Nuclear Import/Export L3 â Protein Targeting L4 - Secretory Pathway
L2 – Nuclear Import/Export Nuclear Localisation Sequence (NLS) - positively charged amino acids (Lys/Arg) Importins Nuclear import receptors (importins/karyopherins) - recognise NLS and carry through NPC Importin α - recognise NLS Importin β - recognise NLS, mediate entry through NPC, low sequence homology but structural similarity to RanGTP (superhelical structure), intrinsic flexibility RanGTP (Ras-related small G-protein) Binds importin β but not importin α RanGTP high in nucleus – binding triggers cargo release Exportins Binding of RanGTP to export – dissociation of Ran-GDP releases cargo in cytosol Interact with NES (nuclear export sequence) - hydrophobic amino acids (Leu/Ile) Leptomycin B – block NES-exportin interaction
L3 – Protein Targeting ER importing of proteins – co-translational Signal sequences – one or more positively charged preceding hydrophobic sequence, relatively polar sequence near carboxyl terminus, short side chains near cleavage site Signal sequence can be transmembrane domain Signal recognition particle (SRP) Six protein subunits, one RNA molecule GTP-binding protein Binding of SRP to signal peptide causes pause in translation – targets to protein translocator, SRP and SRP receptor are released and translation/translocation begins, signal peptidase cleaves signal sequence SRP and SRP receptor interaction strengthened by binding of GTP – both have GTP binding sites Translocator Sec61 – water filled pore in membrane Trimer Plug ensures no unwanted flow Can open laterally to release transmembrane segments into membrane
L4 - Secretory Pathway Vesicles Coat must disassemble to allow vesicle to interact with next membrane COPII - ER to Golgi, arise from smooth ER, fuse to form vesicular tubular clusters (intermediate compartment), bind to motor protein attached to microtubule to move towards Golgi network COPI - bud off to carry ER proteins that have escaped and involved in ER budding (retrieval/retrograde pathway) Clathrin - one type for late Golgi, one type for cell surface
L3 – Protein Targeting ER importing of proteins – co-translational Signal sequences – one or more positively charged preceding hydrophobic sequence, relatively polar sequence near carboxyl terminus, short side chains near cleavage site Signal sequence can be transmembrane domain Signal recognition particle (SRP) Six protein subunits, one RNA molecule GTP-binding protein Binding of SRP to signal peptide causes pause in translation – targets to protein translocator, SRP and SRP receptor are released and translation/translocation begins, signal peptidase cleaves signal sequence SRP and SRP receptor interaction strengthened by binding of GTP – both have GTP binding sites Translocator Sec61 – water filled pore in membrane Trimer Plug ensures no unwanted flow Can open laterally to release transmembrane segments into membrane
L4 - Secretory Pathway Vesicles Coat must disassemble to allow vesicle to interact with next membrane COPII - ER to Golgi, arise from smooth ER, fuse to form vesicular tubular clusters (intermediate compartment), bind to motor protein attached to microtubule to move towards Golgi network COPI - bud off to carry ER proteins that have escaped and involved in ER budding (retrieval/retrograde pathway) Clathrin - one type for late Golgi, one type for cell surface
