Membrane Compartments and Membrane Protein Sorting

Question 1

The precise sorting of macromolecules within cells results in multiple membrane enclosed compartments with discrete functions. How is this accomplished?

Answer 1

sorting signals (consisting of specific amino acid sequences that form a signal peptide) in proteins that direct their cellular localization. NOTE: those proteins lacking specific organelle sorting signals will remain in the cytosol

Question 2

Protein synthesis in the cytosol occurs where?

Answer 2

free ribosomes

Question 3

Proteins made on free ribosomes are destined to be transported where?

Answer 3

Proteins may either remain in the cytoplasm (these lack sorting signals) or can move into the nucleus, peroxisomes or mitochondria.

Question 4

What is the fate of proteins synthesized on the rough endoplasmic reticulum (ER)?

Answer 4

these proteins will either remain in the ER or undergo sorting to the Golgi, lysosomes, secretory vesicles or plasma membrane.

Question 5

What are the three mechanisms for moving proteins between cellular compartments?

Answer 5

1. Gated transport (movement from cytosol to the nucleus)
2. Transmembrane transport (movement from the cytosol into mitochondria, peroxisome and ER)
3. Vesicular transport (movement through the Golgi and movement to lysosomes, secretory vesicles, plasma membrane)

Question 6

Nuclear pore complexes are the ‘gates’ to the nucleus. What is the name of the proteins that compose these gates?

Answer 6

nucleoporins (mediate bi-directional transport

through a water filled channel)

Question 7

How do nuclear pore complexes allow the transport of small molecules to the nucleus?

Answer 7

aqueous channels in the complex

Question 8

Nuclear pore complexes allow large molecules to enter and exit the nucleus only if they are attached to what?

Answer 8

nuclear localization signals or nuclear export signals (NOTE: large molecules typically do so in the folded state)

Question 9

Nuclear localization signals are composed of what?

Answer 9

one or two short stretches of positively charged amino acids

Question 10

The outer membrane of the nucleus is continuous with which organelle?

Answer 10

ER

Question 11

The inner membrane of the nucleus contains sites for binding of which structures?

Answer 11

chromatin and the nuclear lamina

Question 12

Are proteins typically fully folded when transporting through a nuclear pore?

Answer 12

Yes

Question 13

Nuclear localization signals on proteins bind to nuclear import receptors which in turn bind to which structure of the nuclear pore to guide protein transport into the nucleus?

Answer 13

cytoplasmic fibrils of nuclear pores

Question 14

Describe how nuclear transport of large molecules (i.e. proteins and mRNA) is an energy driven event.

Answer 14

Following transport into the nucleus, the nuclear transport receptor and cargo bind to “Ran-GTP”, which stimulates the release of the cargo (in this process, Ran-GTP physically displaces the cargo and forms a complex with the nuclear transport receptor). The transport receptor (still bound to Ran-GTP) then migrates to cytosol where Ran-GTP is hydrolyzed to Ran-GDP, which causes it to release from the receptor. Then, the nuclear transport receptor is free to bind another cargo+nuclear signal complex and repeat the cycle.

Question 15

Describe protein transport to mitochondria.

Answer 15

1. Proteins synthesized in the cytoplasm which are destined for the mitochondria have a signal peptide specific for the mitochondria at the N-terminus (often called a matrix targeting sequence) and are imported post-translationally in an unfolded state.
   a. All proteins imported into the mitochondria have at minimum a matrix targeting sequence (contains positively charged residues and forms an amphipathic helix)
   b. Cytosolic and mitochondrial chaperones maintain the protein in its unfolded state during transport
   c. Unfolded protein binds a receptor in the outer membrane. This complex diffuses (i.e. slides along the outside of the outer membrane) in the membrane until it encounters a translocation channel on the surface. The protein is then transported through the channel all the way to the matrix (i.e. the channel spans both membranes).
   d. The signal peptide is cleaved off the protein in the matrix by a signal peptidase to form a ‘mature protein”
   e. Some proteins never make it all the way into the matrix but rather a second signal peptide anchors them into the inner membrane

Question 16

Are proteins that are synthesized in the cytosol and transported to mitochondria in a folded or unfolded state during transport? Is this pre- or post-translation?

Answer 16

unfolded; post-translation

Question 17

The matrix targeting sequence of proteins bound for mitochondria form what structure?

Answer 17

amphipathic helix (i.e. helix is charged on one side and uncharged on the other side)

Question 18

what is the role of cristae in mitochondria?

Answer 18

cristae are convoluted foldings of the inner membrane which greatly expand its surface area, enhancing the ability to make ATP.

Question 19

What maintains the protein in its unfolded state during transport to mitochondria?

Answer 19

Cytosolic and mitochondrial chaperones

Question 20

Mitochondrial proteins destined for the inter-membrane space or the mitochondrial inner membrane require two signal sequences. How does this work?

Answer 20

These proteins move into the matrix via conventional mitochondrial transport where their matrix-targeting sequence is cleaved and then a second signal peptide is exposed that targets them to the inner membrane or the inter-membrane space.

Question 21

Alcohol dehydrogenase is found where in the mitochondria?

Answer 21

in the matrix (i.e. it does not possess a secondary signal peptide)

Question 22

Peroxisomes are most abundant where in the body?

Answer 22

liver and kidney cells (NOTE: they are still found in all cells in the body)

Question 23

What are the main functions of peroxisomes? (Hint: 3 things)

Answer 23

1. A role in detoxification of toxins, particularly in liver and kidney cells.
   * half the ethanol we drink is oxidized in peroxisomes
2. Breakdown of fatty acids via oxidation (especially long chain FAs)
3. Site of synthesis of some lipids
   a. In animal cells a second site of synthesis (besides ER) of cholesterol

Question 24

How do peroxisomes facilitate the breakdown of fatty acids?

Answer 24

oxidative reactions (NOTE: this also occurs in the mitochondria but fatty acids >20 carbons occurs only in peroxisomes)

Question 25

New peroxisomes always arise from old ones through which mechanisms?

Answer 25

growth and fission

Question 26

Does peroxisomal protein transport require ATP? Are proteins typically transported folded or unfolded?

Answer 26

Yes; folded

Question 27

Describe peroxisomal protein transport

Answer 27

Imported proteins contain targeting sequences that are recognized by peroxisomal transport receptors that bind specific proteins in the peroxisome membrane

Following delivery of protein to the peroxisome matrix,
the peroxisomal transport receptors are recycled to the
cytosol for reuse

Question 28

Peroxisomal transport receptors are called what?

Answer 28

Peroxins

Question 29

What are the two recognized Targeting Sequences for Peroxisomal Matrix Proteins?

Answer 29

• PTS (protein targeting sequence 1): SKL (serine-lysine-leucine) sequence found at the carboxy terminus
• PTS2: 26-36 amino acids at the amino terminus (cleaved off once inside the peroxisome)

Question 30

How does Zellweger syndrome affect peroxisome function?

Answer 30

This results in a lack of functional peroxisomes due to a defect in import of peroxisomal proteins. Individuals affected have defects in the brain, liver and kidneys and die by the time they are a few months old.

Question 31

What is X-linked Adrenoleukodystrophy ?

Answer 31

defective oxidation of very long chain fatty acids. This appears to be caused by the defective import of very long chain acyl CoA synthetase or one of its cofactors into the peroxisome. The activity of very long chain acyl CoA synthetase is required for oxidation of long chain fatty acids.

Question 32

What are the main function of the Smooth Endoplasmic Reticulum?

Answer 32

1. Predominant site of phospholipid synthesis

Question 33

What kinds of cells have abundant smooth ER?

Answer 33

a. cells specializing in lipid metabolism such as adipocytes
b. hepatocytes which make lipoprotein particles and detoxify lipid-soluble drugs

NOTE: Most cells have little smooth ER.

Question 34

Do most cells have predominantly smooth ER or RER?

Answer 34

RER (especially secretory cells)

Question 35

The RER is a main site of what?

Answer 35

protein synthesis (NOTE: ribosomes attached to the RER are no different from ribosomes found free in the cytosol; the protein being synthesized determines whether the ribosome attaches to the RER. (signal sequences) )

Question 36

What does co-translational transport of proteins in the ER mean?

Answer 36

It means that proteins destined for the ER are being transported while they are still be translated. This is guided by a signal sequence (16-30 hydrophobic amino acids) targets the proteins to the RER and initiates transport across the membrane.

Question 37

What are the steps of co-translational transport of proteins to the ER?

Answer 37

1. Binding of signal recognition particle (SRP) to the signal sequence causes a pause in translation.
   
   2. The complex composed of the SRP, ribosome and nascent polypeptide binds to the SRP receptor in the ER membrane.
   3. The signal sequence inserts into the membrane and binds to the protein translocation channel (Sec61 complex). The SRP then dissociates from the complex allowing protein synthesis to resume.
   4. Binding of the signal peptide to the protein translocation channel opens the channel so the protein can move across the membrane as it is synthesized.
   5. The ER signal peptide is removed from lumenal peptides after or during translocation. The signal sequence is cleaved from the peptide by the enzyme signal peptidase.
   6. The signal peptide is then ejected from the channel and the channel closes with the protein now in the lumen of the ER

Question 38

Are transfer sequences in ER bound proteins hydrophobic or hydrophillic?

Answer 38

hydrophobic

Question 39

What happens if a second hydrophobic sequence is encountered on the ER bound protein by a translocation channel during co-translational transport?

Answer 39

the protein will stop the transfer of the protein across the membrane resulting in a simple span integral protein across the membrane of the ER (typically with the C-terminus facing the cytosol and the N-terminus facing the ER lumen). Some proteins end up spanning across the ER membrane several times and their structure is mediated by start and stop transfer sequences as well

Question 40

The orientation of membrane spanning proteins in the ER is preserved when they are transported to downstream compartments of the secretory pathway.

Answer 40

The outside of the cell is equivalent to the lumen of the ER.