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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