Lecture 4 protein targeting

Question 1

What is the default environment of proteins?

Answer 1

Cytosol

Question 2

What are the protein signal for?

Answer 2

The compartmentalisation of eukaryotic cells. Proteins have functional homes and must be sorted to them. Polypeptides contain sorting signals that are different for eukaryotic cell compartments.

Question 3

What are the different translocation mechanisms for?

Answer 3

Different cell compartments. Energy is required for translocation. Translocation involves channels, pores.

Question 4

What does translocation determine?

Answer 4

The correct orientation of membrane proteins.

Question 5

What are the principles of protein targeting to compartments?

Answer 5

Sorting signal on polypeptide required. Folding and unfolding events. Recognition factors for sorting signal. Translocation machinery. Energy (compartmentalisation increases order and decreases entropy known for disorder). Signals to be removed after translocation.

Question 6

What is a signal patch?

Answer 6

A collection of amino acid residues that come together in the 3D structure of a folded protein to create a functional targeting signal. When the protein is unfolded the residues are scattered and non contiguous in the sequence.

Question 7

What are signal peptides?

Answer 7

A continuous stretch of amino acids that serves as a targeting signal for directing the protein to a specific cellular compartment. Found at the N terminus of the protein and cleaved off when the protein reaches its destination.

Question 8

What are contiguous and non contiguous sequences?

Answer 8

A continuous stretch of amino acids in the primary sequence of a protein. Non-contiguous is where functionally important amino acids are scattered throughout the primary sequence but come together in a 3d folded structure.

Question 9

What are the characteristics of an ER signal sequence?

Answer 9

Found at the N-terminus of proteins destined for the ER. Has a hydrophobic core region of 10-15 residues which allows it to interact with the signal recognition particle and insert into the ER membrane. Short polar regions flanked by the hydrophobic region which may help in recognition and processing.

Question 10

What are the characteristics of a mitochondrial signal sequence?

Answer 10

20-30 residues of basic and hydrophobic forming amphipathic helix. Amphiphatic sequences have one polar side and a non polar side which is recognized by mitochondrial import machinery.

Question 11

What are the two different types of nucleocytoplasmic transport?

Answer 11

1. Continuous and bi-directional. Molecules can move in and out of the nucleus. 2. Selective (gated) transport. Requires a receptor/cargo complex for regulated movement. Ensures only specific proteins, RNAs and molecules enter or exit.

Question 12

What is the nuclear pore complex (NPC) channel and what is its structure?

Answer 12

A transport channel located in the nuclear membrane. Large multi-protein structure embedded in the nuclear envelope. 125 mDa (mega daltons) and made of 30 different NPC proteins.

Question 13

How do small proteins move through the transport mechanism?

Answer 13

Move by passive diffusion.

Question 14

How do larger proteins move through the transport?

Answer 14

Require a nuclear transport receptor (e.g., importins, exportins). This ensures only proper tagged proteins (with nuclear localization signals or nuclear export signals) can pass through.

Question 15

What does the nuclear pore complex allow?

Answer 15

The transport of fully folded proteins.

Question 16

How are nucleoporins are arranged in a vertebrate NPC?

Answer 16

Nucleoporins are arranged with striking eightfold rotational symmetry.

Question 17

What cargo signal do nucleocytoplasmic require?

Answer 17

Nuclear localisation signals. Often rich in positively charged residues like lysine and arginine. Can be anywhere in the protein not just the N-terminus. Can be patches of residues that only come together in the folded structure. Only one subunit in a multi protein complex requires an NLS to bring the entire complex into the nucleus.

Question 18

What is an example of a nuclear localisation signal?

Answer 18

SV40 T-antigen is a viral protein that contains a normal NLS required for nuclear import.

Question 19

What is a study that shows importance of nuclear import signal?

Answer 19

Wild type intact NLS sequence. A single amino acid mutation changes Lys to Thr in NLS. Disrupts the nuclear import signal. Prevents transport to nucleus.

Question 20

What are nuclear export signals?

Answer 20

Signals that allow proteins to be actively transported out of the nucleus.

Question 21

What are karyopherins?

Answer 21

Transport requires cargo receptors. Importins and exportins.

Question 22

What do some cargos require to bind?

Answer 22

An adaptor to bind to an important receptor.

Question 23

How do receptor-cargo complexes cross the pore?

Answer 23

Large so have to bind to nuclear transport receptors such as importin-beta through weak, transient interactions to facilitate movement. NPC contains nucleoporins which have phenylalanine-glycine (FG) repeats. They are hydrophobic and form a hydrogel-like meshwork in the NPC. This acts as selectively permeable barrier preventing most molecules from freely diffusing while allowing cargo complexes to pass.

Question 24

What is a hydrogel meshwork?

Answer 24

A soft gel like network composed of hydrated polymers that can trap and regulate the movement of molecules.

Question 25

Why is the nuclear import of proteins energy dependent?

Answer 25

Because it increases order.

Question 26

Where does the energy required for nuclear import come from?

Answer 26

GTP hydrolysis.

Question 27

What is RanGTPase?

Answer 27

A monomeric G-protein that hydrolyses GTP.

Question 28

How does Ran function as a molecular switch?

Answer 28

Two confirmations: 1. Ran-GTP (active form) predominantly in the nucleus. 2. Ran-GDP (inactive form) predominantly in the cytoplasm. The Ran-GTP/Ran-GDP gradient drives import and export of proteins. Import: Importin releases cargo inside the nucleus when bound to Ran-GTP. Exportin binds cargo in the nucleus when associated with Ran GTP facilitating export.

Question 29

What is the role of Ran-GAP?

Answer 29

Ran GTPase activating protein found in cytoplasm stimulates Ran to hydrolyze GTP to GDP to convert RAN-GTP to RAN-GDP. Ensures Ran-GDP is abundant there.

Question 30

What is the role of Ran-GEF?

Answer 30

Guanine nucleotide exchange factor. Found in the nucleus. Exchanges Ran-GDP to Ran-GTP.

Question 31

Why is Ran GTP required in the nucleus and Ran GDP required in the cytoplasm?

Answer 31

Ran GTP binds to importin in the nucleus to release the cargo into the nucleus. Ran GTP is moved to cytoplasm to be recycled. In cytoplasm Ran GTP is hydrolyzed to Ran GDP so cargo can be released into the cytoplasm.

Question 32

What are the steps to nuclear import?

Answer 32

On the cytosolic side importin and cargo/NLS bind to NPC. There is an FG repeat throughout the pore. On the nuclear side Ran GTP promoted by RanGEF releases cargo in the nucleus. Receptor RanGTP is transported to the cytosol. Back in the cytosolic side RanGAP promotes GTP hydrolysis and Ran GDP is released from receptor.

Question 33

What are the steps to nuclear export?

Answer 33

On the nuclear side exportin and cargo/NES binding is promoted by RanGTP. RanGTP-cargo-receptor passes through NPC. On the cytosolic side RanGAP promotes GTP hydrolysis. Cargo and exportin is released and becomes free from the receptor. Exportin is released back into the nucleus. Back in the nuclear side RanGDP is imported into the nucleus bound to its own receptor.

Question 34

What does SUMOylation (small ubiquitin related modifier) of RanGAP do?

Answer 34

Allows its association with the cytosolic face of the nuclear pore.

Question 35

What are the three categories of signals that target and sort signals that direct mitochondria precursor proteins?

Answer 35

Presequences. non cleavable signals of hydrophobic proteins. internal signal for intermembrane space. Look at examples and variations on slide.

Question 36

What is the sorting pathway in mitochondria?

Answer 36

The TOM (translocase of the outermembranes) complex serves as the universal entry point. The SAM (sorting and assembly machinery) complex assembles outer membrane beta-barrel proteins. The MIA (machinery for import and assembly) pathway facilitates disulfide bond formation in intermembrane space proteins. The TIM23 and TIM22 complexes sort proteins into the inner membrane or the matrix. The oxa complex inserts proteins synthesized inside the mitochondria.

Question 37

Why does the TOM complex have seven different subunits?

Answer 37

The receptors Tom20, 22 and 70 recognise precursor proteins and transfer them to the central component, the channel-forming Tom40. Three small Tom proteins 5,6 and 7 are involved in the assembly and dynamics of the TOM complex.

Question 38

What is the classical import route 1 (the presequence pathway)

Answer 38

This the primary route for importing mitochondrial precursor proteins especially matrix proteins and inner membrane proteins.

Question 39

What are the steps to the presequence import pathway?

Answer 39

Precusor proteins contain N-terminal presequences which are recognised by Tom20 and Tom22 receptors. They are translocated through Tom40 the main channel of the TOM complex. Proteins move from TOM to TIM23 in the inner membrane. Membrane potential drives the presequences through TIM23. TIM23 functions in two forms: 1. TIM23SORT inserts proteins into the inner membrane with Tim21 2. TIM23-PAM transports proteins into the matrix with PAM and mitochondrial Hsp70. Mitochondrial processing peptidase cleaves presequences protein folding and assembly occurs in the matrix with chaperones like Hsp70.

Question 40

What is the carrier pathway?

Answer 40

Imports hydrophobic carrier proteins such as ADP/ATP carriers into inner mitochondrial membrane. They lack a presequence but have multiple internal targeting signals. Cytosolic chaperones (Hsp70/90) keep the precursor proteins unfolded. TOM complex (20 and 70) recognises the internal signals. Precursor proteins pass through Tom40 into IMM. Tim9 and 10 complex bind to precursors preventing aggregation. Tim chaperones escort proteins to TIM22 complex in inner membrane. The TIM22 complex facilitates membrane insertion. Membrane potential drives lateral integration.

Question 41

What do OMM outer mitochondrial membrane proteins do?

Answer 41

They are translocated into the mitochondrial intermembrane space where they bind to chaperones that help guide them to the SAM complex. This then inserts these proteins into the outer mitochondrial membrane ensuring their proper folding and function.

Question 42

What are some examples of OMM proteins?

Answer 42

Proteins that contain stop signals so that once the protein travels through TOM can be laterally released into the membrane. Beta-barrel porins bind to chaperones in the IMS such as TinyTIMs which keep them in a partially unfolded state to prevent aggregation. Then they are transferred to the SAM complex for folding and placement in the OMM membrane in a stable, functional configuration.

Question 43

How do IMM proteins translocate there?

Answer 43

Transferred from TOM to TIM. Stop transfer signals are cleaved and released to IMS. Metabolite transporters with internal signals snake through TOM and are released to IMS bound chaperones they bind to TIM22/54 released from TIM22 to the membrane.

Question 44

How are IMM proteins inserted into the IMM?

Answer 44

The polypeptide is inserted into TIM23/17. TIM23/17 recognises the presequence and cleaves it. A stop signal in the polypeptide signals the halting of their translocation. This recognised by TIM23/17 and the proteins are laterally released into IMM instead of continuing in the matrix. A second signal directs the protein to OXA an insertase to insert the protein into the IMM.

Question 45

What are the energy requirements for mitochondrial transport?

Answer 45

Import across OMM driven by ATP hyrolysis by MSF (mitochondrial import stimulating factor) and cytosolic and mitochondrial hsp70. Once bound to TIM further translocation is driven by electrochemical H+ gradient across the IMM acting on positive charged signal sequence. An emerging polypeptide is bound by mitochondrial hsp70 with ATP hydrolysis and often a co-chaperone.

Question 46

How are proteins inserted into the mitochondrial matrix?

Answer 46

Once they have reached the matrix the matrix protease MPP cleaves off the signal sequence. Then the protein becomes a soluble matrix polypeptide. Hsp70 chaperone helps folding and passes it to cpn60 a chaperonin which creates a protected environment for correct folding. It is in the liquid environment ready to function.