Protein Sorting

Question 1

Outline the process of co-translational translocation

Answer 1

Protein synthesis starts. SRp binds to the signal sequence and stops synthesis by blocking the Ef factor site on the ribosome.
SRP binds to the alpha subunit of the SRP receptor.
GTP is hydrolysed, the translocon is opened and SRp detaches.
Signal peptidase cleaves the signal
The protein is ejected into the lumen
translocon closes and ribosome detaches

Question 2

What are the proteins of the translocon in co-translational translocation?

Answer 2

Sec61alpha which is a multi TM protein
Sec61beta which a single Tm domain protein
Sec61gamma which is a single TM domain protein

Question 3

What are the subunits of the translocon in co-translational translocation?

Answer 3

SecY is the channel
SecE is the door
SecG is the plug

Question 4

Outline post translational translocation adn folding in yeast

Answer 4

The polypeptide is imported into the Er by translocon Sec61. The signal sequence is cleaved. BiP ATP is then hydrolysed to BiP ADP by Sec63. BiP binds to the incoming polypeptide to prevent folding. BiP ADp is replaced by ATP causing BiP to leave the protein allowing it to fold.

Question 5

How are TypeI membrane proteins inserted into the membrane?

Answer 5

Co-translational translocation begins. The signal sequence is cleaved in the membrane and the translocation continues. Stop transfer sequence stops in channel. Then synthesis continues

Question 6

How are type IV membrane proteins synthesised?

Answer 6

There are alternating stop and start signals which stick in the membrane

Question 7

How are GPI anchor proteins made?

Answer 7

The precursor protein with a regular TM domain is transferred to the preformed GPI anchor by GPI transamidase.

Question 8

How are tail-anchor proteins made?

Answer 8

A protein is synthesised with a short hydrophobic domain flanked by positively charged amino acids. This end is inserted into the membrane with the aid of cytosolic proteins

Question 9

What are the types of protein modification in the ER?

Answer 9

Glycosylation
Formation of di-sulfide bonds
Functional folding and assembly
Proteolytical maturation

Question 10

Outline O-linked glycosylation

Answer 10

Usually only 1-4 sugar residues
Transfer via glycosyltransferase in the Golgi
Linkage at Serine, Threonine or Tyrosine residues

Question 11

Outline N-linked glycosylation

Answer 11

More complex and conserved
In the ER lumen
Transfer at the amide residues of asparagine
Recognition motif 0 one asparagine seperated via one aa from Serine or Threonine

So The core-mannose oligosachharide usint is linked to dolichol phosphate by a phosphate bound. this is flipped into the ER lumen. More mannose units are added and oligosaccharide transferase removes the mannose oligosaccharide from dolichol phosphate onto the protein at an Asparagine residue.

Question 12

How are disulphie bridges formed in the ER?

Answer 12

Oxidised PDi forms a bond, oxidizes the SH groups on the protein to form a disulphide bond.

Question 13

How does the de novo formation of disulphide bridges work?

Answer 13

Reducded PDi acts as a mediator

Question 14

How is glycoprotein Hemaglutinin folded and assembled in the ER?

Answer 14

the protein is synthesised into the Er and chaperoned by BiP, with mannose oligosaccharide units being added by oligosaccharide transferase. Calnexin and Calreticulin catalyse the removal of the mannose groups,. The monomer is completed with a membrane spanning alpha helix and the associates with two other proteins to form a trimer.

Question 15

Outline the unfolded protein response

Answer 15

It is triggereed by a lack of BiP leading to many unfolded proteins

Ire1 dimeriyes and cleaves Hac1 mRNA
Hac1 mRNA becomes functional and translated
Hac1-TF activates protein folding genes e.g. BiP

Question 16

Where are misfolded proteins degraded?

Answer 16

In the 26S cytoplasmic proteasome

Question 17

How are proteins sorted into mitochondria?

Answer 17

Tom 20 recogniyes the signal sequence of mitochondrial proteins. the protein is then pulled through Tom40 into Tim 23/17 channel. The matrix signal is cleaved by matrix peptidase. To be refolded and HSp

Question 18

How are proteins sorted into plastids?

Answer 18

Signal sequence binds at Toc160, the protein is unfolded by Hsp70. The Toc-75 ring complex is opened and the protein is translocated (GTP dependent). Then there is the ATP dependent formation of a transport intermediate and itneraction with the TIC complex. Pushing the protein through the pore complexes and cleavage of the transit sequence.
The protein is refolded in the stroma by Hsc complexes.

Question 19

Outline the biogenesis of peroxisomes.

Answer 19

Insertion of Pex3 into the sER membrane
Insertion of Pex16 into the sER membrane through farnesylation (CAAX-motif at the C-terminus)
Budding to form the preperoxisome
Pex3/16-mediated import of membrane proteins
Pex5/7 mediated import of matrix proteins -> mature peroxisome.

Question 20

What are the peroxisome target sequences?

Answer 20

PTS1 - YQSKL at the C terminal end

PTS2 - at N terminal end (rare)

Question 21

Outline PTS1 protein import

Answer 21

1. PTS1 recognition by Pex5
2. Prex 5 binds at the Pex14 receptor
3. Translocation (Pex2/10/12)
4. Receptor recycling
5. no removal of recognition sequence