 12

Targeting proteins to inner mitochondrial membrane

Pathway A

Answer 12

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

Question 13

Targeting proteins to inner mitochondrial membrane

Pathway B

Answer 13

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

Question 14

Targeting proteins to inner mitochondrial membrane

Pathway C

Answer 14

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

Question 15

Targeting proteins to the mitochondrial intermembrane space

Pathway A

Answer 15

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

Question 16

Targeting proteins to the mitochondrial intermembrane space

Pathway B

Answer 16

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

Question 17

Targeting proteins to the mitochondrial outer membrane

Answer 17

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

Question 18

Peroxisomes

Answer 18

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

Question 19

Targeting the peroxisomes

Answer 19

Using the C-terminal Peroxisomal Targeting Sequence 1 (PTS1)

• Sequence = Ser-Lys-Leu (SKL)
• First discovered in luciferase (resident protein in peroxisome)

Question 20

Importing into peroxisome matrix

Answer 20

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

Question 21

Different mechanisms for incorporating proteins into the peroxisome membrane or matrix discovered by mutation

Answer 21

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)

Question 22

Importing into peroxisomal membrane

Answer 22

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)

Question 23

Mechanisms of import/export in the Nucleus

Answer 23

Depends on the Nuclear Pore Complex (NPC)

• Macromolecules such as RNA and proteins must associate with transport factors to pass

Question 24

Nuclear Import

Answer 24

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

Question 25

Ran proteins regulating importins

Answer 25

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

Question 26

Nuclear Export

Answer 26

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