Protein Sorting

Question 1

What is the defect that causes cystic fibrosis?

Answer 1

ER exit in CFTR (deltaF508) or golgi exit in CFTR (C-terminal PDZ)

Question 2

Protein is transported to the mitochonrdion (matrix)

1) Location of Sequence within protein
2) Removal of Sequence?
3) Nature of Sequence

Answer 2

1) N-terminus
2) Yes
3) Amphipathic helix, 20-50 residues in length, with Arg and Lys residues on one side and hydrophobic residues on the other

Question 3

Protein is transported to the Endoplasmic reticulum (lumen)

1) Location of Sequence within protein
2) Removal of Sequence?
3) Nature of Sequence

Answer 3

1) N-terminus
2) Yes
3) Core of 6-12 hydrophobic amino acids, often preserved by one or more basic amino acids (Arg, Lys)

Question 4

Protein is transported to the Peroxisome (matrix)

1) Location of Sequence within protein
2) Removal of Sequence?
3) Nature of Sequence

Answer 4

1) C-terminus (most proteins)
N-terminus (few proteins)
2) No
3) PTS1 signal (Serine, Lysine, Leucine) at extreme C-terminus; PTS2 signal at N-terminus (SKL motif)

Question 5

Protein is transported to the Nucleus (nucleoplasm)

1) Location of Sequence within protein
2) Removal of Sequence?
3) Nature of Sequence

Answer 5

1) Varies
2) No
3) Multiple different kinds; a common motif includes a short segment rich in Lysine and Arginine residues

Question 6

Uptake of precursor proteins into mitochondria can be assayed in a ____.

Answer 6

cell-free system

Question 7

Uptake of precursor proteins into mitochondria requires ____.

Answer 7

proton-motive force, ATP, and cytosolic chaperones

Question 8

What are the energy inputs for protein import into the mitochondrial matrix?

Answer 8

Hsc70 ATPase outside, Hsc70 ATPase inside, proton-motive force across IMM

Question 9

Signal peptide for import into the mitochondrial matrix

Answer 9

Signal peptide is an N-terminal, amphipathic alpha-helix; 20-50 residues long, with R and K on one side and delta on the other
Hsc70 in the cytosol keeps protein unfolded
Translocation is post-translational

Question 10

Name a chaperone protein

Answer 10

Hsp70

Question 11

Co-chaperone accessory proteins

Answer 11

DnaJ and Hsp40; GrpE and BAG1
DnaJ and Hsp40 stimulate the hydrolysis of ATP to ADP
GrpE and BAG1 converts the Hsp70 back to the open form

Question 12

Tom20/22

Answer 12

Import receptor; outer mitochondrial membrane complex that recognizes N-terminal mtSP (mitochondrial signaling peptide)

Question 13

Import receptor on the mitochondrial membrane

Answer 13

Tom20/22

Question 14

Tom40

Answer 14

Import pore (on the outer mitochondrial membrane that associates with Tom20/22); part of the translocon (transport proteins into the mitochondrial matrix)

Question 15

The translocon (of mitochondria) is ____.

Answer 15

Tom40 and Tim23/27

Question 16

Tim23/27

Answer 16

located on the inner mitochondrial membrane, part of the translocon (transports proteins into the mitochondrial matrix)

Question 17

Steps of import of protein into mitochondrial matrix.

Answer 17

1. N-terminus signaling peptide recognized by Tom20/22
2. Protein moves across outer membrane through Tom40
3. Protein moves across inner mitochondrial membrane through Tim23/17.
4. Signal sequence cleaved/removed by matrix protease.
5. Matrix Hsc70 binds to the imported polypeptide to facilitate uptake. It is targeted to the translocon through binding Tim44. (Tim 44 is bound to Tim23/17?)

Question 18

Mitochondrial matrix protein must be translocated in the ____ state.

Answer 18

Unfolded

Question 19

Pathways for transporting proteins from the cytosol to the inner mitochondrial matrix

Answer 19

Path A: Most like matrix sorting. Uses a matrix targeting sequence and a stop-transfer sequence. (Tom20/22 and Tom40, Tim23/17, and Tim44 and Hsc70)
Path B: Uses a matrix targeting sequence and an Oxa1-targeting sequence (Tom 40, Tim23/27, Hsc70, Oxa1)
Path C: No N-termination targeting sequence. Different receptor (Tom 70), Intermembrane chaperone (Tim9/10). Different IMM translocon (Tim22/54)

Question 20

function of peroxisomes

Answer 20

oxidation of organic substrates and fatty acids (metabolism of fatty acids)
producing acetyl-CoA

Question 21

Import of peroxisomal matrix proteins is directed by ____.

Answer 21

PTS1 targeting sequence

C-terminal and contains sequence SKL; not cleaved after import

Question 22

PTS1 targeting sequence

Answer 22

sequence SKL, c-terminal, not cleaved after import. Pex5 recognizes the sequence.

Question 23

Import of peroxisomal matrix proteins steps

Answer 23

1. Pex5 (cytosolic) recognizes PTS1 targeting sequence (SKL sequence on C-terminus)
2. Pex14 (membrane protein) associates with Pex5 and may be the translocon. Pex5 and the protein cross the membrane into the peroxisomal matrix.
3. Pex5 and protein disassociate and Pex5 is exported through a Pex2,Pex10, and Pex12 complex.

Question 24

Folded proteins with ___ are also translocated.

Answer 24

metal cofactors

Sorry about this one. It was on the slide.

Question 25

Zellweger syndrome

Answer 25

Group 1: Nearly complete loss of peroxisomal enzymes
Group 2: Loss of only some enzymes

Clinical manifestations:
Lack of peroxisomes in cells of liver, kidney, and brain
Enlarged liver
Elevated levels of copper/iron in blood
Vision disturbances
Mental retardation, seizures, failure to grow, GI bleeding, inability to swallow

Pex protein mutations

If Pex12, is mutated (defective catalase import)
If Pex 3, defective in both PMP70 insertion and in catalase import.

Question 26

True or false: Peroxisomes can arise de novo.

Answer 26

True! They arise de novo from precursor membranes derived from the ER, as well as by division of pre-existing organelles.

Question 27

Proteins involved in synthesis of peroxisomes

Answer 27

Pex19, Pex3, and Pex16 incorporated to form peroxisomal ghost with PMP70 membrane proteins.
PTS1-bearing matrix proteins and PTS2-bearing matrix proteins (Pex5, 7, 14, 10, 12, 2) form a mature peroxisome containing catalase.

Pex 11 divides peroxisomes?

Question 28

Peroxisomal disorders

Answer 28

Zellweger syndrome and Adrenoleukodystrophy

Question 29

Adrenoleukodystrophy

Answer 29

Group 3 peroxisomal disorder

Oxidation of long-chain fatty acids defective in ALD patients
Missing only membrane transporter specific for uptake of long-chain fatty acyl CoA synthase

Clinical Manifestations:
Several forms of ALD:
Classic childhood form: boys ages 4-10 years
Progressive dementia
Learning disabilities, seizures, visual loss
Death 1-10 years