Protein Trafficking Flashcards
The synthesis of all proteins begins where?
cytoplasmic ribosomes
Where do nuclear-encoded proteins function?
-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
What determines site of protein localization?
Information contained on primary structure of the protein
Cytoplasmic protein signal
If cytoplasmic there is no signal
Mitochondrial protein signal
30-50 aa pre-sequence with amphipathic character (hydrophobic and positively charged aa)
Nuclear protein signal
K-K-K-R-K
Membrane/secretory protein signal
20-30 AA signal sequence with core of hydrophobic amino acids preceded by an N terminal Methionine sometimes followed by + charged amino acids
Lysosomal protein signal
mannose-6-phosphate
Co-translational insertion in ER membrane
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
How are integral membrane proteins inserted into the ER membrane?
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
What can happen to proteins inside the ER?
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
What does the Golgi do?
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
Lysosomal Proteins
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
Lysosomal Storage Disease
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
Origin of mitochondrial proteins
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
Pre-sequence in mitochondrial proteins
Longer than signal peptide and has amphipathic character- helix with hydrophobic AA on one side and charged on the other
Folding of mitochondrial proteins
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
Importance of proton motive force
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
NPC
Nuclear pore complex
Form connection between nucleus and cytosol
Nups
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
transmembrane Nups
anchor NPC in the nuclear envelope
structural Nups
stabilize the nuclear envelope at nuclear pores and provide scaffolding for assembling other peripheral Nups
FG Nups
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)
NLS
Nuclear localization signal
Proteins with KKKRG bind to cytoplasmic importins
Molecules
Diffusion
sometimes small proteins require active transport
Import into the Nucleus process
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
Export out of Nucleus
Nuclear export signal on proteins to be moved out are bound to nuclear export receptors (exportins)
Nuclear Lamina
Thick layer of fibrous proteins (intermediate protein)
Function:
- stabilize nuclear structure
- anchoring nuclear pore complexes
