Protein sorting
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where can proteins go once synthesised in the cytosol?
remain in cytosol nucleus ER for trafficking mitochondrion
how are proteins labelled as to where they should go?
sorting signals-specific sequences of amino acids (table on pwp)
sorting signals for nuclear targeting
one or more stretched of positively charged amino acids, lysine, arginine situated on the surface of the protein once it has folded recognised by nuclear import receptors
structure of nuclear pores
each pore is composed of a large number of protein subunits. fibrils protrude from both sides of the complex.
nuclear import receptors
recognise sorting sequence travel down fibres towards nucleus
how do macromolecules get into the nucleus
active transport through nuclear pores
process of protein being imported into nucleus
nuclear import receptors recognise nuclear localisation sequences on prospective nuclear protein. complex of receptor and protein cargo binds to fibrils on cytoplasmic side and is guided towards nuclear pore. binding of nuclear protein to pore opens the pore and the protein receptor complex is actively transported into the nucleus
Ran GTPase
exists in GTP or GDP bound state
Ran-GAP, GTPase activating protein
protein triggering GTP hydrolysis in cytosol converts Ran-GTP to Ran-GDP
Ran-GEF, guanine nucleotide exchange factor
protein causing Ran-GDP to release GDP and take up GTP in nucleus
nuclear import and export
Ran-GEF converts Ran GDP to GTP Ran-GTP binds to import receptors to cause it to release cargo protein receptor-Ran-GTP complex transported back to cytoplasm where Ran-GAP hydrolyses Ran-GTP to Ran-GDP, loses affinity for receptor. receptor can now bind more cargo
where does the energy come form that is needed to drive nuclear import and export
hydrolysis of GTP
mitochondrial targeting sequences
always at N terminus (gets produced first) amphipathic alpha helical sequences-positively charged residues lie on one face of the secondary structure
recognition of mitochondrial targeting sequences
receptors on mitochondrial outer membrane recognise mitochondrial targeting sequences
how do proteins cross the two mitochondrial membranes
TOM: translocator of outer membrane, channel TIM: inner membrane
Hsp70
cytoplasmic chaperone protein prevent protein folding for proteins that are destined for the mitochondria also prevents the protein going back up channel
how do unfolded proteins get through the TOM and TIM
positive residues are attracted to the negatively charged inner membrane energy from hydrolysis of ATP causes chaperones to release protein
targeting to the ER
hydrophobic signal sequence, often at N terminus usually have positive charged N-terminus and a cleavage sequence (alanine)
what recognises the ER targeting sequence as it emerges from the ribosome
ribonucleoprotein complex (RNA and proteins) known as SRP: signal recognition particle. binding of SRP to signal sequence and ribosome halts translation
how ER destined proteins get into the lumen
complex of ribosome, nascent protein chain and SRP binds to SRP-receptor which guides the complex to a translocon. both SRP and SRP receptor hydrolyse their associated GTPs, changing their conformations. SRP is released (loses affinity) and translation resumes. extension of polypeptide chain pushes it through into the ER lumen. the protein is unfolded as this occurs A nascent protein chain-newly synthesized protein, not yet folded
signal sequence of ER proteins once in lumen
signal peptidase in ER lumen cleaves the signal sequence creates new N terminus protein can now fold properly
transmembrane proteins
signal sequence not cleaved signal-anchor sequences longer hydrophobic stretches of amino acids and no signal peptidase cleavage sequence