Endomembrane System Part 2

Question 1

What does the endomembrane system do?

Answer 1

Materials move within the endomembrane system via the biosynthetic, secretory, and endocytic pathways via transport vesicles

Question 2

What is the Endoplasmic Reticulum?

Answer 2

ER is the starting point for both the secretory and biosynthetic pathway
It is the site of protein synthesis, protein folding, and processing

Question 3

What is the structure of the Endoplasmic Reticulum?

Answer 3

A highly complex network of membrane-enclosed, rod-like tubules and sheet-like cisternae (flattened sacs)
It is the organelle with the largest surface area

Question 4

What is the lumen of the Endoplasmic Reticulum?

Answer 4

Aqueous space inside of ER tubules and cisternae

Question 5

What are the tubules and cisternae in the Endoplasmic Reticulum?

Answer 5

They are shapes that are mediated by reticulons

Question 6

What are reticulons?

Answer 6

Unique ER integral membrane proteins that possess a ‘hairpin’ (V-shaped) secondary structure and regulate ER membrane curvature (‘bending’) and overall shape of the ER

Question 7

Why is the Endoplasmic Reticulum special?

Answer 7

It is a highly dynamic network

Question 8

What do the ER tubules and cisternae do?

Answer 8

They are in constant flux

They undergo constant bending, fusion, fission

Question 9

What does the ER consist of?

Answer 9

Distinct regions of the ER network that possess unique morphologies and/or functions
RER and SER are two classic examples of ER subdomains

Question 10

What is the RER?

Answer 10

Mostly cisternae with bound ribosomes, involved in protein and membrane phospholipid synthesis

Question 11

What is the SER?

Answer 11

Mostly curved tubules lacking ribosomes, involved in calcium storage and hormone synthesis –> it is not involved with proteins

Question 12

What does each ER subdomain possess?

Answer 12

Each possesses a unique complement of proteins and membrane lipids that mediate its distinct function(s)

Question 13

What is the nuclear envelope?

Answer 13

It is an ER subdomain

The outer nuclear membrane is continuous with RER, contains Nups, and attached ribosomes

Question 14

What is the Mitochondria and Plasma Membrane-Associated Membranes (MAM & PAM)?

Answer 14

It is an ER subdomain
Regions of ER that make direct contact with mitochondria or the pm, respectively; involved in membrane protein and lipid exchange

Question 15

What is the ER Exit Sites (ERES)?

Answer 15

It is an ER subdomain

Regions where transport vesicles bud off from the ER en route to the Golgi

Question 16

What occurs in the Rough Endoplasmic Reticulum?

Answer 16

One of two main sites for protein synthesis (translation) in the cell
Where proteins are made

Question 17

What two proteins can be synthesized in the RER?

Answer 17

1) Free ribosomes in the cytoplasm

2) RER membrane-bound ribosomes

Question 18

What happens to free ribosomes in the cytoplasm?

Answer 18

The fate of the nascent, properly-folded soluble or membrane protein in the cytoplasm can remain in the cytoplasm OR targets (post-translationally) to the proper intracellular destination (nucleus, mitochondria, chloroplasts, etc.)

Question 19

What happens to RER membrane-bound ribosomes?

Answer 19

The fate of the nascent, properly folded soluble or membrane protein in the RER can remain in the RER or localizes (moves laterally in the RER membrane or lumen) or another ER subdomain OR localizes to other RER derived organelles (peroxisomes-nascent organelles bud off from the ER OR targets (via transport vesicles) from the ER onto other (post ER) compartments in the endomembrane system

Question 20

What is the co-translational translocation of a soluble protein into the RER lumen?

Answer 20

Protein targeting to and across the ER membrane is a complex, multi-step process

Question 21

What are the steps of co-translational translocation of a soluble protein into the RER lumen?

Answer 21

Steps 1 and 2) In the cytoplasm, translocation of an mRNA on a ‘free’ ribosome begins
N terminus of the nascent, growing polypeptide eventually emerges from the ribosome (protein is made)
N terminus contains a signal sequence, which is a stretch of 8-15 hydrophobic amino acids that serve as an RER target signal
The exposed signal sequence is recognized by the signal recognition particle (SRP)
SRP then binds to the ribosome and stops protein translocation
Step 3) SRP targets the complex (ribosome, ‘stalled’ nascent polypeptide, mRNA) to the surface of the RER
SRP binds to the SRP receptor
Interaction between SRP and SRP receptor is strengthened by both binding GTP
Step 4) GTP hydrolysis results in the release of SRP and SRP receptor
Simultaneously, nascent polypeptide and ribosome transferred to the cytoplasmic side of the Sec61 translocon
Transfer of nascent polypeptide-ribosome to Sec61 translocon result in N terminus of polypeptide being inserted into the opening of the translocon channel
Translation resumes and the elongating polypeptide chain continues to pass through the translocon channel towards the ER lumen
–> the passage of the growing polypeptide through the translocon is driven by translation
Step 5 and 6) As the N-terminal signal sequence enters the ER lumen it is cleaved by the signal peptidase
–> ER integral membrane protein (protease) associated with the translocon- catalytic domain of signal peptidase faces the ER lumen
–> Peptidase recognizes cleavage sequence at C-terminal end of the signal sequence
Co-translational translocation of polypeptide into ER lumen continues
Step 7 and 8) Translation is completed and ribosome is released from translocon
Remainder of nascent protein enters ER lumen
Translocon closes - pore plug moves back into (blocks) the channel
Next,
Nascent protein is glucosylated (addition of sugars to the polypeptide) and properly folded by ER lumenal chaperones
Reticuloplasmins: bind to nascent proteins and mediate their proper folding and oligomeric assembly (prevent protein aggregation)

Question 22

What is the SRP?

Answer 22

Ribonucleoprotein protein consisting of 6 proteins and 1 small RNA in the cytoplasm

Question 23

What is the SRP receptor?

Answer 23

A heterodimeric ER integral membrane protein complex

Cytoplasmic-facing domains of the SRP receptor serve as a ‘docking site’ for incoming SRP

Question 24

What is the Sec61 translocon?

Answer 24

A multiprotein complex consisting of several ER integral membrane protein subunits (Sec61alpha, beta, gamma) forming an ‘hourglass’- shaped aqeous channel that contains a pore ring

Question 25

What is the structure of the Sec61 translocon?

Answer 25

It has a ring of 6 hydrophobic amino acids located at the narrowest diameter of the channel Serves as a gate to help seal channel to ions and small molecules (including during protein translocation) The translocon channel is also blocked by a short alpha-helix plug (ensures no leaking) --> This is a second-gate keeping mechanism- maintains ER organelle compartmentalization --> During protein translocation - the growing polypeptide forces 'plug' away from channel

Question 26

What is the co-translation insertion of an integral membrane protein into the RER?

Answer 26

Most membrane proteins are also synthesized on membrane-bound ribosomes at the ER (RER) Including resident membrane proteins of the ER and all other post-ER compartments of the endomembrane system (Golgi, lysosomes, pm) This is the integration of a protein into the membrane ER membrane protein is similar initially to soluble protein import into the ER lumen, except for important mechanistic differences resulting in the mature membrane protein being integrated (anchored) in the ER membrane and with the proper topology

Question 27

What is the membrane protein topology?

Answer 27

Number of membrane-spanning domains and orientation

Question 28

What is a transmembrane domain (TMD)?

Answer 28

Typically an alpha helical, 16-25 stretch of hydrophobic amino acids (energetically favourable within hydrophobic interior of phospholipid bilayer)

Question 29

What happens to integral membrane proteins?

Answer 29

Several different classes of integral membrane proteins are synthesized at the ER

Question 30

How are Type I membrane proteins inserted into the ER?

Answer 30

N lumen - C cytosol Stop transfer anchor sequence moves laterally out of translocon and becomes 'anchored' in the adjacent phospholipid bilayer As translation continues, elongating polypeptide extends into the cytosol Translation completed, ribosome released, protein diffuses away laterally in the membrane bilayer The N terminus drives this process and has the signal Final Membrane Orientation: N lumen - C cytosol

Question 31

How are Type II membrane proteins inserted into the ER?

Answer 31

N cytosol - C lumen No N terminal signal sequence, but possess an internal signal-anchor (SA) sequence First and only TMD functions both as a signal sequence for binding SRP and mediating nascent polypeptide-ribosome complex targeting to the translocation (ER lumenal protein) and as a membrane anchor Proteins internal signal-anchor sequence (TMD) enters translocon

Question 32

How are Type II membrane proteins inserted into the ER? (The steps)

Answer 32

1) Signal anchor sequence is re-oriented (flipped) in translocon so N-terminus of polypeptide chain faces cytosol, the orientation mediated by several +ve charged amino acid residues located just upstream (N-terminal) of the signal anchor sequence 2) As translation continues, elongating polypeptide extends into the ER lumen Also, signal anchor sequence moves laterally out of translocon and becomes 'anchored' in the adjacent membrane bilayer 3) Translation completed, ribosome released, protein diffuses away laterally in the ER membrane bilayer Final membrane orientation: N cytosol - C lumen

Question 33

What is the positive outside rule?

Answer 33

+ve charged amino acids next to signal-anchor sequence determines the orientation of most membrane proteins synthesized at ER +ve charged amino acids indicates that the polypeptide needs to be flipped --> translocon recognizes positive charge, and so N terminus will be flipped.

Question 34

How are Type III membrane proteins inserted into the ER? (The steps)

Answer 34

Same orientation as Type I membrane protein, but, like Type II proteins, possess an internal signal-anchor sequence (no N-terminal signal sequence) 1) SRP-dependent targeting to insertion into translocon similar to Type II membrane protein, but +ve charged amino acids located downstream (C-terminal) of the signal anchor sequence 2) As translocation continues, elongating polypeptide extends into the ER lumen 3) Translation completed, ribosome released, protein diffuses away laterally in the membrane bilayer Final membrane orientation: N lumen - C cytosol

Question 35

What are multi-spanning ER membrane proteins?

Answer 35

ER integral membrane proteins with multiple TMDs lack an N-terminal signal sequence Contain both internal signal-anchor sequences, which serves to a target protein to ER in SRP-dependent manner and anchor protein in ER membrane depending on the positive outside rule, and internal stop-transfer anchor sequences (serve to stop the transfer of protein through and anchor into ER membrane)