Lecture 29: The endomembrane system: central to cell physiology

Zheng 1

endoplasmic reticulum characteristics

• sites for protein (rough) and lipid (smooth) synthesis

• largest single-membrane bilayer organelle

• exists as sheets (rough) and tubules (smooth) that are interconnected

• enclose a space called ER lumen

rough ER

• filled with ribosomes

• used for protein synthesis

• protein target and translocation into the ER

smooth ER

• without ribosomes

• used for lipid synthesis

• have ER exit sites, involved with ER-Golgi trafficking

Which types of the ER are sheets and tubules?

rough ER is interconnected layers of flattened sheets, smooth ER is tubules

what happens when cells are homogenized?

the rough ER breaks into small microsomes, that are still functional (still have ribosomes) and can be easily purified

morphology of ER in animal and plant cells

very similar

ER dynamics

• consistently being reorganized (connections broken/formed)

• ER tubules pulled out and moved on microtubules by motor proteins to make a network

ER tubule growth on microtubules

growing tubules slide along microtubules via motor proteins until the end, and grow with the growing (+) end of the microtubule

what gives the curvature for formation of tubules and edges of sheets?

Reticulons, that have a W-shaped structure

what fuses/connects different ER tubules?

Atlastins, which are a class of dynamin-like large GTPases that undergo oligomerization

what allows for spacing in sheet formation?

CLIMP63, ER luminal spacer

what are proteins targeted to the ER bound with?

ER-bound ribosomes

where do proteins made by ER-bound ribosomes end up?

within the ER lumen

how are secretory proteins targeted to the ER?

ER-signal sequence on the n-terminal of the protein, once emerged, will guide the ribosome-mRNA-nascent peptide complex to the ER

what happens when the ribosome-mRNA-nascent peptide complex is attached to the ER?

the peptide is translated and translocated to the lumen of the ER (co-translational)

what is the ER signal sequence?

~25 amino acid long sequence, with a stretch of ~ 10 hydrophobic amino acids in the centre

what happens when there is no signal sequence on the end of the peptide and what does it mean?

the protein is not incorporated to ER/microsomes. This means that the sequence is necessary for ER import

what happens when imported proteins have the N-terminus removed and what does it mean?

No effect on growing protein, meaning that cleavage of signal sequence once incorporated has no effect on protein synthesis

what happens when proteins that have completed synthesis want to be added to ER/microsomes and what does it mean?

there is no import of proteins. This means that import must occur DURING synthesis (co-translational

What is SRP and what does it do?

Signal Recognition Particle, contains a hydrophobic binding groove (in P54 subunit) that binds the signal sequence

what happens when SRP docks the nascent-polypeptide-ribosome-mRNA?

SRP binds to the alpha subunit of SRP receptor (both in GTP-bound state)

what happens once the nascent polypeptide-mRNA-ribosome is properly docked?

P54 of SRP and alpha subunit of SRP receptor hydrolyze their bound GTP, destabilizing the interaction interface and dissociating them from each other

ER signal sequence opens the translocator and enters the channel

what happens as the polypeptide translocates into the ER and mRNA is translated?

ER signal sequence gets cut off by signal polypeptidase and is degraded

translocator closes, ribosome is released, translocated protein is folded in lumen of ER

entire process of targeting secretory proteins to ER lumen

1. Signal sequence formed from nascent polypeptide-mRNA-ribosome complex

2. signal sequence recognized by P54 of the SRP to form SRP-nascent-polypeptide-mRNA-ribosome complex

3. P54 interacts with alpha subunit of SRP receptor, bringing the complex to the ER

4. complex is docked to the ER translocator, opens it and enters. P54 and alpha subunit of SRP hydrolyze GTP to GDP

5. Signal polypeptides cleaves the signal sequence

6. mRNA translation within ER lumen

7. translocator closes, ribosome is released

8. protein folding begins