NUCLEAR PORE COMPLEX (NPC)
• Gate for molecules to enter or exit the nucleus
• Molecules for import: DNA polymerases, RNA polymerases, histones and transcription factors (transcription takes place in nucleus)
• Molecules for export: mRNA, tRNA and ribosomal subunits (protein synthesis takes place in cytoplasm)
• Passive diffusion: molecules < 20-40 kD pass freely through NPC's
• Facilitated diffusion: molecules > 20-40 kD and RNA are transported selectively using signal dependent processes
• Key features: nuclear and cytoplasmic rings, cytoplasmic fibrils and nuclear basket
Nuclear Transport
KEY COMPONENTS OF TRANSPORT
• Peptide motifs: NLS (nuclear localization signals) and NES (nuclear export signals)
• Nuclear transport receptors: importins and exportins
• Ran GTPase (in cytosol and nucleus; function in import & export)
RAN GTP-ASE MECHANISM
• Ran-GTP = ON; Ran-GDP = OFF
• Ran-GTPase hydrolyzes GTP to GDP and phosphate (it turns itself off)
• Nuclear import: RAN-GTP stimulates cargo release in nucleus
• Nuclear export: Ran-GTP stimulates cargo binding in nucleus
• GEFs (guanine nucleotide exchange factors): enzymes that promote exchange of GDP for GTP
• GAPS (GTPase activating proteins): promote Ran-GTP hydrolytic activity
• Ran-GTP and GEFs are localized in nucleus
• Ran-GDP and GAPs are localized on cytoplasmic side
• Ran-GTP gradient produces directionality in nuclear transport
NUCLEAR IMPORT
1. Importin binds NLS on cargo protein
2. Docking on cytoplasmic fibril
3. Importin transports cargo through NPC
4. Importin releases cargo in nucleus upon Ran-GTP binding
5. Improtin-Ran-GTP complex reenters cytoplasm; hydrolysis occurs and Ran-GDP dissociates from importin
NUCLEAR EXPORT
1. Ran-GTP promotes cargo-exportin binding in nucleus
2. Exportin transports its cargo into the cytosol
3. Hydrolysis occurs and transport complex dissociates (releasing Ran-GDP and phosphate)