MCB 2210 L12: Protein Targeting - Nucleus Part 1

Slides 1-32

Protein Targeting

Biological process cells use to delivery newly made proteins to their correct, functional location to carry out specific functions within cell.

How do proteins get localized to different places in the cell?

Components of organelles in eukaryotic cells

• Organelle = compartment within eukaryotic cell

  • Ex. Nucleus, Golgi apparatus, ER, lysosomes, mitochondria, chloroplasts, etc.

  • Bound by ≥1 membranes

  • Proteins define function of organelles

Potential destinations for proteins

• Cytosol = fluid around organelles (main part of cytoplasm)

• Extracellular/secreted = outside cell

• Plasma membrane = part of membrane

• Membrane of organelles

• Lumen of organelles = space within organelle

Where do the majority of proteins begin to be synthesized?

Cytosolic ribosomes

Pathway of protein targeting in eukaryotes

1. Protein synthesis @ cytosolic/free ribosomes

   1. Has targeting sequence (address)?

      1. NO → stays in cytosol

      2. YES → guided to specific destination (Mitochondrion, nucleus, chloroplast, endoplasmic reticulum)

2. Main pathway = ER/Secretion

   1. HAS signal peptide (address)

   2. Co-translational process

      1. Ribosome → ER WHILE protein synthesis occurs

      2. Protein pushed through pore (translocator) into ER lumen

   3. Protein fold inside ER → vesicles → Golgi apparatus

      1. Golgi sorts protein to final destination (Plasma membrane/lysosome)

Production of cytosolic proteins

1. mRNA leaves nucleus

2. mRNA binds to cytosolic/free ribosome

3. Protein = translated & folds (potentially w/ chaperones/chaperonins)

Which is more complex?

1. Production of cytosolic proteins

2. Production & targeting organellar proteins

Production and targeting organellar proteins = MORE COMPLEX

Cellular topology

Structure connectivity, neighborhood relationships, & arrangement of components within cell or b/w cells in a tissue.

What are the 3 types of transport of proteins across organelle membranes?

• Gated = nuclear ↔ cytosol

  • Folded protein moved through aqueous pores → compartment (topologically similar)

• Transmembrane = ER transport

  • Folded/unfolded protein through non-aqueous transport complexes → compartment (topologically similar)

• Vesicular

  • Protein → lumen or membrane of small vesicles (bubbles) fuse w/ compartment (topology conserved)

Topologically similar compartment = spaces w/ same connectivity & structure (connected by vesicles)

Topology conserved = stay within membranes, never cross membrane

• move through budding/fusion

• Preserve orientation

What are two ways that transport of proteins can occur? + Understandings

• Post-translationally = after protein synthesis = complete

• Co-translationally = @ same time as protein synthesis

• Understandings:

  • Gated transport = post-translational

  • Transmembrane transport = post-/co-translational

  • Vesicular transport ONLY OCCURS AFTER transmembrane transport

• Post-translational transmembrane transport = Cytosolic chaperones

  • Keep protein unfolded to ensure protein can pass through

Explain how amino acid sequences on proteins work?

• No label = Cytosolic protein → stays in cytoplasm

• Targeting signal = short sequences of amino acids

  • Specific labels for specific organelle (Nucleus, mitochondria, ER)

• Contiguous amino acid sequences = combinations of labels/multiple sequences

• Location of label of protein depends on function of organelle protein

  • Recognized by other proteins (receptors) → initiate sequence → protein directed to correct place

What are two forms that targeting signals can take after the protein folds?

• Signal sequence: formed from n-terminal signal sequence

• Signal patch: formed from regions contributing to signal patch

What are the 5 types of targeting signals?

• Endoplasmic reticulum @ N-terminus, REMOVED

  • 6-12 hydrophobic amino acids

  • Preceded by 1+ basic amino acid (Arg, Lys)

• Mitochondrion (matrix) @ N-terminus, REMOVED

  • 20-50 residues form amphipathic helix

  • Basic amino acid (Arg, Lys) and hydrophobic residues on opposite sides

• Chloroplast (stroma) @ N-terminus, REMOVED

  • No common motifs

  • Rich in Ser, Thr, & small hydrophobic residues

  • Poor in Glu & Asp

• Peroxisome (matrix) most @ C-terminus, few @ N-terminus NOT REMOVED

  • PTS1 signal @ extreme C-terminus

  • PTS2 signal @ N-terminus

• Nucleus (nucleoplasm) usually @ C-terminus, NOT REMOVED

  • Multiple kinds

  • Short segment of Lys & Arg residues (+ charged)

5 components of Nuclear Structure

• Nuclear envelope = double membrane around nucleus

  • Outer membrane ↔ Rough Endoplasmic Reticulum

  • Space b/w membranes topologically = outside/interior of ER

  • Nuclear pore complex controls entry & exit

• Nuclear lamina = network of intermediate filaments (Lamins) under nuclear envelope → support

• Nucleoplasm = fluid in nucleus

• Chromatin = DNA/protein complex

• Nucleolus = DNA/protein complex hold rRNA genes for ribosomal RNA production

  • Where ribosomal subunits assembly

Where are nuclear proteins from?

• Imported bc protein synthesis = cytoplasm

  • Nuclear proteins = DNA polymerase, histones, lamins, etc.

  • All nuclear proteins MUST be reacquired → targeting signals

    • Nucleus breaks down & reforms after each cell division

What happens to many RNAs produced in nucleus?

• Exported

• Some reimported w/ associated proteins

  • mRNA → nucleus → cytoplasm

  • Ribosomal subunits assembled in nucleolus = in nucleus → out to cytoplasm

  • Small nuclear RNAs (snRNAs) → cytoplasm → assembled w/ proteins → nucleus

    • SnRNPs = regulate mRNA splicing

• Nuclear Pore Complex = controls entry & exit

Nuclear Pore Complex

• Large aqueous channel/pore though double nuclear membrane

  • Movement = selective barrier in both directions

• Structure = ring, basket, plug

• 50 proteins

• 3000-4000 per nucleus in typical cell

  • 30x mass of ribosome

  • Can help cell transport ribosome through it

SEM of nuclear membranes

TEM of nuclear membrane

Nuclear Pore Diffusion & size of molecule

• Molecule < 5000 MW (small) → passive diffusion

• Molecule = large → slower passive diffusion

• Molecule > 40-60,000 MW (600 amino acids) cannot freely cross

  • Size of average protein = 40,000 MW → active transportation

• Import/export = gated transport → proteins transported(post-translation)

• Nuclear transport = bidirectional

• NPC selectivity + nuclear targeting signals = different proteins in cytoplasm & nucleus

What diameter of particles can diffuse passively through nuclear pores?

• Particles < 9 nm = 9 × 10^-6 mm

• Particles > 9 nm → active transport

What is the direction of different substrates through the nuclear membrane?

Bidirectional

• Import: Histones, nonhistone proteins, ribosomal proteins

• Export: Ribosomal subunits, mRNA

Nuclear Localization Signal (NLS) + Experiment used to determine

Targeting sequences allowing for nuclear transport through the nuclear pore

• Xenopus oocyte (frog egg) = LARGE CELLS (1 mm)

• Inject substances into nucleus/cytoplasm → see Δ compartments

• Protein = nucleoplasmin = pentameric nuclear protein

Describe steps of preparing Nucleoplasmin targeting signal + 4 types of experiments

1. Purify nucleoplasmin protein

2. (Fluorescently/radioactively) Tag protein to determine localization

Experiment 1

1. Inject nucleoplasmin pentamer → oocyte (frog egg) cytoplasm

2. Nucleoplasmin taken up into nuclei

Experiment 2

1. Inject pentameric core + 1 tail

2. Nucleoplasmin taken up into nuclei

Experiment 3

1. Inject tail

2. Tail taken up into nucleus

Experiment 4

1. Inject pentameric heads into cytoplasm/nucleus

2. No movement

What are 3 conclusions that can be made from the nucleoplasmin injection experiments

1. Entire protein can be taken up into nuclei

2. Tail is required AND sufficient for uptake

   1. Head is NEITHER required nor sufficient

3. Start cutting off amino acids @ end of tail (C-terminal) until the shortened protein no longer enters the nucleus (= NLS = REMOVED → protein no longer enters nucleus)

   1. Defines nuclear localization signal (NLS)

What is the role of the Nuclear Localization Signal in nuclear import? + Where is it attached on the cytoplasmic protein?

• NLS = necessary and sufficient for nuclear targeting

• Attaches to C-TERMINUS → transported to nucleus

  • Necessary = NEEDED for protein → nucleus

  • Sufficient = ONLY SIGNAL NEEDED for protein → nucleus

What are type of mutation in a protein can demonstrate that NLS is necessary?

• Compare wild-type protein (positively charged amino acid - Lys, Arg)

  • Replace Lys for polar uncharged amino acid (Thr) → DESTROYS signal

• Proves that NLS is necessary

What protein can be targeted to nucleus to prove that NLS is sufficient?

• Pyruvate Kinase = enzyme involved in last step of glycolysis

Summary of Nuclear Localization Signals (NLS)

• Proteins imported to nucleus ALL HAVE NLS = (+) charged group of 5-6 amino acids near C-terminal end of protein

  • NLS sequence ≠ same for all nuclear proteins (similar, + charged)

• Transport after translation (post-translational) & proteins = folded

• (+) charged region of protein MUST be on surface of folded protein → interact with transport proteins/pore

• Signal = necessary + sufficient → protein → nucleus

  • Signal recognized by transport-regulating proteins

• Signal ≠ removed upon entering nucleus, stay attached, REUSED

  • Reused after each cell division (localization lost during cell division)

    • Nuclear envelope breaks down completely → proteins → cytoplasm

    • NLS remaining attached allows nuclear proteins to return to nucleus

• Transported through aqueous pore (passive or active depending on size)

Nuclear Export Signal (NES)

Leucine-rich short amino-acid sequence that acts as a signaling directs protein to be transported to cytoplasm