29 – Morphology of the ER & protein targeting to the ER

Question 1

Endoplasmic reticulum
/
major function

structure?

Answer 1

Sites for protein & lipid biosynthesis

Single membrane bilayer organelle – largest in eukaryotic cell
-<50% of total membrane

->10% cell volume

Question 2

ER lumen

Answer 2

Membrane bound tubules & sheets enclose a single internal space (ER lumen)
->10% cell volume

-where most prtoeins are folded

Question 3

Endoplasmic reticulum exist as

Answer 3

interconnected tubules & sheets as an extensive network stretching throughout the cell

Question 4

2 subdomain of ER

Answer 4

rough & smooth ER

Question 5

what is Rough ER?

function?

Answer 5

coated with ribosomes

-Protein biosynthesis

-Protein target & translocation into ER
–Cytosol to ER

interconnected layers of flattened sheets

Question 6

Smooth ER

function?

Answer 6

No ribosomes coated

-Lipid synthesis

-Contain ER exit sites
–Involved in ER-Golgi trafficking
–Protein sent to Golgi

exists as tubules

Question 7

When cells are homogenized (want to purify something) what happens to rough ER and why?

is it easy or no?

Answer 7

Rough ER breaks up into small microsomes

-Still functional – ribosomes still on
–used for In vitro protein translation

Easily purified
-Density > other membranous organelles

Question 8

Morphology of ER in animal & plant cells …., so…

Answer 8

VERY similar

Function of ER in animal & plant cells is conserved

mechanism of making ER is same in both

Question 9

ER motility

Answer 9

ER = highly dynamic

Consistently being reorganized (change shapes)
-Some connections broken while new ones reformed

ER tubules are pulled out & moved on microtubules by motor proteins to fuse with another one to make a network

Question 10

2 models for ER motility on microtubules:

Answer 10

Slide along microtubule – toward + or – end

OR

Growth on + end (tip)

Question 11

… drives motility

Answer 11

force

Question 12

ER shaping: Curvature for ER tubules & edges of sheets

Answer 12

Reticulons have W-shaped structure

important for generating curvature necessary for formation of ER tubules & edges of ER

Question 13

ER shaping: Fusion of ER tubules

Answer 13

Alastins = class of dynamin-like large GTPases
-undergo GTP-dependent oligomerization important for fusion of different tubules
-can pull 2 different tubules tgt

Question 14

ER shaping: ER sheet formation

Answer 14

CLIMP63 = ER luminal spacer for sheet formation
-Ribosome to flatten sheet
-you have lumen & space = sheet

Question 15

Proteins are targeted to ER with

Answer 15

ER bound ribosomes

Question 16

Proteins made by ER-bound ribosomes end up

Answer 16

end up within ER lumen

Question 17

Targeting of secretory proteins to ER

Answer 17

1. mRNA protein for secretory protein
2. Mix mRNA with Rough ER
   -protein translated - want to see where proteins end up
3. Homogenization – purify
   -microsomes purified
4. Treat with detergent – disrupt lipid bilayer (membranes)
   -product is degraded - Digestion of secretory protein

OR

5. Add only protease
   -NO Digestion of secretory protein

Question 18

How secretory proteins are targeted to ER?

Answer 18

Signal sequence in proteins - ER signal sequence

1. ER signal sequence, once merged- guide ribosome-mRNA-nascent peptide complex to ER
2. When ribosome-mRNA-nascent peptide complex properly attached to ER, peptide is translated & translocated into lumen of ER
   * Co-translational

Question 19

ER signal sequence

Answer 19

~25aa long, stretch of ~10 hydrophobic amino acids in center – key feature

-Target protein to ER

Question 20

How secretory proteins are targeted to ER? step by step schematic:

Answer 20

1. ER signal sequence determines where your proteins will go
   -If present = go to ER
2. mRNA targeted to ER
3. if no ER signal sequence = go to become cytosolic protein
   -most protein made by ribosome stay in cytosol

Question 21

Discovery of co-translational ER signal sequence in secretory proteins

Answer 21

Without N terminal signal peptide = no incorporation = signal sequence necessary for ER import

Imported proteins has their N-terminus signal peptide removed=ER signal sequence cleaved off

Import only occurred when microsomes were present while synthesis took place = co translational

Question 22

3 important factors needed to targeting & transportation of secretory proteins into ER lumen.

Answer 22

SRP (Signal recognition particle)
-recognizze signal sequence

SRP receptor on ER

translocator Sec61
-translocate protein from cytosol to ER

Question 23

how does SRP recognize the signal sequence?

Answer 23

P54 = key subunit of SRP

Recognize signal w/ hydrophobic binding group

Question 24

subunit of SRP receptor and what does it do?

Answer 24

⍺ subunit - Physical interaction with P54
-they cna interact because both GTP binding proteins

β subunit - anchors SRP on ER membrane

Question 25

Step-by-step targeting & transportation of secretory proteins into ER lumen.

Answer 25

1. ER signal sequence recognized & bound by SRP (RNA based protein) 2. SRP docks the nascent polypeptide-ribosome-MRNA onto the ER by interacting with SRP receptor 3. Once nascent polypeptide-ribosome mRNA properly docked to translocator -Both P54 of SRP & ⍺ subunit of SRP receptor hydrolyze their bound GTP that destabilize interaction interface -They dissociate – SRP recycles back to sytosol ---GTP to GDP : Tightly controlled by GTP hydrolysis --ER signal sequence opens translocator & proteins enters the channel 4. Polypeptide translocated into ER lumen as mRNA = translated 5. Once translocation completes --Sec61 channel closes --Ribosomes released – recycled back to cytosol --Translocated protein folded within lumen of ER

Question 26

ER signal sequence = cleaved off by... when?

Answer 26

by signal peptidase & degraded when polypeptide are translocated into ER lumen

Question 27

Summary: 8 steps

Answer 27

1&2. ER signal sequence emerges from ribosome – recognized by P54 of the SRP = form SRP-nascent polypeptide-ribosome-mRNA complex 3. P54 of SRP interact with ⍺ subunit of SRP receptor to bring complex to ER 4. Once complex is properly docked to ER translocator (Sec61) -ER signal sequence opens translocator & enters -Both P54 & ⍺ subunit of SRP receptor hydrolyze GTP to GDP --They dissociate -SRP recycles back for re-use 5&6. Polypeptide chain enters & continues to elongate into Er lumen as mRNA is translated -ER signal sequence is cleaved by signal peptidase 7&8. Once entire polypeptide crosses into ER lumen -Translocator closes -Ribosome released -Folding of polypeptide starts = to make it functional