Targeting of Proteins to the Organelles. Targeting of Proteins: Nucleus and Mitochondria.
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Transcript of Targeting of Proteins to the Organelles. Targeting of Proteins: Nucleus and Mitochondria.
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Targeting of Proteins to the Organelles
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Targeting of Proteins: Nucleus and Mitochondria
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• Central Dogma
• Functions
The Nucleus
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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
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Structures
Nuclear Envelope
Nuclear Lamina
Nuclear Matrix
Chromosomal Domains
Nuclear Pore Complex
Houses DNA
Organized
Regulated movements
The Nucleus
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• 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
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• Chromatin
• Discrete DNA localization
The Nucleus:DNA organization
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• Supportive mesh on the inside of the nuclear envelope
• Nuclear Matrix
• Provides a structured space
The Nucleus:Nuclear Lamina
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• The Nuclear Envelope is a continuous membrane that forms a double bilayer
• Outer:
• Inner:
The Nucleus
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• How do molecules get into the nucleus?• Nuclear Pore complex
The Nucleus:Nuclear Pore Complex
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Nuclear Pore ComplexCytoplasmic Nucleoplasm
Regulates movement of proteins between cytoplasm and nucleus
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• The NPC is one of the largest protein complexes in the cell
• Composed of:
• Limited diffusion:
• Membrane bound:
The Nucleus:Nuclear Pore Complex
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• Basket portion on the nucleoplasm side is joined by filaments
The Nucleus:Nuclear Pore Complex
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– Center of the nuclear pore is aqueous
– Imported proteins move through the NPC via a brownian gate model
The Nucleus:Nuclear Pore Complex
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– Some channel and basket nucleoporins form hydrophobic stretches
– Structural Nups:
The Nucleus:Nuclear Pore Complex
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NPC Structure
Cytoplasmic Nups
Symmetric Nups
Nuclear Nups
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Nuclear Import and Export
• Many proteins are too large to pass through the NPC alone
• How does this occur?
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PLAYERSCargo Proteins
Import Proteins
Ran
Ran-GEFRan-GAP
Nuclear Import
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Nuclear Import
• Importins• Exportins• FG repeats
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Nuclear Import• Importins can form a heterodimeric nuclear
import receptor
• Ran
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Nuclear Import
• Free Importin binds to NLS in the cytoplasm
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Nuclear Import
• Upon entering the nucleus, importin interacts with Ran-GTP
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Nuclear Import• Ran-GTP/Importin
complex then diffuses out of the nucleus
• Ran-GAP
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Nuclear Import
• Ran GDP returns to nucleus through NPC
• Ran-GEF
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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
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Nuclear Import
• Diffusion through the pores is random, but transport is directional
• How is this achieved?
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Nuclear Export
• Exportin binds to cargo
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Nuclear Export• Ran-GTP makes contact
with Ran-GAP in cytoplasm
• Complex dissociates
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Nuclear Import and Export
• Both use Ran-GTP– Import:
– Export:
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Nuclear Import
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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?
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Signal Sequence: Cellular Address
Barack Obama1600 Pennsylvania AvenueWashington, D.C. 20003
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Signal Sequences
NLS
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Signal Sequences
• Signal sequences direct the final protein destination
• Chemical properties of the amino acids in the sequence direct interactions
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• These sequences are read by specific transport receptors
Signal Sequences
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Nuclear Localization Signal
• Nuclear localization signal– Pro-Lys-Lys-Lys-Arg-Lys-Val
• Digitonin
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Nuclear Export Signal
• Used to transport proteins, tRNA, ribosomal subunits out of the nucleus
• Exportins bind the NES to start the process
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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
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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
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Mitochondria
• Mitochondria harness energy
• Glycolysis (pyruvate)
• Generate ATP
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Mitochondria• cellular respiration
– “Breathing” on a cellular level
• Occurs in the inner membrane and the inner membrane space– membrane surface area
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Electron Transport Chain
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Mitochondria• Matrix contains all necessary components for
mitochondrial replication
• Mix of mitochondrial encoded proteins and those from the nuclear genome
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Signal Sequences
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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
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Mitochondrial Protein Transport
• Transport of proteins in to the mitochondria requires:
• Transport occurs:
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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
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Mitochondrial Protein Transport
• Chaperones (HSC 70) keep proteins unfolded
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Mitochondrial Protein Transport
• Precursor binds to the import receptor on the outer membrane
• Import receptors: Tom 20/22
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Mitochondrial Protein Transport• Transport to the matrix
occurs simultaneously• Tim23/17 mediate transport
to the matrix
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Juxtaposition of Inner and Outer Membranes at Transport Sites
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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
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Mitochondrial Transport Movie
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Transport of Mitochondrial Membrane Proteins
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Stop-Transfer
Players
Tom20/22
Tom40
Tim23/17
Tim44
Hsc70
Matrix Protease
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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
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Path B• Proteins contain a matrix targeting
sequence and internal hydrophobic domains
• Oxa1• Tom40, Tim23/17 are involved in
transport
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Path C• Proteins do not contain a
matrix targeting sequence
• Recognized by the Tom70/22 complex
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Intermembrane Space Localization
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Path A• Major Pathway
• Proteins contain a matrix targeting sequence
• Translocation to the matrix begins
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Path B
• Some proteins can just move through Tom40
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Outermembrane proteins
• Like Tom40 itself• Proteins interact with Tom40• Then transferred to the SAM complex (sorting
and assembly machinery)
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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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