Protein Sorting Flashcards
Secretory pathway examples
Insulin, enzymes, hormones, neurotransmitters (need to get outside cell to cause effects)
Protein sorting w/o signal
Stays in cytoplasm
Cytoplasmic pathway
Signal for proteins that need to go to mitochondria
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”
Signal for proteins that need to go to nucleus
Lots of lysine/argenine residues
Tells cell it’s nuclear protein
Secretory pathway
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.
What signal is used to tell proteins to go into lumen of mitochondria
KDEL
Proteins with mannose-6-phosphate go to….
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
i-cell disease
Inclusion cell disease
Failure of adding M6P…These enzymes do not enter the lysosome and are released into the blood
Proteins that are released outside cell use which signal?
Trp-rich domain
Cytoplasmic pathway:
for proteins destined for cytosol, mitochondria, nucleus, and peroxisomes
Secretory pathway:
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
Where does translation begin and where does it end?
Begins on free ribosomes and ends on ribosomes sent to ER
Protein signals are built into the protein
True
Signal sequence for Mitochondrial
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
Signal sequence for Lysosomal proteins
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
Signal sequence for Secretory proteins
Trp-rich
Released outside the cell
Chaperone proteins
Help protect the linear structure of the proteins
e.g. Heat shock proteins 70 (HSP70). (type of chaperone)
Mitochondrial Protein Import
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
Nuclear import
Imported via nuclear pores
Large proteins require nuclear localization signals
Four continuous basic residues (Lys and Arg)
Nuclear fibrils on nuclear envelope
Protein interacts with fibrils and they push the protein into the pore and inside the nucleus
Signal sequence for Membrane destined proteins
N-terminal apolar region
transporters
Secretory Pathway
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
Signal Recognition Particle
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
Is protein folding easier for small or large proteins?
Small-can fold into native conformations spontaneously
Chaperonins
HSP60
Have barrel shaped compartments that admit unfolded proteins and catalyze their folding in an ATP-dependent manner
Help large proteins
Chaperonins vs chaperones
Chaperonins: HSP60
Chaperones: HSP70
Proteolytic Cleavage
- 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)
Post-translational Modifications
Covalent modifications
- Glycosylation: sugar attached
- Phosphorylation: kinases that add phosphate to activate/deactivate
- Disulfide bond formation: cystines will form the bond
Kinase vs phosphotases
Kinases: stick phosphate on
Phosphotases: take it off
Glycosylation forms
- 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)
Protein glycosylation
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)
