Protein Trafficking

Question 1

The synthesis of all proteins begins where?

Answer 1

cytoplasmic ribosomes

Question 2

Where do nuclear-encoded proteins function?

Answer 2

-Cytoplasm
-Mitochondria
-Nucleus
^cytoplasmic mitochondrial and nuclear proteins –> cytoplasmic ribosomes

-Cell membrane
-Secreted from the cell
-Lysosomal
^Membrane, secretory and lysosomal proteins –> ER bound for completion thru N terminus signal

Question 3

What determines site of protein localization?

Answer 3

Information contained on primary structure of the protein

Question 4

Cytoplasmic protein signal

Answer 4

If cytoplasmic there is no signal

Question 5

Mitochondrial protein signal

Answer 5

30-50 aa pre-sequence with amphipathic character (hydrophobic and positively charged aa)

Question 6

Nuclear protein signal

Answer 6

K-K-K-R-K

Question 7

Membrane/secretory protein signal

Answer 7

20-30 AA signal sequence with core of hydrophobic amino acids preceded by an N terminal Methionine sometimes followed by + charged amino acids

Question 8

Lysosomal protein signal

Answer 8

mannose-6-phosphate

Question 9

Co-translational insertion in ER membrane

Answer 9

For secretory, membrane, lysosomal proteins

• as secretory proteins are being translated onto cytoplasmic ribosomes, translation is interrupted after the 1st 50-70 amino acids are added to growing chain
• SRP binds the signal peptide as it exits the ribosome
• ribosome-mRNA-SRP complex directed to the ER membrane where SRP binds to SRP receptor
• SRP and SRP receptor bind GTP leading to GTP hydrolysis and release of SRP from the complex leaving the mRNA-ribosome complex on the ER membrane
• ribosome associates with translocon
• protein synthesis resumes and the polypeptide chain is inserted through the channel co-translationally
• once polypeptide is inside ER membrane, signal peptidase cleaves it

Question 10

How are integral membrane proteins inserted into the ER membrane?

Answer 10

In the exact conformation that they will assume in the plasma membrane using a combination of hydrophobic start and stop sequences which either start or stop insertion into ER membrane

Question 11

What can happen to proteins inside the ER?

Answer 11

1) Folding by Hsp70

2) Glycosylation with N-linked glycans added on asparagine residues
Asn-X-Ser/Thr

3) Formation of disulfide bonds
4) Folding by peptidyl prolyl isomerase for proline containing proteins

Question 12

What does the Golgi do?

Answer 12

Bunch of sorting

• O-linked glycosylation of Ser/Thr residues
• trimming of N-linked glycans
• phosphorylation of sugar residues and sulfation of both tyrosine residues and some sugar chains

Question 13

Lysosomal Proteins

Answer 13

Lysosomal hydrolase enzymes are tagged with mannose-6-phosphate in the Golgi

1) GIcNAC phosphotransferase (in cis Golgi) and enzyme in trans golgi removes the GIcNAC residue masking the mannose-6-phosphate
2) A mannose-6-phosphate receptor in the trans Golgi segregates these proteins in vesicles which bud off and fuse with endosomes

Question 14

Lysosomal Storage Disease

Answer 14

I-cell Disease

Results from a defect in the GIcNAC phosphotransferase and the subsequent inability to direct lysosomal hydrolases to endosomes

Accumulation of lysosomal hydrolases in plasma as a result

Question 15

Origin of mitochondrial proteins

Answer 15

most proteins encodes in the nuclear genomes and not mitochondrial genomes –> proteins are translated on cytoplasmic ribosomes and inserted into the mitochondrial membrane where translation is completed

Question 16

Pre-sequence in mitochondrial proteins

Answer 16

Longer than signal peptide and has amphipathic character- helix with hydrophobic AA on one side and charged on the other

Question 17

Folding of mitochondrial proteins

Answer 17

Most proteins targeted to the matrix

Proteins are maintained in an unfolded state in the cytoplasm, assisted by the binding of chaperone proteins Hsp70

Mitochondrial targeting sequence interacts with outer membrane receptor TOM 20/22

Binds to TOM40, the import channel

Once inside the matrix, targeting sequence is cleaved and the protein associates with several mitochondrial chaperone proteins which lets it assume its native conformation

Question 18

Importance of proton motive force

Answer 18

Proton motive force is required for entry of proteins into the mitochondria

Membrane potential across the inner mitochondrial membrane aids in the insertion of the + charged residues in presequence

Question 19

NPC

Answer 19

Nuclear pore complex

Form connection between nucleus and cytosol

Question 20

Nups

Answer 20

Nucleoproteins (type of NPC)
8 Nups form a ring-like structure through which molecules can pass thru

Also stabilize the complex and facilitate transport through the pore

Question 21

transmembrane Nups

Answer 21

anchor NPC in the nuclear envelope

Question 22

structural Nups

Answer 22

stabilize the nuclear envelope at nuclear pores and provide scaffolding for assembling other peripheral Nups

Question 23

FG Nups

Answer 23

Phenlyalanine-Glycine Nups
Form diffusion barrier and bind transport receptors

FG repeats line the pore and also create basket into cytosol –> restrict diffusion of larger moelcules (>40kDa)

Question 24

NLS

Answer 24

Nuclear localization signal

Proteins with KKKRG bind to cytoplasmic importins

Question 25

Molecules

Answer 25

Diffusion

sometimes small proteins require active transport

Question 26

Import into the Nucleus process

Answer 26

NLS on molecules is recognized by cytosolic proteins importins

Molecule bound importin also bind the F Nups and the filaments that extend into the cytosol from the NPC

Importins repeatedly bind dissociate and bind to F repeats to facilitate movement thru pore

Once inside, importin releases and moves back into cytoplasm

Question 27

Export out of Nucleus

Answer 27

Nuclear export signal on proteins to be moved out are bound to nuclear export receptors (exportins)

Question 28

Nuclear Lamina

Answer 28

Thick layer of fibrous proteins (intermediate protein)

Function:

• stabilize nuclear structure
• anchoring nuclear pore complexes