Targeting of Proteins to the Organelles
Targeting of Proteins: Nucleus and Mitochondria
• Central Dogma
• Functions
The Nucleus
Central dogma:
DNA--> RNA--> Protein
Nucleus
Cytoplasm
Proteins are made in cytoplasm-transported to other locations
nucleus mitochondria Rough endoplasmic reticulum (ER)
Fig 13.1 animation
Structures
Nuclear Envelope
Nuclear Lamina
Nuclear Matrix
Chromosomal Domains
Nuclear Pore Complex
Houses DNA
Organized
Regulated movements
The Nucleus
• Nucleus holds DNA
• Keeps DNA organized throughout the cell life cycle
• The nucleus is organized similar to a mini-cell within a cell
The Nucleus
• Chromatin
• Discrete DNA localization
The Nucleus:DNA organization
• Supportive mesh on the inside of the nuclear envelope
• Nuclear Matrix
• Provides a structured space
The Nucleus:Nuclear Lamina
• The Nuclear Envelope is a continuous membrane that forms a double bilayer
• Outer:
• Inner:
The Nucleus
• How do molecules get into the nucleus?• Nuclear Pore complex
The Nucleus:Nuclear Pore Complex
Nuclear Pore ComplexCytoplasmic Nucleoplasm
Regulates movement of proteins between cytoplasm and nucleus
• The NPC is one of the largest protein complexes in the cell
• Composed of:
• Limited diffusion:
• Membrane bound:
The Nucleus:Nuclear Pore Complex
• Basket portion on the nucleoplasm side is joined by filaments
The Nucleus:Nuclear Pore Complex
– Center of the nuclear pore is aqueous
– Imported proteins move through the NPC via a brownian gate model
The Nucleus:Nuclear Pore Complex
– Some channel and basket nucleoporins form hydrophobic stretches
– Structural Nups:
The Nucleus:Nuclear Pore Complex
NPC Structure
Cytoplasmic Nups
Symmetric Nups
Nuclear Nups
Nuclear Import and Export
• Many proteins are too large to pass through the NPC alone
• How does this occur?
PLAYERSCargo Proteins
Import Proteins
Ran
Ran-GEFRan-GAP
Nuclear Import
Nuclear Import
• Importins• Exportins• FG repeats
Nuclear Import• Importins can form a heterodimeric nuclear
import receptor
• Ran
Nuclear Import
• Free Importin binds to NLS in the cytoplasm
Nuclear Import
• Upon entering the nucleus, importin interacts with Ran-GTP
Nuclear Import• Ran-GTP/Importin
complex then diffuses out of the nucleus
• Ran-GAP
Nuclear Import
• Ran GDP returns to nucleus through NPC
• Ran-GEF
Ran: GTPase in Nuclear TransportG protein switchesTwo confirmations GTP-bound GDP-bound
GTPase hydrolyzes GTP to GDP; slow enzyme
Modifying ProteinsGuanine nucleotide exchange factors (GEF)GDP GTP Nuclear
GTPase Activating Protein (GAP) ***this protein is not a GTPase!!GTP GDP Cytoplasmic
Nuclear Import
• Diffusion through the pores is random, but transport is directional
• How is this achieved?
Nuclear Export
• Exportin binds to cargo
Nuclear Export• Ran-GTP makes contact
with Ran-GAP in cytoplasm
• Complex dissociates
Nuclear Import and Export
• Both use Ran-GTP– Import:
– Export:
Nuclear Import
Nuclear Transport Proteins
Imported ProteinsDNA replicationDNA repairTranscriptionUnassembled ribosome
Exported ProteinsmRNA (bound to proteins)tRNA (bound to proteins)Assembled ribosome
How are proteins targeted to the nucleus?
Signal Sequence: Cellular Address
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Signal Sequences
NLS
Signal Sequences
• Signal sequences direct the final protein destination
• Chemical properties of the amino acids in the sequence direct interactions
• These sequences are read by specific transport receptors
Signal Sequences
Nuclear Localization Signal
• Nuclear localization signal– Pro-Lys-Lys-Lys-Arg-Lys-Val
• Digitonin
Nuclear Export Signal
• Used to transport proteins, tRNA, ribosomal subunits out of the nucleus
• Exportins bind the NES to start the process
Mitochondria
Power house of cell Outer membrane• simple bilayer • permeable to ions and small molecules
Inner membrane• IMPERMEABLE to all material except through carrier channels• Complex in conformation• Contains all electron transport chain machinery• Contains all ATP synthesis machinery
Mitochondria
Power house of cell• Intermembrane space• Complex shape (follows
contours of inner membrane• pH ~7
• Matrix• all enzymes required for Krebs
cycle• Hold mitochondrial genome,
ribosomes, enzymes for mitochondrial division
• pH ~8
Mitochondria
• Mitochondria harness energy
• Glycolysis (pyruvate)
• Generate ATP
Mitochondria• cellular respiration
– “Breathing” on a cellular level
• Occurs in the inner membrane and the inner membrane space– membrane surface area
Electron Transport Chain
Mitochondria• Matrix contains all necessary components for
mitochondrial replication
• Mix of mitochondrial encoded proteins and those from the nuclear genome
Signal Sequences
Mitochondrial targeting sequences
• The signal for matrix mitochondrial proteins is part sequence, but also part structure
• Amphipathic
• Receptors that bind the sequences can bind to more than one specific sequence
Mitochondrial Protein Transport
• Transport of proteins in to the mitochondria requires:
• Transport occurs:
Mitochondrial Protein Transport
Players
Hsc70 Chaperones
Outer membrane Translocon
Outer membrane receptors- Tom 20/22 or Tom 70/22
Outer membrane channel-Tom 40
Inner membrane Translocon
Inner membrane channel- Tim 23/17
Matrix Proteases
Mitochondrial Protein Transport
• Chaperones (HSC 70) keep proteins unfolded
Mitochondrial Protein Transport
• Precursor binds to the import receptor on the outer membrane
• Import receptors: Tom 20/22
Mitochondrial Protein Transport• Transport to the matrix
occurs simultaneously• Tim23/17 mediate transport
to the matrix
Juxtaposition of Inner and Outer Membranes at Transport Sites
Mitochondrial Protein Transport
Cytosolic Hsc70 keeps precursor Protein in partially unfolded state
Tom20/22 Receptor recognized matrix-targeting sequence
Transfer of Precursor through Tom40 channel
Passes through Tim23/17 channel
Matrix Hsc70 helps pull precursor through channel
Matrix protease cleaves off signal sequence
Mitochondrial Transport Movie
Transport of Mitochondrial Membrane Proteins
Stop-Transfer
Players
Tom20/22
Tom40
Tim23/17
Tim44
Hsc70
Matrix Protease
Stop-Transfer
Process same as with Matrix-targeted protein
Have Matrix targeting sequence
BUT
Internal Stop-Transfer sequence recognized by Tim23/17 channel
Sequence is hydrophobic
Precursor is transferred into the inner membrane bilayer
Path B• Proteins contain a matrix targeting
sequence and internal hydrophobic domains
• Oxa1• Tom40, Tim23/17 are involved in
transport
Path C• Proteins do not contain a
matrix targeting sequence
• Recognized by the Tom70/22 complex
Intermembrane Space Localization
Path A• Major Pathway
• Proteins contain a matrix targeting sequence
• Translocation to the matrix begins
Path B
• Some proteins can just move through Tom40
Outermembrane proteins
• Like Tom40 itself• Proteins interact with Tom40• Then transferred to the SAM complex (sorting
and assembly machinery)
Section assignments for next week
UndergraduateRead article on Ran in Nuclear Transport
GraduateRead article on Tubular ER network
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Problem Set 1
Undergraduates
Problem Set 1 will be posted on Friday
Due Feb 28th by
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