Targeting of Proteins to the Organelles

Targeting of Proteins: Nucleus and Mitochondria

The Nucleus Central Dogma Functions

Proteins are made in cytoplasm-transported to other locations Central dogma: DNA--> RNA--> Protein Nucleus Cytoplasm nucleus mitochondria Rough endoplasmic reticulum (ER) Fig 13.1 animation

The Nucleus 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: DNA organization Chromatin Discrete DNA localization

The Nucleus: Nuclear Lamina Supportive mesh on the inside of the nuclear envelope Nuclear Matrix Provides a structured space

The Nucleus The Nuclear Envelope is a continuous membrane that forms a double bilayer Outer: Inner:

The Nucleus: Nuclear Pore Complex How do molecules get into the nucleus? Nuclear Pore complex

Nuclear Pore Complex Cytoplasmic Nucleoplasm Here is an electron micrograph of the NPC. Regulates movement of proteins between cytoplasm and nucleus

The Nucleus: Nuclear Pore Complex 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:

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?

Nuclear Import PLAYERS Cargo Proteins Import Proteins Ran Ran-GEF Ran-GAP

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 Transport G protein switches Two confirmations GTP-bound GDP-bound GTPase hydrolyzes GTP to GDP; slow enzyme Modifying Proteins Guanine 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

How are proteins targeted to the nucleus? Nuclear Transport Proteins Imported Proteins DNA replication DNA repair Transcription Unassembled ribosome Exported Proteins mRNA (bound to proteins) tRNA (bound to proteins) Assembled ribosome How are proteins targeted to the nucleus?

Signal Sequence: Cellular Address Barack Obama 1600 Pennsylvania Avenue Washington, D.C. 20003

Signal Sequences NLS

Signal Sequences Signal sequences direct the final protein destination Chemical properties of the amino acids in the sequence direct interactions

Signal Sequences These sequences are read by specific transport receptors K—Lysine R--Arginine

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 Undergraduate Read article on Ran in Nuclear Transport Graduate Read article on Tubular ER network

Problem Set 1 Undergraduates Problem Set 1 will be posted on Friday Due Feb 28th by