Lecture 11: Protein Sortin II

Question 1

Protein translocators

Answer 1

In cell membranes typically unfold proteins to move from cytosol to ER or mitochondria

Question 2

Structure of Mitochondria and Nucleus similarities

Answer 2

Have double membrane (2 lipid bilayer)
Have outer membrane, inner membrane, and intermembrane space

Question 3

Structure of Mitochondria

Answer 3

Double membrane
Outer mitochondrial membrane
Intermembrane Space
Inner mitochondrial membrane

Question 4

To initiate transport into mitochondria, what needs to happen

Answer 4

• Signal sequence on mitochondrial protein is recognized by receptor on outer mitochondrial membrane (TOM complex)
• Mitochondrial import signal sequence is at N terminus

Question 5

Where are protein translocators found in Mitochondria

Answer 5

On both outer and inner mitochondrial membranes; form complex to transport proteins into matrix

TOM= translocator for outer membrane
TIM= translocator for inner membrane

Question 6

How can proteins be embeded to different parts in mitochondria?

Answer 6

Use different sorting signals and different protein translocators to reach destination within mitochondria

Question 6

Proteins encoded by mtDNA also use translocators, nearly all nucleus encoded proteins use ___ for import

Answer 6

TOM complex

Question 7

Critical steps for mitochondrial import

Answer 7

1. cytosolic Hsp70 chaperone proteins bound to unfolded protein, TOM20 receptor binds to signal sequence
2. Protein bypasses outermembrane using translocation channel in TOM complex
3. Unfolded protein translocated into mitochondrial matrix by TIM complex
4. Hsp70 chaperones in matrix fold proteins into correct shape and move into matrix, signal sequence is cleaved by signal peptidase

Question 8

Endoplasmic Reticulum (ER)

Answer 8

most extensive membrane system in a eukaryotic cell
Entry point for proteins destined for Golgi apparatus, lysosomes, and plasma membrane

Question 9

TIM23

Answer 9

Translocator for inner membrane that provides a pore for an unfolded protein
Does not use energy for directional transport

Question 10

Smooth ER

Answer 10

lacks ribosomes, synthesis of lipids and hormones

Question 11

Rough ER

Answer 11

has ribosomes that make proteins which are being translocated into ER during translation

Question 12

To initiate transport into ER

Answer 12

signal sequence on ER protein is recognized by receptor; receptor will recruit protein to ER membrane

Question 13

ER import signal sequence is usually located at

Answer 13

N terminus

Question 14

Membrane Bound vs. Free ribosomes

Answer 14

Structurally identical, they only differ in the protein they are translating

Question 15

Free ribosomes are formed for via:

Answer 15

common pool of ribosomal subunits in cytosol –> mRNA without ER signal sequence remains free in cytosol and is translated by multiple free ribosomes forming a polyribosome

Question 16

Membrane bound ribosomes are formed for ER transport via

Answer 16

common pool of ribosomal subunits in cytosol, mRNA with ER signal sequence translated by membrane bound ribosomes because signal sequence sends protein to ER

force of growing amino acid chain provides energy for translocation into ER lumen

Ribosomes become membrane bound when they attach to ER membrane (ribosomes have binding site for translocator)

Question 17

Critical steps for ER import

Answer 17

1. SRP binds to signal sequence on protein; binding of SRP to ribosome causes protein translation to slow (recognition)
2. SRP ribosome complex binds to SRP receptor on ER membrane (targeting)
3. SRP releases signal sequence; signal sequence inserts into protein translocator (release)
4. Protein with ER signal sequence is transported into ER lumen during translation (translation continues and translocation begins), SRP and SRP receptor dissociate from each other and are recycled for another round of protein sorting

Question 18

Once translocation into ER is complete, what occurs

Answer 18

Signal sequence is removed using signal peptidase
Cut signal sequence is released from translocator into lipid bilayer and rapidly degraded
BiP: (ER version of Hsp70) chaperone in ER lumen that helps protein fold into correct shape

Question 19

Protein Disulfide Isomerase (PDI)

Answer 19

ER protein that catalyzes formation of disulfide bonds
ER is oxidizing environment
Oxidized PDI and reduction protein –> Reduced PDI and oxidized protein with disulfide bond

Question 20

Oligosaccharyl transferase

Answer 20

ER transmembrane protein that attaches multiple sugars (glycosylation) to asparagine residue in ER protein during translation

Question 21

Most ER proteins undergo what type of glycosylation

Answer 21

N linked glycosylation: sugar attached to nitrogen of asparagine residue

Question 22

Glucose sugars are important to ER proteins as

Answer 22

it monitors folding

Question 23

Calnexin

Answer 23

ER chaperone protein that binds to glucose; prevents partially folded ER protein from aggregating
(ER does not have Hsp60, so Calnexin is second line of defense)

Question 24

After N linked glycosylation to ER protein, what occurs

Answer 24

1. After N linked glycosylation occurs, 2 glucose sugars are removed (glycose trimming) from ER protein
2. Calnexin binds to protein with 1 glucose, helps with folding, glucose is eventually removed and protein is released
3. If correct protein shape, ER protein can go to Golgi apparatus and exit from ER
   If incorrect protein shape, glucose added by enzyme (glucosyl transferase) , ER protein binds to calnexin again
   If ER protein can not fold into correct shape, it is exported to cytosol for degradation by proteasome, chaperones prevent unfolded proteins from aggregating and disulfide isomerase eliminates disulfide bridges

Question 25

Water soluble ER proteins

Answer 25

have ER signal sequence at N terminus
Sequence cut and left in membrane
BiP and disulfide isomerase are water soluble ER membrane proteins
Not embedded in ER membrane

Question 26

Embedded ER proteins

Answer 26

Embedded in ER membrane during translation
Calnexin stays in ER
All transmembrane proteins (plasma and organelle membrane proteins) are embedded in ER membrane during translocation

Question 27

Single pass transmembrane protein

Answer 27

Has signal sequence and one transmembrane segment: region of hydrophobic amino acids that will form an alpha helix in lipid bilayer
Transmembrane segment stops translocation; protein released from translocator; translation finished in cytosol

Question 28

Multipass transmembrane proteins

Answer 28

Have start transfer and stop transfer membrane segments
Start transfer sequence initiates transport and stop transfer sequence halts transport and releases protein from translocator
ER signal sequence (start transfer sequence) is not on N terminus for this transmembrane protein
MP transmembrane proteins have pairs of start and stop transfer sequences that allow them to traverse membrane multiple times