Lecture 8: Protein sorting to organelle (I)

Question 1

A typical mammalian cell contains up to

Answer 1

10 000 different kinds of proteins.

Question 2

For a cell to function properly each protein must be

Answer 2

localized to the correct membrane-bound organelle.

Question 3

Name proteins and there location (6)

Answer 3

1- Na+/K+: plasma membrane.
2- RNA polymerase: nucleus.
3- Proteases: Lysosomes.
4- Catalase: Peroxisomes.
5- ATP synthase: Mitochondria.
6- Hormones: Extracellular space.

Question 4

In eukaryotic cells a few proteins______ and most proteins______.

Answer 4

• a few proteins (~5%): encoded by the DNA present in m/c, synthesized on ribosomes in m/c, incorporated directly into m/c.
• most proteins (~95%): encoded by nuclear DNA, synthesized on ribosomes in cytosol, delivered to destined organelle from cytosol.

Question 5

Define protein sorting and sorting signal

Answer 5

• protein sorting: is the process of directing newly made proteins to a particular membrane-bounded organelle.
• sorting signal: a continuous sorting sequence of 3-60 amino acids responsible for directing protein to destined organelle.

Question 6

Sorting sequence for nucleus and peroxisomes

Answer 6

nucleus= Lys-lys-lys-lys-arg-lys (all pos).
peroxisomes= Ser (polar)-lys (+)- leu (hydrophobic)

Question 7

Which proteins lack a signal sequence?

Answer 7

proteins that remain in the cytosol.

Question 8

What happens if a large enough ER signal sequence is attached to a cytosolic protein?

Answer 8

• signal sequence is removed from ER protein.
• altered protein ends up in abnormal location in cell, therefor ER signal sequence is both necessary and sufficient to direct a protein to the ER.

Question 9

Steps in protein sorting

Answer 9

1- recognition of sequence by shuttling cytosolic receptor.
2- target to outer surface of organelle membrane.
3- insertion (or transport) of target protein into membrane.

Question 10

What’s a general problem for import of proteins into destined organelles?

Answer 10

how to transport hydrophilic (charged and polar molecules) which are normally impermeable to interior of membranes.

Question 11

Name three mechanisms which membrane-bound organelles import proteins

Answer 11

1) through nuclear pores
2) across membranes
3) by vesicles

Question 12

Define protein transport through nuclear pores

Answer 12

• proteins sorted from cytosol to nucleus.
• nuclear pores function as selective gates which actively transport specific proteins.
• proteins remain folded.

Question 13

Define protein transport across membranes

Answer 13

• proteins sorted from cytosol to mitochondria, chloroplast, ER, or peroxisomes.
• transported by protein translocators.
• proteins remain unfolded due to narrow space of translocators.

Question 14

Define protein transport by vesicles

Answer 14

• proteins sorted throughout endomembrane system (ER and onward).
• transported by transport vesicles.
• vesicles loaded with proteins from interior space, lumen, of one compartment as they pinch off from its membrane.
• discharge vesicle into second compartment by fusion.
• proteins remin folded.

Question 15

NPCs selectively transport which macromolecules and in what quantity through nuclear envelope?

Answer 15

• large uncharged polar, ions, globular proteins up to 60 kDa (~1 nm in d) diffuse.
• proteins over 60 kDa (~1-25 nm in d) do not diffuse.

Question 16

Describe the structural components of an NPC

Answer 16

1) NPC nuclear ring supports eight 100nm filaments, ends joined by terminal ring forming a nuclear basket.
2) cytosolic ring supports eight 50nm cytosolic filaments.
3) central plug actively transport proteins (1-25nm in d) since they cannot diffuse in water-filled channel.

Question 17

Signal sequence specific to nucleus is called

Answer 17

nuclear localization signal (NLS).

Question 18

Steps in the mechanism of active protein transport through NPC

Answer 18

Step 1:
- importins (nuclear import receptors) in cytosol bind to NLS, driven by GTP hydrolysis.

Step 2:
- importins directed to NPC by interacting with cytosolic filaments.

Step 3:
- binding to NPC opens pore and cargo protein with importin is transported into nucleus, driven by ATP hydrolysis.

Step 4:

• importing-cargo protein complex dissociates.
• importing are exported back to cytosol for reuse.

Question 19

Steps in the mechanism of protein import into mitochondrial matrix

Answer 19

Step 1:

• cytosolic shuttling receptors: MSF (mitochondrial import stimulating factor) and Hsp70 (heat shock protein 70)
• chaperones use energy of ATP hydrolysis to maintain unfolded, import competent protein, drive force to channel-linked receptors located on outer membrane of mitochondria.

Step 2:

• shutting receptors released, protein passes through.
• translocation into matrix occurs at “contact sites” where inner and outer membrane are in close proximities.

Step 3:
- matrix Hsp70 binds to protein, uses ATP hydrolysis to pull protein inside and prevent premature folding.

Step 4:

• matrix Hsp70 is released.
• matrix protease breaks peptide bond between sorting sequence and protein.

Step 5:

• some proteins fold spontaneously (15%).
• most proteins bind to Hsp60 which uses ATP hydrolysis to fold protein.

Question 20

Difference between proteins transported across membranes and proteins transported throughout endomembrane system

Answer 20

• proteins are transported before it is completely synthesized.
• this required membrane bound ribosomes synthesizing the protein attached to rough ER.
• co-translocation.

Question 21

Name two types of ribosomes in cytosol

Answer 21

1) membrane-bound: attached to cytosolic surface of rough ER, synthesize proteins in ER.
2) free polyribosome: unattached to any membrane, synthesize all other proteins.

Question 22

mRNA encoding a cytosolic protein remains

Answer 22

free in the cytosol.

Question 23

mRNA encoding a protein targeted to the ER by the

Answer 23

ER targeting sequence remains bound to the rough ER.

Question 24

Steps in the mechanism of soluble protein across the rough ER into lumen

Answer 24

Step 1:
- SRP (signal recognition particle), cytosolic protein, binds to ER signal sequence and ribosome slowing down translation, moves to rough ER surface.

Step 2:
- SRP-ribosome complex binds to SRP receptor.

Step 3:
- SRP and receptor dissociate, ER signal sequence and growing ribosome polypeptide chain binds and loops to central cavity of translocon.

Step 4:
- ER signal sequence cleaved by signal; peptidase in lumen.

Step 5:
- ER Hsp70 use ATP hydrolysis to pull protein into lumen, and folding of protein.

Question 25

Differences and similarities from import of proteins in mitochondria to ER

Answer 25

differences: in ER a)sorting signal is first removed then pulled in by Hsp70, b) no proteins fold spontaneously, c) no Hsp60, d) use different enzymes to remove sorting signal.
similarities: a) both use family of Hsp to pull in protein, b) use ATP hydrolysis, c) are transported through translocons.