Protein Targeting Flashcards
process of making type-II single-pass proteins
BIP
chaperone that keeps protein chain from folding
where do secretory proteins go shortly after synthesis?
secretory proteins are localized in the ER lumen shortly after synthesis
name of guy responsible for understanding secretory pathway
Blobel
what if a protein is going to mitochondria somewhere other than the matrix?
most mitochondrial proteins go to the matrix, but if a protein is going elsewhere then it will first go to the matrix and then to its destination from the matrix
location of N-term and C-term in single-pass transmembrane protein
N-term: ER lumen
C-term: cytosol/cytoplasm
hydrophobic regions in secretory and single pass proteins
- signal peptide
- stop-transfer sequence
- signal-anchor sequence
NLS
nuclear localization signal; signal that is put on in the middle of a protein to indicate that the protein belongs in the nucleus
specificity of importin
importin is not cargo specific
process of making single-pass transmembrane protein
page 3 notes
how are sugar moieties added onto proteins in the golgi?
there are different enzymes for different sugars, and there are different enzymes in each cisterna so sugars getted added in in different cisternae of the golgi complex
protein synthesis in nucleus
all proteins that are found in the nucleus are synthesized in the cytoplasm and transported to the nucleus via nuclear pore complexes
experiment: requirement of cytosolic proteins for nuclear transport
- treatment with digitonin (detergent) makes the plasma membrane permeable such that the cytosolic constituents leak out but leave the nuclear envelope and pore intact
- accumulation of transport substrate in nucleus occurred only when cytosol was included in the incubation
trans-face of golgi also called:
leaving face
In cotranslational translocation, what is the source of energy that drives protein into the ER lumen?
the energy comes from the translation process – translation drives the process
Hydrophobicity Plot
evaluates moving averages of hydrophobicity of sections
process: endocytic pathway for internalizing LDL
- cell-surfcae LDL receptors bind to apoB protein which is embedded into the phospholipid outer layer of LDL
- cell-surface receptors located in Clathrin-coated pit
- clathrin-coated pits with receptor LDL complex pinch off of plasma membrane to become coated vesicle
- vesicle coat sheds off; early endosome fuses with late endosome
- pH difference in late endosome causes receptor to release LDL
- late endosome fuses with lysosome
- LDL receptor recycled to cell srface; neutral pH of exterior returns to receptor to active conformational change
function of rough ER
synthesis of secreted proteins and post-translational modification
importance of NLS considering the cell cycle
in the cell cycle the nuclear envelope breaks down in Prophase and releases nuclear proteins; it is important that these proteins have NLS to indicate that they should go back into the nucleus
process: nuclear import of protein
- importin binds to NLS of a cargo protein in the cytoplasm
- importin-protein complex diffuses through the nuclear pore complex (NPC) via interaction of F-G repeats
- in nucleoplasm, Ran-GTP binds importin –> this causes a conformational change so import releases protein
- import-RanGTP complex diffuses through NPC to cytoplasm
- Ran-GAP (GTPase activating protein) hydrolyzes bound GTP of Ran-GTP to GDP –> conformational change makes release of importin
- cycle repeats
SRP functions
- binds to signal peptide
- arrest translation
viruses and NPCs
viruses can take advantage of NPCs to use cell nucleus components (dangerous!)
how does a ribosome get recruited to the ER membrane?
there is an ER signal sequence on the translated protein
how does a vesicle fuse with its target membrane?
after being released and shedding its coat, a vesicle fuses with its target membrane using interaction of SNARE proteins
import of proteins into mitochondria: cotranslational or post-translational?
post-translational
experiment that showed post-translational import to mitochondria
- yeast mitochondrial proteins made by cytoplasmic ribosomes in cell-free environment
- add mitochondria and sample without mitochondria
- add trypsin – sample without mitochondria led to degraded proteins and sample with mitochondria were protected
clathrin
- coat protein that is involved in the making of vesicles
- collectively multiple clathrin cause the formation of a depression that deforms a membrane allowing formation of a vesicle
- spherical in shape
Fuctions of ER and Golgi
- post-translational modification (such as adding GPI anchor)
- putting sugar moieties onto proteins
difference between membrane-bound and cytosolic ribosomes
membrane-bound and cytosolic ribosomes are the same – ribosomes are recruited to the ER during protein synthesis when there’s an ER signal sequence
what is cotranslational translocation?
normal translation at first but as protein is translated it is guided into the ER lumen
SRP acronym
signal recognition particle
Experiment: in vitro translation of secretory protein in the absence and presence of microsomes
no microsomes present
- no incorporation into microsomes
microsomes present
- mature protein made its way into microsome and signal sequence was not present
conclusion
- during translation there is cotranslational transport of protein into the microsome and removal of the signal sequence
Process: protein import into the mitochondrial matrix
- proteins synthesized on cytosolic ribosomes; kept unfolded via chaperones (Hsc70)
- precursor protein binds to import receptor
- protein transferred to general import pore
- protein moves through TOM and TIM
- in matrix, protein binds Hsc70 which helps move protein along
- uptake-targeting sequence removed by matrix protease and Hsc70 released
- protein folds into mature, active conformation within matrix
cis-face of golgi also called:
forming face
GPI anchor
anchors proteins to extracellular face
experiment that found that secretory proteins are in microsomes
labeled secretory proteins in rough ER homogenized microsomes with attached ribsomes
added detergent and protease
- protein degraded
added only protease
- protein safe – protease couldn’t get to the protein because it was in a microsome
nuclear pore transport rate
60,000 proteins transferred per minute per pore
describe the ER signal sequence
- N-term
- hydrophobic
- gets cut off in the microsomal fraction (ER)
experiment demonstrating significance of NLS for nuclear protein transport
NLS was added to a protein that’s normally found in the cytoplasm; the protein was imported into the nucleus
import of peroxisomal matrix proteins
- c-term uptake-targeting sequence binds to cytosolic receptor Pex5
- Pex5 forms complex with Pex14 receptor which is on the peroxisome membrane
- Pex5 with protein transferred into peroxisomal matrix then Pex5 dissociates and goes back to cytosol
all type-I transmembrane proteins possess:
- N-term signal sequence that targets them to ER
- internal hydrophobic sequence –> becomes the membrane-spanning alpha-helix
how many flattened sacs do golgi typically have?
5-6
process of cotranslational translocation
- ER signal sequence translated; emerges from ribosome
- SRP (signal recognition peptide) binds signal sequence
- SRP delivers ribosome and polypeptide to SRP receptor on ER membrane
- GTP helps bind/hold these
- ribsome/polypeptide connect to translocon; translocon opens
- polypeptide with signal sequence pushed into central pore
- SRP and SRP receptor dissociate from translocon and hydrolyze their bound GTP to GDO
- as polypeptide elongates, passes through translocon into the ER lumen; signal sequence cleaved by signal peptidase and degraded
- when traslation complete: ribosome released, rest of protein drawn into ER lumen, translocon closes, protein assumes native conformation
post-translational translocation
- translocating polypeptide chain to translocon; signal sequence cleaved
- protein chain moves up and down randomly –> BIP binds to chain on down movement (then can’t backtrack)
Type IV transmembrane proteins
there are multiple transmembrane portions and each has a stop-transfer sequence
two notable features of process of peroxisomal matrix protein import
- signal sequence is left on the protein
- signal sequence is on the C-term
organelles that protein passes through in secretory pathway
ER –> Golgi –> plasma membrane
orientation of protein in type-II single-pass proteins
C-term is located in the ER lumen (orientation is reversed by type I)
signal peptide for keeping protein in the ER
KDEL
GTP-ase ARF and coat vesicles
GTPase ARF involved in the formation of coat proteins (whether assembling or disassembling)
sorting signal for protein to be taken to lysosome
mannose-6-phosphate
how a cell knows to stop dividing
integrin can sense the surroundings of a cell; can sense if a dish is confluent
receptor that can sense mechanical force
integrin receptor
