Module 4: Protein targeting

Question 1

Characteristics of peroxisome

Answer 1

1. Bound by single membrane
2. Responsible for oxidative and synthetic reactions in the cell.
3. Does not have its own genetic information (i.e. genome).
4. Reproduction by fission

Question 2

Where is the protein catalase located in the peroxisome?

Answer 2

In the peroxisome, there are dense regions called protein aggregates which is almost exclusively composed of a protein called catalase.

Question 3

Role of peroxisome in animal cells

Answer 3

cholesterol synthesis

Question 4

Role of peroxisome in nerve cells

Answer 4

synthesize plasmalogen for cell membranes

Question 5

Role of peroxisome in liver cells

Answer 5

site of oxidation of toxins (ex. alcohol)

Question 6

Role of peroxisome in plant cells

Answer 6

convert fatty acids to carbohydrates which will then power seedling germination

Question 7

Major function of peroxisome in all cells

Answer 7

breakdown of very long chain fatty acids through beta oxidation

Question 8

What role does fatty acids serve in the body?

Answer 8

energy source

Question 9

What is the unfortunate by-product of fatty acid metabolism by peroxisome?

Answer 9

hydrogen peroxide

Question 10

Function of catalase

Answer 10

converts the toxic hydrogen peroxide into non-toxic oxygen and water molecules.
The abundance of catalase serves as a visual marker for peroxisomes in the cell

Question 11

Advantage of using fluorescence over TEM

Answer 11

Fluorescence allows us to look a living cells

Question 12

How are peroxisomes formed?

Answer 12

Through the division and incorporation of new proteins (division occurs through a process called fission)

Question 13

Describe how peroxisomes are formed in detail.

Answer 13

1. Peroxisomal proteins are synthesized in cytosol and then transported to peroxisome.
2. Peroxisomal membrane proteins join precursor membrane to create a peroxisomal ghost.
3. Once membrane proteins are present, they are used to transport peroxisomal matrix proteins (ex. catalase) into interior of its organelle.
4. As more proteins are synthesized, the peroxisome can grow and new peroxisomes can form by a process called fission.

Question 14

Luciferase

Answer 14

• an enzyme that serves as an excellent model for deciphering the mechanism of peroxisomal protein transport
• enzyme that allows bioluminescence that we see in fireflies
• found in peroxisomes of cells in the abdomen

Question 15

What was the conclusion derived from the study in which luciferase was expressed in mammalian cells?

Answer 15

The luciferase went into the peroxisome of mammalian cells telling us that pathways for protein transport to peroxisome are conserved in fireflies to mammals.

Question 16

Five rules for protein transport

Answer 16

1. A signal sequence is on transported protein
2. Receptor for that signal sequence is on target organelle
3. Translocation channel is required to get the protein across membrane into organelle
4. There is a requirement for energy at some step in the process.
5. Needs to be a way of targeting a protein to specific and different locations within organelle

Question 17

PTS1

Answer 17

• stands for perioxsomal-transport sequence 1
• signal sequence that was identified in luciferase and in many peroxisomal proteins in different species
• consists of tripeptide: serine, lysine and leucine or can be called SKL
• found at C-terminus of translated protein

Question 18

Is PTS1 necessary for transport of proteins into peroxisome?

Answer 18

Yes

Question 19

Is PTS1 sufficient to direct a protein in the peroxisome?

Answer 19

Yes

Question 20

Pex5

Answer 20

• cytosolic protein that acts as a receptor and to the PTS1 sequence at the C-terminus of the target protein
• contains 7 TPR motifs that together form a PTS1 binding pocket

Question 21

Wha makes up a TPR motfi?

Answer 21

two alpha helices and a loop

Question 22

Can Pex5 bring the protein to the peroxisomal membrane on its own?

Answer 22

No it must associate with transmembrane Pex14 protein

Question 23

How does the peroxisomal protein on the surface of the membrane get into the organelle?

Answer 23

It uses a translocation channel. There are two proteins that can form this channel.

Question 24

Does Pex 14 have any other function than recognition for Pex5?

Answer 24

Yes. It forms a translocon channel that transports Pex5 with target protein into peroxisomal matrix. Pex5 then releases peroxisomal matrix protein and Pex5 is recycled back out of cytosol to be reused.

Question 25

Peroxisomal matrix

Answer 25

interior of peroxisome

Question 26

In what part of the process is energy used in peroxisomes?

Answer 26

- Pex5 is uniquitinylated (requires energy) to release the matrix protein and deubiquitinylated to recycle Pex5 out to cytosol

Question 27

Two places that proteins can go to in peroxisome

Answer 27

matrix and membrane

Question 28

Why is it that when there is a mutation in PMP70, catalase also remains in cytosol?

Answer 28

Because if system to transport peroximal membrane is faulty, as a side effect, the cell cannot transport proteins into matrix.

Question 29

Zellweger's syndrome

Answer 29

- autosomal recessive disorder caused by mutations in genes that encode peroxisomal transport proteins (Pex2, Pex3, Pex5, Pex 10, and Pex12) - mutations in these lead to same phenotypes - causes impaired peroxisome function which accumulates very long chain fatty acids (since it is normally degraded by peroxisomes) - no treatment is available.

Question 30

Characteristics of Mitochondria

Answer 30

1. Bound by double membrane 2. Primary site of ATP production 3. Have their own DNA 4. Reproduction by fission

Question 31

Outer membrane

Answer 31

simply surrounds the entire organelle

Question 32

Inner membrane

Answer 32

much more complex with involutions that create cristae

Question 33

Cristae

Answer 33

- distinct regions within the matrix of mitochondria | - increases the surface area of inner membrane where ATP synthesis occurs

Question 34

Intermembrane space

Answer 34

- region that lies between the inner membrane and outer membrane

Question 35

Where is ATP created in the mitochondria?

Answer 35

Inner membrane using ATPase and proteins of electron transport chain

Question 36

______ of mitochondria enables high levels of ATP synthase

Answer 36

Structure

Question 37

How does mitochondria usually appear in images?

Answer 37

as an elaborated network of tubules

Question 38

Fission

Answer 38

- one mitochondrion becomes two new mitochondria | - process in which new mitochondria is created

Question 39

Fusion

Answer 39

- process in which two mitochondria become one mitochondrion

Question 40

Four destinations in which proteins can be located

Answer 40

outer membrane, intermembrane space, inner membrane, and matrix

Question 41

Two places where proteins come from

Answer 41

1. Mitochondrial genome and are synthesized by mitochondrial ribosomes.' 2. Coded by nuclear genes - genes are transcribed in nucleus, mRNA is translated by cytoplasmic ribosomes, and then transported to mitochondria in post-translational pathway. (major method)

Question 42

Protease

Answer 42

enzyme that degrades proteins in solution

Question 43

What is the signal peptide sequence for mitochondrial protein transport into the mitochondrion from the cytosol?

Answer 43

matrix-targeting motif

Question 44

What receptor does the matrix-targeting motif bind to?

Answer 44

matrix-targeting motif is recognized by an import receptor on the outer membrane of the mitochondria

Question 45

Tom40

Answer 45

- general import pore embedded in the outer membrane | - known as translocon of outer membrane

Question 46

Tim44, Tim23, Tim17 complex

Answer 46

- translocon of inner membrane

Question 47

Contact sites

Answer 47

allow direct movement through the translocons

Question 48

What is the purpose of cytosolic Hsc70?

Answer 48

- keeps polypeptides unfolded because the mitochondrial proteins must be unfolded to fit through the pored of the Tom and Tim translocons

Question 49

What process of post-translational protein transfer into mitochondria requires ATP?

Answer 49

the Hsc70 function requires ATP

Question 50

What is the purpose of matrix Hsc70?

Answer 50

- to bring proteins to the matrix | - it binds to the polypeptide and helps pull it out of the translocon and prevents it from moving backwards

Question 51

Two compartments in which proteins travel to in the mitochondria?

Answer 51

mitochondrial matrix and inner membrane

Question 52

what are the two targeting motifs that are needed when a protein goes into the inner membrane?

Answer 52

1. N-terminal matrix-targeting motif | 2. Hydrophobic stop-transfer sequence

Question 53

What is the function of the stop-transfer sequence?

Answer 53

1. Stops translocation so that protein is no longer being pulled through translocon 2. It directs transfer of polypeptide out of translocon and into membrane

Question 54

Three things that defective import of mitochondrial proteins can arise from?

Answer 54

- mutations in targeting signal - mutations that disrupt import machinery - deficiencies in the chaperones

Question 55

Protein transport to the mitochondria and peroxisomes occur ________.

Answer 55

post-translationally

Question 56

Characteristics of RER

Answer 56

1. Bound by single membrane 2. Does not have its own genome 3. Site of protein synthesis and modification 4. First destination for secreted proteins

Question 57

Why is a portion of the RER labelled "rough ER" instead of just ER?

Answer 57

it is labelled rough because a portion of the ER has a punctuate appearance created by associated ribosomes

Question 58

Are proteins transported to the lumen of the ER immediately upon translation so that they are not exposed to the cytosol?

Answer 58

Newly synthesized proteins can move directly into the microsome without being exposed to external environment

Question 59

Microsomes

Answer 59

created by disrupting the ER membranes of the cell in a process called homogenization

Question 60

Does translocation and translation need to occur together?

Answer 60

Yes, they must occur co-translationally