ER translocation, general mechanisms of protein trafficking (part 1)

Question 1

What does “translocation” mean in protein trafficking ?

Answer 1

Translocation means “a change of location”.

In the context of protein trafficking, translocation refers to a protein crossing a membrane.

Question 2

What does “nascent” mean ?

Answer 2

Nascent means “coming into being” (nascent from Latin nascens, “being born”). A nascent chain is therefore a newly synthesized polypeptide chain.

Question 3

What is glycosilation ?

Answer 3

Glycosylation refers to the addition of sugars to a protein or lipid.

Question 4

Receptors and channels are made of proteins, with
one notable exception.
Which one ?

Answer 4

The signal recognition particle (SRP) is an abundant, cytosolic, universally conserved ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes.

Question 5

What does a typical signal for protein import into the endoplasmic reticulum (ER) contain ?

Answer 5

A typical ER signal sequence has one or more positively charged amino acids followed by a stretch of 6-12
hydrophobic amino acids.
An example of an ER signal sequence (this is prelysozyme):
+H3N-MRSLLILVLCFLPLAALG/K–
/ = signal peptidase cleavage

Question 6

What is special about about protein import into the ER ?

Answer 6

It can cotranslational or post-translational.

Question 7

How are proteins imported into the ER ?

Answer 7

The polypeptide, w/ its signal sequences, is recognized by SRP, which binds to the SRP receptor on the ER membrane and is then translocated through the membrane by the sec61 complex.

Question 8

How does co-translation protein import into the ER occur ?

Answer 8

• binding of SRP to the signal causes a pause in translation
• the SRP bounds ribosome attaches to SRP receptor in the ER membrane
• translation continues and translocation continues
• SRP and SRP receptor are displaced and recycled

Question 9

What is the role of GTP in co-translational protein import in the ER ?

Answer 9

GTP hydrolysis powers assembly of the nascent chain/translocon complex and release of the SRP/SRP receptor.

Question 10

What is the SRP made of ?

Answer 10

6 proteins + 1 small RNA molecule

Question 11

Which proteins does post-translational translocation require?

Answer 11

The sec61 complex (again)
Sec 62,63,71,72 complex
BiPs (Binding Proteins) which keep the polypeptide unfolded in the ER (ATP hydrolysis required)

Question 12

How does translocation of a soluble protein into the ER lumen occur ?

Answer 12

The polypeptide is translocated through an inactive protein translocator, until the signal peptide (now in the membrane) is cleaved by a signal peptidase and the mature soluble protein is released in the ER lumen.

Question 13

How does translocation of membrane protein into the ER membrane occur ?

Answer 13

The polypeptide possess start- and stop-transfer sequences (hydrophobic regions)
When both these regions are within the membrane, the start-transfer sequence is cleave by a signal peptidase and the mature transmembrane protein is now in the ER membrane

Question 14

What is N-linked glycosylation ?

Answer 14

N-linked glycosylation, is the attachment of the sugar molecule oligosaccharide known as glycan to a nitrogen atom (amide nitrogen of asparagine (Asn or N) residue of a protein).
It can be N-X-S or N-X-T

Question 15

What is dolichol ?

Answer 15

Dolichol refers to any of a group of long-chain mostly unsaturated organic compounds that are made up of varying numbers of isoprene units terminating in an α-saturated isoprenoid group, containing an alcohol functional group.

Question 16

How does N-linked glycolysation happen ?

What is the oligosaccharide composed of ?

Answer 16

In oligossacharide is tranferred from dolichol-P-P to the N residue of a growing polypeptide chain by the enzyme oligosaccharyl transferase (gpes from lipid linked oligosaccharide to N-linked saccharide).
The oligosaccharide is often :
N (Asn) - N-acetylglucosamine - manose - glucose

Question 17

How are polypeptides processed in the ER ?

Answer 17

• Inside the ER, polypeptides are glycosylated and helped to fold by “chaperone” proteins
• Chaperones help proteins to fold correctly, and also mark incorrectly folded proteins for degradation
• The ER is also the site of disulphide bond formation (S-S).
• Disulphide bonds (between cysteine residues) help stablise the tertiary and quarternary structure of proteins.

Question 18

What is the fate of misfolded proteins in the ER ?

What is this process called ?

Answer 18

Misfolded proteins are transported back out of the ER (a process involving accessory exit factors) and degraded in the cytosol “retrotranslocation” or “dislocation”
The process of degrading misfolded proteins is called ER-associated degradation or ERAD

Question 19

How does ERAD work ?

Answer 19

• a misfolded protein in the ER is marked by a chaperon
• the misfolded protein is translocated out of the ER by an ER protein translocator with accessory proteins
• N-glycanase cleaves the N-oligosaccharide
• ubuquitin tags the misfolded protein to direct it to the proteasome where it is degraded

Question 20

What are the 3 main methods used to study protein translocation across membranes ?

Answer 20

1. Transfection approach. Does a particular amino acid sequence act as a signal?
2. Biochemical approach. Does a protein co-purify with a particular organelle?
3. Genetic approach.
   Yeast as a model organism
   • widely used in protein trafficking studies
   • yeast screens have identified, for example, the Sec61 ER translocon

Question 21

What happens to proteins one they have been translocated into the ER ?

Answer 21

• Once translocated into or through the ER membrane, proteins are glycosylated and folded. If they pass ER quality control, they can be transported to the Golgi complex
• Some proteins are maintained in the ER “resident proteins” eg. BiP

Question 22

What are the 2 main relationships that proteins can have w/ the different compartments of the cell ?

Answer 22

Proteins can be “resident to a compartment” or “en route to another compartment.

Question 23

What are the 3 main pathways in the cell between nucleus and plasma membrane ?

Answer 23

• Biosynthetic/ secretory pathway
• Endocytic pathway
• Retrograde/ recycling pathways

Question 24

Signals direct proteins to the correct place.

What can these signals be ?

Answer 24

Amino acid sequences : signal sequences or signal patches

Protein modifications : glycosylation, ubiquitination, lipid modifications

Question 25

How is a protein transported from one membrane-bound compartment to another ?

Answer 25

1. Vesicular transport e.g. ER to Golgi

2. Direct fusion e.g. late endosome/lysosome fusion

Question 26

What do vesicles look like under the electron microscope ?

Answer 26

Many transport vesicles appear to have electron dense “coats” –> due to several proteins :

• clathrin
• COPI
• COPII

Question 27

How does a transport vesicle sort proteins ?

Answer 27

With other protein located inside the vesicle :

• GTP-binding protein
• v-SNARE protein
• membrane cargo protein
• membrane cargo-receptor protein
• coat protein (~the vesicle “membrane”)

Question 28

How do the vesicles know how to take which proteins where ?

Answer 28

Different types of coats sort proteins at different transport steps :
- COPI –> cis-Golgi to ER
- COPII –> ER to cis-Golgi
- clathrin and adaptor proteins –> trans-Golgi to endosome + p membrane to endosome
• The GTPases ARF and Sar1 control coat recruitment.
• They also control disassembly of COPI and II coated vesicles.
• Uncoating of clathrin-coated vesicles requires an Hsp70
family ATPase.

Question 29

Does every step of protein trafficking require vesicles ?

Answer 29

Not every step requires vesicles: some steps are better described as “direct fusion” eg. late endosome to lysosome fusion