Protein Sorting

Question 1

Secretory pathway examples

Answer 1

Insulin, enzymes, hormones, neurotransmitters (need to get outside cell to cause effects)

Question 2

Protein sorting w/o signal

Answer 2

Stays in cytoplasm

Cytoplasmic pathway

Question 3

Signal for proteins that need to go to mitochondria

Answer 3

N terminal (NH2). AA’s form alpha helical structure and are recognized by receptors on mitochondria and they pull the protein in

Signals can either be on ends of protein or in the sequence itself.

When protein folds, the sequences come together to make a signal “patch”

Question 4

Signal for proteins that need to go to nucleus

Answer 4

Lots of lysine/argenine residues

Tells cell it’s nuclear protein

Question 5

Secretory pathway

Answer 5

Proteins are synthesized on ribosomes that are on the surface of RER. All of these proteins go into the lumen of the ER first, then other signals are added to tell proteins to go to their final destinations.

Question 6

What signal is used to tell proteins to go into lumen of mitochondria

Answer 6

KDEL

Question 7

Proteins with mannose-6-phosphate go to….

Answer 7

lysosomes

M6P was added on the cis part of golgi and there are receptors on the trans part and so they go to the trans part and they are put into vesicles and then into the lysosomes.

M6P is sorting signal for all soluble lysosomal proteins

Failure of adding M6P results in i-cell disease

Question 8

i-cell disease

Answer 8

Inclusion cell disease

Failure of adding M6P…These enzymes do not enter the lysosome and are released into the blood

Question 9

Proteins that are released outside cell use which signal?

Answer 9

Trp-rich domain

Question 10

Cytoplasmic pathway:

Answer 10

for proteins destined for cytosol, mitochondria, nucleus, and peroxisomes

Question 11

Secretory pathway:

Answer 11

Proteins are made on ribosomes that are on the Rough ER. They will go into lumen of ER first and then other signals are added to get them to their final destination

for proteins destined for ER, lysosomes, plasma membranes, or for secretion

Question 12

Where does translation begin and where does it end?

Answer 12

Begins on free ribosomes and ends on ribosomes sent to ER

Question 13

Protein signals are built into the protein

Answer 13

True

Question 14

Signal sequence for Mitochondrial

Answer 14

N-terminal hydrophobic α-helix signal peptide

NH2 (form alpha helical structure)

N terminal sequence w/ hydrophobic α-helix. This sequence helps them interact w/ chaperone proteins

Question 15

Signal sequence for Lysosomal proteins

Answer 15

Mannose 6-phosphate (M6P) (added in cis-part of golgi)

cis–>trans part

Failure of adding M6P–> I-cell disease (inclusion disease). These enzymes in lysosome do not enter lysosome but are released into the blood

Question 16

Signal sequence for Secretory proteins

Answer 16

Trp-rich

Released outside the cell

Question 17

Chaperone proteins

Answer 17

Help protect the linear structure of the proteins

e.g. Heat shock proteins 70 (HSP70). (type of chaperone)

Question 18

Mitochondrial Protein Import

Answer 18

TOM: transporter of outer membrane (mitochondrial membrane)

TIM: “ “ inner membrane (inner mitochondrial membrane)

Protein w/ mitochondrial targeting sequence goes from cytoplasm to mitochondrial membrane, TOM recognizes and then TIM will help if it needs to go further inside cell

If it goes into matrix, past both TOM and TIM, then peptidase that cleaves off the signal and traps protein in matrix

Question 19

Nuclear import

Answer 19

Imported via nuclear pores

Large proteins require nuclear localization signals

Four continuous basic residues (Lys and Arg)

Question 20

Nuclear fibrils on nuclear envelope

Answer 20

Protein interacts with fibrils and they push the protein into the pore and inside the nucleus

Question 21

Signal sequence for Membrane destined proteins

Answer 21

N-terminal apolar region

transporters

Question 22

Secretory Pathway

Answer 22

Every protein going through this pathway goes through ER

You need ER targeting signal peptide (at N terminus of protein)…Lys/Arg rich

Very hydrophobic sequence on C terminus of basic residues

• Signal recognition particle (SRP) (has RNA backbone)binds to ER-targeting signal and the ribosome during translation
• SRP wraps itself around ribosome-mRNA peptide complex, tethering it to ER membrane and halting translation temporarily
• Translation resumes when protein directed into the ER lumen
• Enzymes on luminal side cleave the signal to release the protein
• Undergoes additional post-translational modifications in ER and/or Golgi apparatus

Question 23

Signal Recognition Particle

Answer 23

Recognizes every protein that goes to ER. Protein with ER is being translated, as soon as it pops out of ribosome, it is recognized by SRP, so SRP binds and hugs it, and halts protein synthesis to make sure everything is ok, and so it brings whole assembly close to ER membrane, particle has a receptor in the ER membrane, so receptor recognizes SRP and when it binds there is a translocation channel on ER membrane that opens up and then ER protein goes through channel into lumen of ER and particle and receptor are released to do the process again

Question 24

Is protein folding easier for small or large proteins?

Answer 24

Small-can fold into native conformations spontaneously

Question 25

Chaperonins

Answer 25

HSP60

Have barrel shaped compartments that admit unfolded proteins and catalyze their folding in an ATP-dependent manner

Help large proteins

Question 26

Chaperonins vs chaperones

Answer 26

Chaperonins: HSP60

Chaperones: HSP70

Question 27

Proteolytic Cleavage

Answer 27

• Converts inactive forms to active enzymes (e.g. trypsinogen and chymotrypsinogen to trypsin and chymotrypsin)
• Converts nascent precursor proteins to mature ones (e.g. proinsulin to insulin)

Question 28

Post-translational Modifications

Answer 28

Covalent modifications

• Glycosylation: sugar attached
• Phosphorylation: kinases that add phosphate to activate/deactivate
• Disulfide bond formation: cystines will form the bond

Question 29

Kinase vs phosphotases

Answer 29

Kinases: stick phosphate on

Phosphotases: take it off

Question 30

Glycosylation forms

Answer 30

• O-linked: formed with the hydroxyl groups of Ser or Thr residues
• N-linked: always with the amino group (NH2) of Asn acid amide side chain (NH group on Asn that gets glycosylated)

Question 31

Protein glycosylation

Answer 31

N-glycosidic likage is the more common form

Core oligosaccharide residue (made on ER membrane) is made on a phosphorylated form of dolichol

Enzymes that sticks this oligo group on protein is called a glycosyltransferase protein

Different changes can be made to core residue to make different oligosaccharides on protein

Tunicamycin: inhibits the addition of the oligosaccharide chain

(Inhibits the initial step in making the core oligosaccharide attached to the phosphorylated form of dolichol)