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Molecular Cell Biology > Protein Sorting Pathways 1 > Flashcards

Protein Sorting Pathways 1 Flashcards

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1
Q

What is the non-secretory pathway of protein sorting? (1)

A

In the absence of a signal sequence the protein will remain a cytosolic protein.

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2
Q

What is the non-secretory pathway of protein sorting? (2)

A

If there is a signal sequence the protein will be targeted to the respective organelle.

Respective organelle being Mitochondrion, Chloroplast, Peroxisome, Nucleus

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3
Q

What is the non-secretory pathway of protein sorting? (3)

A

If the protein contains an ER signal sequence it will be targeted to the ER and follow the secretory pathway.

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4
Q

How do proteins and RNA molecules move between the cytosol and the nucleus?

A

The type of transport that facilitates the movement is gated transport

however, what helps the molecules move are nuclear pore complexes

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5
Q

What does the nuclear transport?

A

Bidirectional traffic via Nuclear Pore Complexes (NPC)
Selective permeability
Uses specific receptors (importin / exportin)
Requires a GTPase (Ran)

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6
Q

What does the nuclear envelope consist of?

A

Inner Nuclear Membrane:
anchoring sites for chromatin, nuclear lamina

Outer Nuclear Membrane
continuous with ER membrane
Studded with ribosomes

Perinuclear space
Continuous with ER lumen

Nuclear Pores
Nuclear Pore Complexes (NPCs)

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7
Q

How many Nuclear pore complex are in a nucleus?

A

2000 – 3000 NPCs in a nucleus

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8
Q

What are some structural features of the nuclear pore complex

A

60.000 – 80.000 KDa
Span both membranes

Nuclear Basket formed by filaments
Made up of multiple copies (50-100 molecules) of approx. 30 different proteins (nucleoporins)
Fibrils protruding from both sides (different)

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9
Q

Can the NPC diffuse molecules?

A

It can carry out simple diffusion for ions, small metabolites and small proteins up to 40kDa

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10
Q

What do some proteins contain?

A

Proteins destined for the nucleus contain a Nuclear Localisation Signal sequence (NLS)

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11
Q

What do Nuclear Localisation Signals do? (1)

A

NLS will interact with importins (nuclear import receptor) in cytoplasm and direct the proteins to the nucleus

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12
Q

What do Nuclear Localisation Signals do? (2)

A

Cargo protein will enter into the nucleus in a folded state

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13
Q

What do Nuclear Localisation Signals do? (3)

A

In nucleus, dissociation of cargo protein mediated by Ran

Importin will be recycled back into the cytosol

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14
Q

Where can NLS be found?

A

Can be anywhere in the amino acid sequence.
If one protein subunit of a complex contains a NLS sequence the whole complex can be transported in to the nucleus
NLS sequence is rich in lysine, arginine, usually contain proline

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15
Q

What is the role of Ran? (1)

A

Importin will recognize NLS on cargo protein/complex

The complex will pass through the NPC by interactions between importin and nucleoporins

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16
Q

What is the role of Ran? (2)

A

Ran-GTP will bind importin

Conformational change of importin, will result in the release of the cargo protein.

17
Q

What is the role of Ran? (3)

A

Importin/Ran-GTP will be recycled to the cytoplasm

GTP hydrolysis (Ran-GDP) will result in importin being released.

18
Q

What two states does Ran exist in?

A

Ran-GTP - active
binds importin/exportin and moves it out of the nucleus

Ran-GDP - inactive
releases importin/exportin in the cytosol

19
Q

What mediates the transition from Ran- GTP to Ran-GDP?

A

GTPase Activating Proteins (GAP):
Increase the rate by which G- proteins can hydrolyse GTP

Guanine Exchange Factors (GEF):
exchange GDP for GTP

20
Q

How does the nuclear export work? (1)

A

Exportin will recognize Nuclear Export Signal (NES) on cargo protein/complex

Ran-GTP will bind exportin/cargo complex

21
Q

How does the nuclear export work? (2)

A

The complex will pass through the NPC by interactions between exportin and nucleoporins

22
Q

How does the nuclear export work? (3)

A

GTP hydrolysis (Ran-GDP) will result in exportin and cargo being released in the cytosol.

Exportin will be recycled back in the nucleus

23
Q

Briefly explain the Role of Ran in protein transport in and out of the cell nucleus?

A

Importin binds to Ran-GTP in nucleus - cargo is released
Exportin binds to Ran-GTP in nucleus - binds to the cargo
Importin binds to cargo protein in the cytosol
Exportin binds to cargo protein in the nucleus
Hydrolysis of GTP (Ran-GDP) releases exportin/importin

(Look at the slide, it will help)

24
Q

How does the protein sorting pathway occur in the mitochondria?

A

Matrix Targeting sequence (MTS)
Receptor recognises MTS
Protein passes through the translocon (TOM/TIM) in an unfolded state
Signal sequence is cleaved

25
Q

How does protein targeting to the mitochondrial matrix occur?

A
  1. The signal sequence binds to Import receptors (which was attached to the TOM complex)
  2. Protein is translated through the TIM 23 complex, so that it transiently spans both mitochondrial membranes
  3. Signal sequence cleaved off a signal peptidases in the matrix space to form the matrix protein.
26
Q

How do proteins enter the mitochondria?

A

Proteins will enter the mitochondria in an unfolded state- maintained by chaperons (Hsp70)

27
Q

What does TOM20/22 do when it detects the protein?

A

TOM20/22 (Translocon of Outer Membrane) will recognise the signal sequence and the chaperon and will guide the complex to the general import pore TOM40

28
Q

By interacting with TIM, what does the protein initially create?

A

A channel, in which results in Proteins passing simultaneously through both membranes

29
Q

What is the signal sequence cleaved by?

A

Once the signal sequence enters the matrix it is cleaved by a protease

30
Q

What are chaperones?

A

Hsp70, Hsp90,
Maintain the unfolded state of the proteins,
Hydrolyse ATP to be released from the protein.

31
Q

What does mitochondrial Hsp70 do?

A

Mitochondrial Hsp70 will drive the import of the protein by the hydrolysis of ATP
When Hsp70 is released the protein will fold to its mature conformation

32
Q

What does the Outer membrane in Mitochondria have?

A

Porins, which are permeable to inorganic ions and metabolites

33
Q

What can’t the TOM complex do?

A

They cannot integrate porins into the lipid bilayer

34
Q

How are porins inserted into the Outer membrane?

A

The porins are transported unfolded into the inter-membrane space
This is where they bind to the specialised chaperon proteins - which help them aggregate
They then bind to SAM complex in outer membrane which inserts them into outer membrane and helps them fold properly

35
Q

How does the protein target the inter-membrane space?

Pathway 1

A

Two targeting sequences
MTS will be cleaved in the matrix by a matrix protease (same as matrix pathway)

Protein will diffuse laterally in the membrane
A protease embedded in the inner membrane will cleave the IST sequence.
Protein will fold.

36
Q

How does the protein target the inter-membrane space?

Pathway 2

A

Proteins do not contain a MTS
They will pass through the general pore TOM40 without
the interaction with a receptor

Examples are Tim10, Tim9 that will form disulphide bonds once in the intermembrane space (Erv1, Mia40)

By the bonds being formed the proteins will be trapped in the intermembrane space

37
Q

How does the protein target to inner membrane?

Pathway 1

A

Proteins have two signal sequences

  • MTS
  • Stop-transfer sequence

The protein will pass through TOM40
N-terminus of protein will pass through TIM and MTS will be cleaved in the matrix
Stop-transfer sequence will anchor the protein to the inner membrane

38
Q

How does the protein target to inner membrane?

Pathway 2

A

Proteins have two signal sequences

  • MTS
  • Oxa1-targeting sequence

The protein will pass through TOM40
The protein will pass through TIM into the matrix where
The MTS will be cleaved and
Protein will be redirected to the membrane
Oxa1 will recognise Oxa1-targeting sequence

39
Q

How does the protein target to inner membrane?

Pathway 3

A

Specific for multipass proteins
Do not have the MTS sequence but contain internal targeting sequences

The protein will pass through TOM40.
The protein will interact with Tim22/54 that will incorporate the protein into the membrane.

Molecular Cell Biology (18 decks)

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