Topic 4B - Protein Transport In Mitochondria/Chloroplasts

Question 1

Give an example of a plastid which is not a chloroplast

Answer 1

Chromoplast

Question 2

Describe the way that mitochondria reproduce

Answer 2

Division by fission - similar to bacteria

Question 3

Most proteins acting in the mitochondria are synthesized in…

Answer 3

The ER/cytosol - transported into the mitochondria

Question 4

Approximately how many proteins does the mitochondria synthesize that act in the organelle? What are they encoded by?

Answer 4

13, encoded by mtDNA

Question 5

Translated proteins are relocated to the mitochondria through the process of…

Answer 5

Post-translational translocation

Question 6

TOM, TIM, SAM and OXA are all examples of…

Answer 6

Beta-barrel transmembrane proteins

Question 7

Once a proteins has passed through the TOM complex of the outer mitochondrial membrane, where are the three destinations it can go from there?

Answer 7

1. To the SAM complex
2. To the TIM23 complex
3. Can be inserted laterally through the TOM complex into the outer mitochondrial membrane

Question 8

What is the role of the SAM complex?

Answer 8

Insert beta barrel proteins into the outer mitochondrial membrane

Question 9

When a protein passes through the TIM22 complex, where is its final location?

Answer 9

Inner mitochondrial membrane

Question 10

When a protein passes through the OXA complex, where is its final location?

Answer 10

The inner mitochondrial membrane

Question 11

What is the difference between TIM22 and OXA?

Answer 11

They both insert proteins into the inner mitochondrial membrane, but the proteins are coming from different locations
TIM22 = from intermembrane space
OXA = from matrix

Question 12

Describe the state of proteins destined for the mitochondria during transport: are they unfolded or folded?

Answer 12

They are unfolded because the folded protein cannot pass through the TOM complex folded

Question 13

This kind of protein keeps unfolded proteins unfolded to pass through the TOM complex

Answer 13

Chaperones

Question 14

Do TOM and TIM work sequentially or simultaneously?

Answer 14

Depends, can do both

Question 15

Is transport through TOM and TIM active or passive?

Answer 15

Active - concentrating proteins inside the mitochondria

Question 16

What 2 processes drive active transport of proteins into the mitochondria?

Answer 16

1. ATP hydrolysis (outer membrane)

2. Membrane potential (inner membrane)

Question 17

Describe how “membrane potential” can drive a protein into the matrix from the inner mitochondrial membrane

Answer 17

Protons are pumped out of the matrix due to oxidative phosphorylation, therefore the inside of the membrane is more negative than the outside of the membrane. The N-terminal signal sequence is drawn towards the inside of the inner mitochondrial membrane because it is attracted the the negative charge

Question 18

Is stripping chaperones an active or passive process?

Answer 18

Active - requires ATP to take the chaperones away

Question 19

What is Hsp60?

Answer 19

A heat shock protein contained within the mitochondria

Unfolded proteins are contained within the protein and the protein is capped; the cap is released with ATP hydrolysis and the correctly folded protein emerges

Question 20

In gram-negative bacteria, the equivalent to the mitochondrial SAM complex is…

Answer 20

BAM complex

Question 21

In gram-negative bacteria, the TOM equivalent is…

Answer 21

Sec

Question 22

In a multi-pass transmembrane protein with an internal signal sequence, how does the signal peptidase cleave the sequence?

Answer 22

It doesn’t - sequence is spared and is a part of the mature protein

Question 23

What is the Mia40 complex?

Answer 23

Aids in proper assembly of mitochondrial proteins

Oxidizes proteins after they pass through the TOM complex by forming disulfide bonds on the protein, mia is reoxidized and oxidizes the next incoming protein

Question 24

What are the divisions of the chloroplast from external to internal?

Answer 24

Outer membrane, intermembrane space, inner membrane, stroma, thylakoid membrane, thylakoid

Question 25

Why do proteins destined for the chloroplast have 2 signal sequences? What are they

Answer 25

Both N-terminal, have chloroplast signal sequence followed by thylakoid signal sequence, chloroplast sequence is cleaved in the stroma

Question 26

How many pathways are there for a protein to be transported from the stroma to the thylakoid?

Answer 26

4

Question 27

To cross the inner membrane in chloroplasts (from intermembrane to stroma), what process is used?

Answer 27

ATP or GTP hydrolysis

Question 28

What are the 3 major functions of the ER?

Answer 28

1. Site of lipid biosynthesis 2. Protein biosynthesis of transmembrane proteins 3. Storage place for Ca++

Question 29

Are all transmembrane proteins synthesized in the ER?

Answer 29

No, the mitochondria and chloroplast also synthesize some of their own TM proteins

Question 30

What is the difference between the smooth and rough ER?

Answer 30

Rough = presence of ribosomes

Question 31

The major modification made to most proteins in the ER is the addition of...

Answer 31

Sugars

Question 32

How can the ER be isolated?

Answer 32

Through a centrifuge - will be in microsome form

Question 33

Is it possible (realistic) to assume all microsomes from a centrifuged sample will be the ER?

Answer 33

No - there are many other membranes in the cell

Question 34

The 2 major types of protein translocation are...

Answer 34

1. Cotranslational translocation | 2. Post-translational translocation

Question 35

Are ribosomes which produce cotranslational proteins different from ribosomes which produce posttranslational proteins?

Answer 35

No - only difference is in the signal sequences

Question 36

Transport into which organelles require chaperones?

Answer 36

Chloroplasts and mitochondria

Question 37

Describe the composition of a signal recognition particle (SRP)

Answer 37

6 polypeptides wrapped around an RNA molecule

Question 38

Describe the binding pocket of an SRP

Answer 38

Largely hydrophobic to interact with hydrophobic signal sequences. Composed of methionines which have simple side chains to accommodate different, bulkier sequences within the binding pocket

Question 39

The signal sequence of a protein destined for the ER usually has a minimum of ( ) hydrophobic amino acids

Answer 39

8

Question 40

How many binding sites does an SRP have?

Answer 40

2 - one for the signal sequence and one which binds to the ribosome

Question 41

During cotranslation, when the SNP is bound to the signal sequence and the ribosome of a polypeptide, other than relocating to the ER what happens?

Answer 41

Translation of the protein pauses

Question 42

When the SNP is bound and headed towards the ER - describe its binding to the ER

Answer 42

Binds to SRP receptor which is associated with a protein translocator

Question 43

What is the role of the protein translocator?

Answer 43

Acts as a channel for the protein to be synthesized through, transports the growing polypeptide into the ER lumen

Question 44

What are the two ways the protein translocator can open?

Answer 44

1. Opens a pore for hydrophilic parts of the protein | 2. Opens laterally to insert TM regions into the ER membrane

Question 45

Proteins that are meant to stay within the ER contain this:

Answer 45

ER retention signal

Question 46

What 2 elements does the ER signal sequence bind to?

Answer 46

1. SRP | 2. Protein translocator

Question 47

Once a protein reaches the protein translocator in the ER, the signal sequence is cleaved and...

Answer 47

Degraded, if no longer needed

Question 48

If "start" and "stop" segments of a transmembrane protein are swapped out, does the protein still assemble properly?

Answer 48

Yes - as long as they are relatively similar hydrophobic regions with similar functions

Question 49

Where are ER retention signals on a protein?

Answer 49

At the c-terminus

Question 50

What is the function of the Get1-Get2 complex?

Answer 50

Insert C-terminal-anchored membrane proteins into the ER membrane

Question 51

What is the function of the Get3 ATPase?

Answer 51

Targeted by the Get1-Get2 complex to form the Get1-Get2-Get3 complex. It hydrolyses its ATP to disassemble the whole complex

Question 52

In order for a c-terminal ER retention signal to be brought to Get3, what intermediate is required?

Answer 52

A pre-targeting complex recognizes the signal and brings it to Get3

Question 53

How many proteins produced in the ER are glycosylated (%)

Answer 53

50%

Question 54

What is the role of oligosaccharyl transferase?

Answer 54

Transfers lipid-linked oligosaccharides onto proteins to be glycosylated

Question 55

The interface between a protein to be glycosylated and the oligosaccharide is...

Answer 55

An asparagine side chain

Question 56

What is the function of the oligosaccharides added in the ER?

Answer 56

Aid in protein folding

Question 57

The first sugars to be cleaved from a "young" oligosaccharide are...

Answer 57

Glucoses

Question 58

Mannose sugar groups are cleaved from an oligosaccharide chain in this organelle

Answer 58

The golgi

Question 59

The asparagine and oligosaccharide chains of a glycosylated protein are ( )-linked

Answer 59

N-linked

Question 60

What is the role of glycosylation?

Answer 60

Ensure proper folding of a protein and determine its final 3D structure

Question 61

What is the difference between calnexin and calreticulin?

Answer 61

``` Calnexin = membrane-bound Calreticulin = soluble ER resident protein ``` Both are thought to perform the same role

Question 62

What is the role of calnexin? What kind of protein is it?

Answer 62

Chaperone protein Bind oligosaccharides of incompletely-folded protein to retain them within the ER, prevent unfolded proteins from clumping together irreversibly

Question 63

Which ER protein checks proteins leaving the ER to see if they have been correctly folded?

Answer 63

Glucosyl transferase

Question 64

What does glucosyl transferase do when it encounters an incorrectly-folded protein?

Answer 64

Adds a glucose group to it to retain it in the ER

Question 65

Glucoses are added by glycosyl transferases by taking them from...

Answer 65

UDP (Uridine diphosphate) bound to glucose

Question 66

A complex involving what elements bind to a misfolded protein to export it to the cytosol?

Answer 66

Chaperones, disulfide isomerase, lectin

Question 67

In the export of misfolded proteins to be degraded, lectin binds to...

Answer 67

The remainder of the oligosaccharide chain of a misfolded protein

Question 68

Why are chaperones necessary in the export of misfolded proteins?

Answer 68

Proteins need to be exported through a pore, so chaperones help keep it in its primary stage

Question 69

Is the transport of misfolded proteins out of the ER active or passive?

Answer 69

Active - goes thru a channel!

Question 70

What enzyme is responsible for removing sugar groups off a misfolded protein?

Answer 70

N-glycanase

Question 71

What helps a proteasome recognize proteins which need to be degraded?

Answer 71

Ubiquitin - misfolded proteins are ubiquinated as they leave the ER

Question 72

If a protein is leaving the ER to go to the cytosol, what can we say about this protein?

Answer 72

It is likely misfolded/to be degraded - almost NO cytosolic proteins are synthesized outside of the cytosol

Question 73

How can the degradation mechanisms for cellular proteins be helpful for the survival of viruses?

Answer 73

Can use this machinery to degrade immune proteins

Question 74

The process of sending proteins out of the ER into the cytosol is called...

Answer 74

Retrotranslocation

Question 75

What is an example of a situation where splicing mRNA does not happen in the nucleus?

Answer 75

The IRE1 pathway in the misfolded/unfolded protein response - splicing happens in the cytosol